Search

Background: Platinum is a commonly used drug for ovarian cancer (OvCa) treatment, but drug resistance limits its clinical application. This study intended to delineate the effects of adipocytes on platinum resistance in OvCa., Methods: OvCa cells were maintained in the adipocyte-conditioned medium. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, separately. Proliferation and apoptosis-related protein expression were assayed by western blot. The IC 50 values of cisplatin and carboplatin were determined using CCK-8. IGF1 secretion and expression were assayed via ELISA and western blot, respectively. A xenograft model was established, and pathological changes were detected by H&E staining. Proliferation and apoptosis-associated protein expression was assessed via IHC., Results: Adipocytes promoted the viability and repressed cell apoptosis in OvCa, as well as enhancing platinum resistance, while the addition of IGF-1 R inhibitor reversed the effects of adipocytes on proliferation, apoptosis, and drug resistance of OvCa cells. Treatment with different concentrations of Ojeok-san (OJS) inhibited the adipocyte-induced platinum resistance in OvCa cells by suppressing IGF1. The combined treatment of OJS and cisplatin significantly inhibited tumour growth in vivo with good mouse tolerance., Conclusion: In summary, OJS inhibited OvCa proliferation and platinum resistance by suppressing adipocyte paracrine IGF1 secretion.

Recent studies have shown that dysregulation of transglutaminase 3 (TGM3) is related to the aggressive progression of several cancer types. Our study aimed to determine the function of TGM3 in cervical cancer (CC) tumorigenesis. Gene expression profiles GSE63514, GSE9750, GSE46857 and GSE67522 were obtained from the Gene Expression Omnibus (GEO) database. Overlapping differential expressed genes (DEGs) in CC were screened using GEO2R online tool and Venn diagram software. The Kaplan-Meier plotter was used to determine overall survival. TGM3 expression was analyzed based on GEO and The Cancer Genome Atlas (TCGA) databases, qRT-PCR and western blot analyses. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. The half-maximal inhibitory concentration (IC50) value of cisplatin and cell apoptosis was assessed by CCK-8 and TUNEL assays, respectively. P-glycoprotein (P-gp) expression and the changes of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway were examined using western blot analysis. We identified 3 overlapping DEGs, including TGM3, glutathione peroxidase 3 (GPX3), and alpha B-crystallin (CRYAB), which were downregulated in CC tissues. TGM3 expression was reduced in CC cells and related to the poor prognosis of CC patients. TGM3 overexpression retarded the proliferation, reduced IC50 value of cisplatin, accelerated cisplatin-induced apoptosis, and inhibited cisplatin-induced P-gp level in CC cells. Furthermore, TGM3 overexpression suppressed the PI3K/Akt pathway in CC cells. Moreover, treatment with 740Y-P, a PI3K activator, abolished the effect of TGM3 overexpression on proliferation and cisplatin resistance in CC cells. In conclusion, overexpression of TGM3 suppressed proliferation and cisplatin resistance in CC cells by blocking the PI3K/Akt pathway., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: This study aimed to evaluate the protective effects of gentiopicroside against lipopolysaccharide-induced chondrocyte inflammation., Methods: SW 1353 chondrosarcoma cells were stimulated with LPS (5 μg/ml) for 24 h and treated with different concentrations of gentiopicroside (GPS) for 24 h. The toxic effects of GPS on chondrocytes were determined using a CCK-8 assay and EdU staining. Western blotting, qPCR, and immunofluorescence analysis were used to examine the protective effect of GPS against the inflammatory response in chondrocytes induced by lipopolysaccharide (LPS). One-way ANOVA was used to compare the differences between the groups (significance level of 0.05)., Results: The CCK-8 results showed that 10, 20 and 40 μM GPS had no significant toxic effects on chondrocytes; GPS effectively reduced the production of IL-1β and PGE2, reversed LPS-induced extracellular matrix degradation in cartilage by inhibiting the Stat3/Runx2 signaling pathway, and suppressed the hypertrophic transformation of SW 1353 chondrosarcoma cells., Conclusion: Our study demonstrated that GPS significantly inhibited the LPS-induced inflammatory response and hypertrophic cellular degeneration in SW 1353 chondrosarcoma cells and is a valuable traditional Chinese medicine for the treatment of knee osteoarthritis., (© 2024. The Author(s).)

Ethnopharmacological Relevance: Modified Biejia Jianwan (M-BJJW), a Traditional Chinese Medicine (TCM) decoction, has exhibited great potential in treating hepatocellular carcinoma (HCC). However, its underlying functional mechanism still remains unknown., Aim of the Study: The study aimed to explore the anti-hepatocarcinogenic effects of M-BJJW, specifically its influence on PD-L1-mediated immune evasion in hypoxic conditions, and elucidate the related molecular mechanisms in HCC., Materials and Methods: To investigate the therapeutic efficacy and mechanisms underlying M-BJJW's effects on HCC, we employed a diethylnitrosamine (DEN)-induced rat model maintained for 120 days. Following model establishment, flow cytometry was utilized to assess the distribution of immune cell populations in peripheral blood, spleens, and tumor tissues after M-BJJW administration. Simultaneously, enzyme-linked immunosorbent assays (ELISA) were conducted to analyze cytokine profiles in serum samples. Immunohistochemistry was employed to determine the expression levels of crucial proteins within tumor tissues. Furthermore, HCC cells exposed to CoCl 2 underwent Western blot analysis to validate the expression levels of HIF-1α, PD-L1, STAT3, and nuclear factor kappa B (NF-κB) p65. The modulatory effects of STAT3 and NF-κB p65 were investigated using specific inhibitors and activators in wild-type cell lines. High-performance liquid chromatography coupled with mass spectrometry (HPLC/MS) was utilized to identify the chemical constituents present in M-BJJW-medicated serum. The immunomodulatory properties and the anti-tumor activities of M-BJJW were evaluated by co-culturing with peripheral blood mononuclear cells (PBMC) and the CCK-8 assay. Additionally, we assessed M-BJJW's impact on hypoxia-induced alterations in HCC cell lines using immunofluorescence and Western blot assessments., Results: M-BJJW exhibited substantial therapeutic advantages by effectively alleviating pathological deterioration within the HCC microenvironment. In the DEN-induced rat model, M-BJJW administration notably reduced tumor growth. Flow cytometry analyses revealed an increased proportion of Cytotoxic T lymphocytes (CTLs) accompanied by a simultaneous decrease in regulatory T cells (Tregs). ELISA data supported a marked decrease in pro-inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor α (TNF-α). Immunohistochemistry confirmed the suppressive effect of M-BJJW on the expression of HIF-1α and PD-L1. Notably, western blotting unveiled the role of HIF-1α in regulating PD-L1 expression via the STAT3 and NF-κB signaling pathways in HCC cell lines, which was validated using activators and inhibitors of STAT3 and NF-κB. The CCK-8 assay and co-culture techniques demonstrated the anti-tumor activity of M-BJJW. Immunofluorescence and western blotting further confirmed that M-BJJW-containing serum dose-dependently inhibited HIF-1α, PD-L1, p-STAT3, and p-p65 in hypoxic HCC cell lines., Conclusions: M-BJJW demonstrates significant therapeutic potential against HCC by influencing the hypoxic microenvironment, thereby regulating the immunosuppressive milieu. Specifically, M-BJJW modulates the HIF-1α/STAT3/NF-κB signaling pathway, leading to reduced PD-L1 expression and an elevated ratio of cytotoxic T lymphocytes (CTLs), while concurrently decreasing T regulatory cells (Tregs) and immunosuppressive factors. These synergistic effects aid in countering PD-L1-mediated immune evasion, presenting compelling pharmacological evidence supporting the clinical application of M-BJJW as a therapeutic approach for HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: As a common chronic musculoskeletal condition, osteoarthritis (OA) presently lacks particular treatment strategies. The aim of this study was to examine how AT-III therapies affected macrophage repolarity in order to slow down the advancement of OA., Methods: RAW264.7 macrophages were polarized to M1 subtypes then administered with different concentrations of AT-III. Immunofluorescence, qRT-PCR and flow cytometry were used to assess the polarization of the macrophages. The mechanism of AT-III repolarize macrophages was evaluated by western blot. Furthermore, the effects of macrophage conditioned media (CM) on the migration, proliferation, and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were investigated using CCK-8 assays, the scratch test, and alcian blue staining. The effects of macrophage CM on chondrocyte proliferation and degeneration were investigated using CCK-8 and qRT-PCR. In vivo micro-CT and histological observations were performed on rats with anterior cruciate ligament transection and partial medial meniscectomy, either with or without AT-III treatment., Results: AT-III repolarized M1 macrophages to M2 phenotype. Mechanistically, AT-III reduced the expression of Toll-like receptor(TLR) 4 induced by lipopolysaccharide in RAW264.7 and lowered nuclear factor-κB (NF-κB) signaling molecules p-p65 and p-IκBα. The TLR4 agonist RS09 reversed the effects of AT-III on macrophage repolarization. AT-III-induced macrophages CM stimulated BMSCs migration, proliferation and chondrogenic differentiation. AT-III-treated macrophage CM promoted chondrocyte proliferation while inhibiting chondrocyte degeneration. In vivo, AT-III treatment alleviated the degree of synovitis, inhibited subchondral bone remodeling and reduced cartilage destruction in the rat OA model., Conclusions: AT-III attenuates OA by repolarizing macrophages through inactivating TLR4/NF-κB signaling. These data suggest that AT-III may be an effective therapeutic candidate for OA treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Antioxidant 1 copper chaperone (Atox1) may contribute to preventing DDP cochlear damage by regulating copper transport family and cell cycle proteins. A rat model of cochlear damage was developed by placing gelatin sponges treated with DDP in the cochlea. HEI-OC1 cells were treated with 133 μM DDP as a cell model. DDP-induced ototoxicity in rats was confirmed by immunofluorescence (IF) imaging. The damage of DDP to HEI-OC1 cells was assessed by using CCK-8, TUNEL, and flow cytometry. The relationship between Atox1, a member of the copper transport protein family, and the damage to in vivo / vitro models was explored by qRT-PCR, western blot, CCK-8, TUNEL, and flow cytometry. DDP had toxic and other side effects causing cochlear damage and promoted HEI-OC1 cell apoptosis and cell cycle arrest. The over-expression of Atox1 (oe-Atox1) was accomplished by transfecting lentiviral vectors into in vitro / vivo models. We found that oe-Atox1 increased the levels of Atox1, copper transporter 1 (CTR1), and SOD3 in HEI-OC1 cells and decreased the expression levels of ATPase copper transporting α (ATP7A) and ATPase copper transporting β (ATP7B). In addition, the transfection of oe-Atox1 decreased cell apoptosis rate and the number of G2/M stage cells. Similarly, the expression of myosin VI and phalloidin of cochlea cells in vivo decreased. Atox1 ameliorated DDP-induced damage to HEI-OC1 cells or rats' cochlea by regulating the levels of members of the copper transport family., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background and Objectives: The utilization of natural products to enhance the function of periodontal ligament cells (PDLCs) has emerged as a popular area of research. Recent investigations have demonstrated that sappanchalcone (SC) possesses pharmacological properties such as anti-inflammatory and osteoprotective effects. This study aims to explore the impact of SC on the in vivo and in vitro osteogenic differentiation ability of PDLCs., Materials: Cell proliferation was quantified using the CCK-8 assay, while gene expression levels were assessed through qRT-PCR analysis. Osteoblast differentiation capacity was evaluated by employing Alizarin red staining (ARS), alkaline phosphatase (ALP) staining and western blot (WB) analysis. A rat model of periodontitis was established utilizing the tether-wire method. Micro-CT imaging and hematoxylin and eosin (HE) staining were employed to evaluate alveolar bone resorption. Masson's trichrome staining was utilized to observe fiber alignment, whereas immunohistochemistry (IHC) techniques were applied for detecting osteogenic and inflammatory factors., Results: The results from the CCK-8 assay indicate no observed cytotoxicity for concentrations of 1, 5, or 10 nM for SC treatment (p < .05), while qRT-PCR analysis demonstrates a significant decrease in inflammatory factors such as MMP-1 and IL-6 with treatment by SC (p < .05). Additionally, western blotting reveals an increase in protein expression levels of Runx2 and OPN within PDLCs treated with SC compared to control groups (p < .05), which is further supported by ARS and ALP staining indicating an increase in mineralized nodules formation along with elevated ALP content within these cells following treatment with this compound (p < .05). Finally, both HE staining as well as micro-CT imaging suggest potential benefits associated with using this compound including slowing alveolar bone resorption while simultaneously promoting junctional epithelium proliferation., Conclusions: Our in vitro and in vivo findings suggest that SC can effectively enhance the inflammatory response of PDLCs and promote their osteogenic differentiation ability under inflammatory conditions, indicating its potential as a promising therapeutic agent for improving periodontal inflammation and bone formation., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: To clarify the effect of genistein(GEN) on osteogenic differentiation and explore the effect of GEN loaded by platelet-rich fibrin (PRF) on the repair process of bone defects in obese mice., Methods: In in vitro experiments, the effect of GEN(0, 0.1, 1, 10, 50 μmol/L) on the proliferation of mouse embryonic osteoblast precursor cells (MC3T3-E1) was determined by CCK 8. Alkaline phosphatase(ALP) staining and quantitative detection of ALP activity were performed to determine the changes of ALP activity in cells; RNA and protein expression levels of ALP, osteopontin (OPN) and osteocalcin (OCN) were detected by quantitative real-time PCR(qRT-PCR) and Western blot. Alizarin red staining was used to define the effect of GEN on mineralization of MC3T3-E1. To verify the feasibility of the PRF drug loading, the ultrastructure of PRF was subsequently observed under SEM. In in vivo experiments, obese C57 mouse models were established by high-fat diet feeding. On this basis, skull defect models with a diameter of 2.8 mm were established, and the prepared GEN/PRF complexes were placed into the bone defect area. The effects of GEN on skull defect repair in obese mice were evaluated by Micro-CT scanning and hematoxylin-eosin(H-E) staining. Statistical analysis was performed with GraphPad Prism 5.0 software package., Results: CCK 8 results showed that 0.1, 1 μmol/L GEN promoted cell proliferation within 7 days(P＜0.05); 10 μmol/L GEN had no significant effect on the process of cell proliferation. From the second day, 50 μmol/L GEN significantly inhibited cell growth and showed cytotoxicity(P＜0.05). These two concentrations had similar effects in promoting cellular osteogenic differentiation. SEM results showed that PRF presented a 3-dimensional network structure, providing space for loading drug molecules. In in vivo experiments, the body weight of mice in the high-fat diet (HFD) group was 27.7% greater than that in the normal diet group(P＜0.05) and had abnormal glucose tolerance (P＜0.05). Micro-CT showed that compared with the normal diet group, the number of bone trabeculae in the femur of obese mice was decreased(P＜0.05), the distance between bone trabeculae was widened(P＜0.05), and the bone density was decreased (P＜0.05). In addition, GEN (0.1, 1.0 μmol/L) loaded by PRF increased bone volume fraction in the skull of obese mice (P＜0.05). H-E results showed that GEN/PRF promoted the healing of the bone defects., Conclusions: GEN promotes osteogenic differentiation of MC3T3-E1, and it can effectively accelerate the healing of cranial bone defects after loading with PRF in obese mice.

This study aimed to explore the molecular mechanism of LCN2 regulating aerobic glycolysis on abnormal proliferation of HCC cells. Based on the prediction of GEPIA database, the expression levels of LCN2 in hepatocellular carcinoma tissues were detected by RT-qPCR analysis, western blot, and immunohistochemical staining, respectively. In addition, CCK-8 kit, clone formation, and EdU staining were used to analyze the effect of LCN2 on the proliferation of hepatocellular carcinoma cells. Glucose uptake and lactate production were detected using kits. In addition, western blot was used to detect the expressions of aerobic glycolysis-related proteins. Finally, western blot was used to detect the expressions of phosphorylation of JAK2 and STAT3. We found LCN2 was upregualted in hepatocellular carcinoma tissues. CCK-8 kit, clone formation, and EdU staining results showed that LCN2 could promote the proliferation in hepatocellular carcinoma cells (Huh7 and HCCLM3 cells). Western blot results and kits confirmed that LCN2 significantly promotes aerobic glycolysis in hepatocellular carcinoma cells. Western blot results showed that LCN2 could significantly upregulate the phosphorylation of JAK2 and STAT3. Our results indicated that LCN2 activated the JAK2/STAT3 signaling pathway, promoted aerobic glycolysis, and accelerated malignant proliferation of hepatocellular carcinoma cells., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ethnopharmacological Relevance: Hemsleya amabilis Diels, belongs to cucurbitaceae, was traditional Chinese medicine (TCM). It is widely used to treat various diseases. However, these diseases may contribute to the development of RCC., Aim of the Study: investigated the anticancer activities of root extract of Hemsleya amabilis Diels (HRE), and elucidated the underlying molecular mechanism in vivo and in vitro., Materials and Methods: Dried Hemsleya amabilis Diels roots were extracted by ethyl acetate and used to treat RCC4, OS-RC-2 and ACHN cells. UHPLC-MS was used to analyze the chemical composition of the extract. CCK-8 and colony formation assay were used to investigate proliferation. PI staining was used to detect cell cycle. Annexin-V-FITC, AO/EB and TEM were used to evaluate apoptosis. Transwell and wound healing assays were used to evaluate migration and invasion. RNA-seq, Network pharmacology, autodocking for virtual screening and molecular dynamics simulation were used to analyze potential molecular mechanisms and active components of HRE inhibiting proliferation of RCC. LY294002 and UC2288 were used to inhibit PI3K and P21 expression, respectively. IGF-1 was used to activate PI3K. Xenograft tumor model was established to evaluate its anti-tumor potential in vivo. Immunohistochemistry and Western blot were used to test protein expression levels. H&E staining was used to explore the side effects of HRE in vivo. Applying bioinformatics to analyze the effect of P21 on RCC., Results: HRE consists of 739 compounds. CCK-8 and colony formation assay showed that HRE significantly inhibited RCC cells proliferation. PI staining indicated that HRE caused G 2 /M phase arrest. Annexin-V-FITC, AO/EB and TEM experiments revealed that HRE significantly promoted apoptosis of RCC cells. Transwell and wound healing assays showed that HRE can inhibit the migration and invasion of RCC cells. RNA-seq showed that HRE induced 230 gene changes. Network pharmacology analysis found the relationship between HRE-component-target-RCC. Auto-docking found that Epitulipinolide diepoxide in HRE can stably bind to PIK3CA (-7.22 kJ/mol), and molecular dynamics simulation verified the combination between Epitulipinolide diepoxide of PIK3CA. In RCC4 cells, pretreatment with IGF-1, attenuated HRE-induced apoptosis and G 2 /M arrest. When pretreated with PIK3 inhibitor LY294002, the opposite result appears. Pretreatment with CDKN1A (P21) inhibitor UC2288 attenuated HRE-induced G 2 /M arrest. Xenograft tumor model showed that HRE inhibited tumor growth. Western blot analysis indicated that HRE can regulating Bax, Bcl-2, PARP, cleared-PARP, Caspase-9, Caspase-8, Caspase-3, Survivin, Cyclin-B1, CDK1, N-cadherin, snail, slug, E-cadherin, MMP-9. Immunohistochemical staining showed that in the treated group, expression of E-cadherin, Bax, P21 was up-regulated, while N-cadherin, PI3K, AKT and Bcl-2 were down-regulated. H&E staining showed that compared to control groups, the main organs in the HRE-treated groups showed no histological abnormalities. The overall survival rate of RCC patients in the high-expression group of P21 was higher than in the low-expression group of P21 on bioinformatics analysis., Conclusions: HRE inhibited RCC migration and invasion through EMT, and inhibited proliferation in vivo and in vitro. In addition, HRE inhibited proliferation through promoting apoptosis and P21-induced G 2 /M phase arrest via PI3K/AKT signaling pathway. Overall, these results suggest that HRE may be a promising chemotherapy agent for RCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cholecystokinin (CCK) is a peptide that has been implicated in pain modulation. Acid sensitive ion channels (ASICs) also play an important role in pain associated with tissue acidification. However, it is still unclear whether there is an interaction between CCK signaling and ASICs during pain process. Herein, we report that a functional link between them in rat dorsal root ganglion (DRG) neurons. Pretreatment with CCK-8 concentration-dependently increased acid-evoked ASIC currents. CCK-8 increased the maximum response of ASICs to acid, but did not changed their acid sensitivity. Enhancement of ASIC currents by CCK-8 was mediated by the stimulation of CCK2 receptor (CCK2R), rather than CCK1R. The enhancement of ASIC currents by CCK-8 was prevented by application of either G-protein inhibitor GDP-β-S or protein kinase C (PKC) inhibitor GF109203×, but not by protein kinase A (PKA) inhibitor H-89 or JNK inhibitor SP600125. Moreover, CCK-8 increased the number of action potentials triggered by acid stimuli by activating CCK2R. Finally, CCK-8 dose-dependently exacerbated acid-induced nociceptive behavior in rats through local CCK2R. Together, these results indicated that CCK-8/CCK2R activation enhanced ASIC-mediated electrophysiological activity in DRG neurons and nociception in rats. The enhancement effect depended on G-proteins and intracellular PKC signaling rather than PKA and JNK signaling pathway. These findings provided that CCK-8/CCK2R is an important therapeutic target for ASIC-mediated pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Prostate cancer (PC) is the most common malignancy in male reproductive system. Sennoside A (SA) is an anthraquinone active ingredient extracted from Rheum officinale Baill., which exerts anti-tumor activity on different tumors. In the present study, the toxicity of SA on PC3 and DU 145 cells was detected via CCK-8. The effects of SA on growth, apoptosis, and autophagy were determined through CCK-8, Hoechst stain, flow cytometry, western blot, and immunofluorescence examinations. An in vivo experiment was performed in xenografted mice with intraperitoneal introduction of 10 mg/kg SA and validated via TUNEL, immunohistochemistry and western blot. The results showed that SA inhibited the cell viability with a IC50 value of 52.36 and 67.48 µM in DU 145 and PC3 cells respectively, and enhanced the apoptosis of PC3 and DU 145 cells. Additionally, SA elevated the relative LC3B expression, and the relative protein expression of LC3II/LC3I and Beclin-1, but diminished the P62 protein expression. The relative protein level of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR was reduced with SA treatment, which was verified by the 740 Y-P application. The 740 Y-P treatments also restored the SA-induced the cell viability, apoptosis rate and relative LC3B expression. Meanwhile, SA inhibited the growth of PC cell and the relative protein level of PI3K/AKT/mTOR axis in vivo. Taken together, SA regulated the proliferation, apoptosis and autophagy via inactivating the PI3K/AKT/mTOR axis in PC., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Objective: Osteosarcoma is a rare primary malignant tumor of the bone characterized by poor survival rates, owing to its unclear pathogenesis. Rho GTPase-activating protein 44 (ARHGAP44), which belongs to the Rho GTPase-activating protein family, has promising applications in the targeted therapy of tumors. Therefore, this study aimed to investigate the biological function of ARHGAP44 in osteosarcoma and its possible application as a therapeutic target., Methods: The expression level of ARHGAP44 in osteosarcoma and its relationship with tumor prognosis were detected using Gene Expression Omnibus database analysis and immunohistochemical staining of clinical specimens. The cell model of ARHGAP44 knockdown was constructed, and the effects of this gene on the malignant biological behavior of osteosarcoma cells were investigated using CCK-8, clone formation, transwell invasion, wound healing, and flow cytometry assays. Western blotting was performed to detect the expression of ARHGAP44, p53, C-myc, and Cyclin D1 in osteosarcoma., Results: Biogenic analysis showed that ARHGAP44 was highly expressed in osteosarcoma. This result was associated with poor tumor prognosis and negatively correlated with the expression of the tumor suppressor gene p53. Immunohistochemistry and western blotting revealed significantly upregulated expression of ARHGAP44 in osteosarcoma tissues. Additionally, Kaplan-Meier analysis of clinical specimens suggested that ARHGAP44 was negatively correlated with tumor prognosis. CCK-8, clone formation, transwell invasion, wound healing, and flow cytometry assays showed that downregulation of ARHGAP44 expression significantly reduced the malignant biological behavior of osteosarcoma cells. Furthermore, western blotting showed that the expression level of p53 in osteosarcoma cells was significantly increased after the downregulation of ARHGAP44 expression, whereas the expression of C-myc and Cyclin D1 was significantly decreased compared with that in the control group., Conclusion: ARHGAP44 was highly expressed in osteosarcoma and was negatively correlated with its prognosis. The downregulation of ARHGAP44 expression reduced the malignant biological behavior of osteosarcoma cells. These findings suggest that the downregulation of ARHGAP44 expression inhibits the malignant progression of osteosarcoma by regulating the p53/C-myc/Cyclin D1 pathway, demonstrating the potential of ARHGAP44 as a therapeutic target for osteosarcoma., (© 2023. The Author(s).)

Background: Runt-related transcription factor 1 (RUNX1), also called acute myeloid leukaemia 1, is a member of RUNX family of transcription factors. This family is composed of evolutionarily conserved transcription factors that function as critical lineage determinants in various tissues, however its function in cancer development and clinical significance in RCC are still unknown., Methods: We used paraffin-embedded tumor tissues from 100 patients and fresh-harvested and paired adjacent normal renal tissues from 15 RCC patients who underwent primary surgical resection in Xijing Hospital between 2018 and 2022. The expression level of RUNX1 was evaluated by immunohistochemistry and Western Blot. RUNX1 promoted tumor cells proliferation, migration and invasion were verified by CCK-8, wound-healing and transwell assays. Finally, we constructed a xenografts model of the 786-O cell lines to observe the effect of RUNX1 on tumorigenesis in vivo., Results: TCGA database showed higher RUNX1 expression levels in KIRC (kidney renal clear cell carcinoma). In overall survival analysis, RCC patients with higher RUNX1 expression level would have a shorter survival period than those with lower expression. Similarly, immunohistochemical results of our cohort also showed that RUNX1 was over-expression in cancer tissues than in corresponding non-cancer tissues. We also proved this result at protein level by western-blot. Meanwhile, prognostic and OS analyses of our cohort showed that the RUNX1 expression level was an individual prognostic factor in RCC patients. CCK-8, wound-healing and transwell assays proved that the overexpression of RUNX1 in Caki-1 cells promoted the proliferation, migration and invasion of the cells. Knocking down RUNX1 in 786-O cells inhibited the proliferation, migration and invasion of cells. The experimental results of xenografts model in nude mice showed that the knockdown of RUNX1 in 786-O cells slowed down the growth of tumor., Conclusion: RUNX1 is a poor prognostic factor of clear cell renal carcinoma, which may provide a novel therapeutic target for ccRCC., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

In a fasting gastrointestinal tract, a characteristic cyclical rhythmic migrating motor complex (MMC) occur that comprises of three phases: I, II, and III. Among these, phase III contractions propagate from the stomach to the lower intestine in mammals, including humans, dogs, and Suncus murinus (suncus). Apart from the phase III of MMC propagating from the stomach, during the gastric phase II, small intestine-originated strong contractions propagate to the lower small intestine; however, the mechanism of contractions originating in the small intestine has not been clarified. In this study, we aimed to elucidate the role of cholecystokinin (CCK) in small intestinal motility. Administration of sulfated CCK-8 in phase I induced phase II-like contractions in the small intestine, which lasted for approximately 10-20 min and then returned to the baseline, while no change was observed in the stomach. Contractions of small intestine induced by CCK-8 were abolished by lorglumide, a CCK1 receptor antagonist. Gastrin, a ligand for the CCK2 receptor, evoked strong contractions in the stomach, but did not induce contractions in the small intestine. To examine the effect of endogenous CCK on contractions of small intestinal origin, lorglumide was administered during phase II. However, there was no change in the duodenal motility pattern, and strong contractions of small intestinal origin were not abolished by treatment with lorglumide. These results suggest that exogenous CCK stimulates contractions of small intestine via CCK1 receptors, whereas endogenous CCK is not involved in the strong contractions of small intestinal origin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Triple-negative breast cancer (TNBC) is a heterogeneous carcinoma characterized by the most aggressive phenotype among all breast cancer subtypes. However, therapeutic options for TNBC patients have limited clinical efficacy due to lack of specific target and efficient targeted therapeutics., Aim: To investigate the biological characteristics of a novel estrogen receptor (ER)-α splice variant ER-α30 in breast cancer cells, and its possible role in the anticancer effects of calycosin, a typical phytoestrogen derived from the herbal plant Astragalus membranaceus, against TNBC. This may also provide a better understanding of the inhibitory activity of calycosin on TNBC progression., Methods: Breast cancer tissues and para-cancer tissues were collected and analyzed for the expression levels of ER-α30 using immunohistochemistry (IHC), and its expression in two TNBC cell lines (MDA-MB-231 and BT-549) was detected by western blot and qRT-PCR assays. Then the alteration of cell viability, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in response to overexpression or knockdown of ER-α30 was separately determined by CCK-8, Hoechst 33258, wound healing, transwell and western blot assays in two TNBC cell lines. Next, the anticancer effects of calycosin on MDA-MB-231 cells were evaluated through CCK-8, colony formation, flow cytometry, Hoechst 33258 and western blot assays, along with the role of ER-α30 in these effects and the possible downstream targets of ER-α30. In addition, the in vivo experiments were carried out using MDA-MB-231 xenograft model intraperitoneally treated with calycosin. The volume and weight of xenograft tumor were measured to evaluate the in vivo anticancer activities of calycosin, while the corresponding changes of ER-α30 expression in tumor tissues were detected by IHC., Results: It was demonstrated that the novel ER-α splice variant ER-α30 was primarily distributed in the nucleus of TNBC cells. Compared with normal breast tissues, ER-α30 expression was found in significantly higher levels in breast cancer tissues of ER- and progesterone receptor (PR)-negative subtype, so did in TNBC cell lines (MDA-MB-231 and BT-549) when compared to normal breast cell line MCF10A. Moreover, ER-α30 overexpression strikingly enhanced cell viability, migration, invasion and EMT progression and reduced apoptosis in TNBC cells, whereas shRNA-mediated knockdown of ER-α30 revealed the opposite results. Notably, calycosin suppressed the expression of ER-α30 in a dose-dependent manner, accompanied with the inhibition of TNBC growth and metastasis. A similar finding was observed for the xenografts generated from MDA-MB-231 cells. The treatment with calycosin suppressed the tumor growth and decreased ER-α30 expression in tumor tissues. Furthermore, this inhibition by calycosin was more pronounced in ER-α30 knockdown cells. Meanwhile, we found a positive relationship between ER-α30 and the activity of PI3K and AKT, which could also be inactivated by calycosin treatment., Conclusion: For the first time, it is demonstrated that the novel estrogen receptor-α splice variant ER-α30 could function as pro-tumorigenic factor in the context of TNBC by participating in cell proliferation, apoptosis, invasion and metastasis, thus it may serve as a potential therapeutic target for TNBC therapy. Calycosin could reduce the activation of ER-α30-mediated PI3K/AKT pathway, thereby inhibited TNBC development and progression, suggesting that calycosin may be a potential therapeutic option for TNBC., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.

Background: Cervical cancer (CC) is a common gynecological malignancy with high morbidity worldwide. Butyrate, a short-chain fatty acid produced by intestinal flora, has been reported to inhibit cervical carcinogenesis. This study aimed to investigate the pro-apoptotic effects of butyrate on CC and the underlying mechanisms., Methods: Human HeLa and Ca Ski cells were used in this study. Cell proliferation, cell migration and invasion were detected by CCK-8 and EdU staining, transwell and wound healing assay, respectively. Cell cycle, mitochondrial membrane potential and apoptosis were evaluated by flow cytometry. Western blot and RT-qPCR were carried out to examine the related genes and proteins to the mitochondrial complex Ι and apoptosis. Metabolite changes were analyzed by energy metabolomics and assay kits. The association between G protein-coupled receptor 41, 43, 109a and CC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA)., Results: CCK-8 results showed significant inhibition of CC cell proliferation induced by butyrate treatment, which was confirmed by EdU staining and cell cycle detection. Data from the transwell and wound healing assay revealed that CC cell migration was dramatically reduced following butyrate treatment. Additionally, invasiveness was also decreased by butyrate. Western blot analysis showed that cleaved Caspase 3 and cleaved PARP, the enforcers of apoptosis, were increased by butyrate treatment. The results of Annexin V/PI staining and TUNEL also showed an increase in butyrate-induced apoptotic cells. Expression of Cytochrome C (Cytc), Caspase 9, Bax, but not Caspase 12 or 8, were up-regulated under butyrate exposure. Mechanistically, the decrease in mitochondrial NADH and NAD + levels after treatment with butyrate was observed by energy metabolomics and the NAD+/NADH Assay Kit, similar to the effects of the complex Ι inhibitor rotenone. Western blot results also demonstrated that the constituent proteins of mitochondrial complex Ι were reduced by butyrate. Furthermore, mitochondria-dependent apoptosis has been shown to be initiated by inhibition of the complex Ι., Conclusion: Collectively, our results revealed that butyrate inhibited the proliferation, migration and invasion of CC cells, and induced mitochondrial-dependent apoptosis by inhibiting mitochondrial complex Ι., (© 2023. The Author(s).)

Ethnopharmacological Relevance: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H 2 O 2 and its potential molecular mechanisms need larger studies., Aim of the Study: We established an IPEC-J2 cell damage model induced by H 2 O 2 to explore the protective mechanism of koumine on intestinal injury., Materials and Methods: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H 2 O 2 -induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H 2 O 2 -trigged IPEC-J2 cell damage., Results: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H 2 O 2 -induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1β, IL-6, IL-8, TNF-α and TGF-β), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H 2 O 2 . Moreover, koumine dramatically attenuated H 2 O 2 -triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-ΚB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H 2 O 2 -stimulated IPEC-J2 cell inflammation., Conclusions: This in vitro experimental study suggests that koumine suppresses the H 2 O 2 -induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: The treatment of chronic myeloid leukemia (CML) is facing the dilemma of tyrosine kinase inhibitors (TKIs) resistance and disease recurrence. The dysfunctional DNA damage repair mechanism plays an essential role not only in the initiation and progression of hematological malignancies but also links to the development of TKI resistance. Deciphering the abnormally regulated DNA damage repair and proteins involved brings new insights into the therapy of leukemias. As a G2/M phase checkpoint kinase and a DNA damage repair checkpoint kinase engaged in the DNA damage response (DDR), along with an oncogenic driver present in various cancers, the particular involvement of Wee1 in DNA damage is far from clear. Deciphering its function and targeting it via modulating DNA repair pathways is important for improving our understanding of cancer treatment., Methods: Wee1 expression was assessed in cell lines using RT-qPCR and western blot, and Wee1 knockdown efficacy was validated using RT-qPCR, western blot, and immunofluorescence. Wee1 function was investigated by CCK-8, colony formation, and flow cytometry assay in vitro. Wee1 role in DNA repair and its interactions with other proteins were then studied using western blot, immunofluorescence, and double plasmid-repair studies. Finally, the CCK-8 and flow cytometry assay was utilized to investigate Wee1 and imatinib's synergistic effect, and a CML mouse model was constructed to study Wee1's role in carcinogenesis in vivo., Results: Wee1 was reported to respond quickly to DDR in an ATM-γH2AX-MDC1-dependent way upon DNA double-strand breaks (DSBs) occurrence, and it regulated homologous recombination by stimulating the recruitment of critical proteins RAD51/BRCA1 upon DSB sites. Wee1 was also revealed to be abnormally upregulated in CML cells. Further suppression of Wee1 not only causes cell cycle arrest and inhibits the proliferation of cancer cells but also enhances CML cell sensitivity to Imatinib in vitro and in vivo, possibly through an excessive accumulation of overall DSBs., Conclusion: Wee1 is extensively involved in the DRR signaling and DSB repair pathway. Inhibiting abnormally elevated Wee1 benefits CML therapy in both IM-resistant and IM-sensitive cells. Our data demonstrated that Wee1 participated in promoting cell proliferation and imatinib resistance in chronic myeloid leukemia via regulating DNA damage repair dependent on ATM-γH2AX-MDC1. In the fight against CML, Wee1's dysregulation in the DNA damage repair mechanism of CML pathogenesis makes it a viable therapeutic target in clinical applications., (© 2022. The Author(s).)

Objectives: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus. Oxidative stress resulting from high glucose promotes accumulation of ECM and development of DN. Activation of Nrf2 could attenuate oxidative stress and following accumulation of ECM. To find novel therapy for DN, we explored the effects of swinhoeic acid from Potentilla fragarioides on mesangial cells under high glucose and underlying mechanisms., Methods: CCK-8 and BrdU incorporation assays for survival of mesangial cells gave the concentration of swinhoeic acid in following investigations. ROS, MDA, SOD and CAT were determined. And ECM proteins and their upstream regulators TGF-β 1 and CTGF were detected using ELISA assays. Activation of Nrf2 was explored by immunofluorescence staining together with luciferase reporter assay. To demonstrate the role of Nrf2 activation, siRNA interference was performed. And co-immunoprecipitation assay was used to elucidate swinhoeic acid affects the interaction between Keap1 and Nrf2., Results: Swinhoeic acid at 10 and 20 μM attenuated oxidative stress and accumulation of ECM in mesangial cells under high glucose. Itactivated Nrf2 in a Keap1-dependent manner, which was involved in its effects., Conclusion: Swinhoeic acid ameliorates oxidative stress and accumulation of ECM resulting from high glucose in mesangial cells via activating Nrf2 in Keap1-dependent manner.

Background: Based on extensive research on cytotoxicity of exogenous compounds in vitro, it is essential to develop a cell model that better mimics environment in vivo to explore cytotoxic mechanisms of exogenous compounds., Methods: A co-culture system was established using a transwell system with Beas-2B and U937 cells. Cells were treated with fine particulate matter (PM2.5; 25, 50 and 100 μg/mL), nicotine-derived nitrosamine ketone (NNK; 50, 100 and 200 μg/mL) and benzo(a)pyrene diol epoxide (BPDE; 0.5, 2 and 8 μM) for 24 h. Cell proliferation, apoptosis and cell cycle, DNA damage were detected by CCK-8 and EdU, flow cytometry, and comet assay, respectively. Differentially expressed transcript and cytokine concentrations were determined by transcriptome sequencing and Cytokine Array, respectively., Results: Compared with mono-culture, cell proliferation increased, apoptosis decreased, and DNA damage decreased in a dose-response relationship in co-culture. Gene expression profile was significantly different in co-culture, with significantly increased expression levels of 48 cytokines in co-culture., Conclusion: Cytotoxic damage to Beas-2B cells induced by exogenous carcinogens, including PM2.5, NNK and BPDE, was significantly reduced in a co-culture system compared with a mono-culture system. The mechanism may be related to changes in expression of cytokines, such as LIF, and activation of related pathways, such as TNF signaling pathway. Cytotoxic damage to Beas-2B induced by PM2.5, NNK and BPDE, was significantly reduced in co-culture. The mechanism may be related to changes in expression of cytokines and activation of related pathways. These findings provide new insights into cytotoxicity and experimental basis for safety evaluations of exogenous carcinogens., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: Oral squamous cell carcinoma (OSCC) is characterized by high recurrence and metastasis and places a heavy burden on societies worldwide. Cancer cells thrive in a changing microenvironment by reprogramming lipidomic metabolic processes to provide nutrients and energy, activate oncogenic signaling pathways, and manage redox homeostasis to avoid lipotoxicity. The mechanism by which OSCC cells maintain lipid homeostasis during malignant progression is unclear., Methods: The altered expression of fatty acid (FA) metabolism genes in OSCC, compared with that in normal tissues, and in OSCC patients with or without recurrence or metastasis were determined using public data from the TCGA and GEO databases. Immunohistochemistry was performed to examine the carboxylesterase 2 (CES2) protein level in our own cohort. CCK-8 and Transwell assays and an in vivo xenograft model were used to evaluate the biological functions of CES2. Mass spectrometry and RNA sequencing were performed to determine the lipidome and transcriptome alterations induced by CES2. Mitochondrial mass, mtDNA content, mitochondrial membrane potential, ROS levels, and oxygen consumption and apoptosis rates were evaluated to determine the effects of CES2 on mitochondrial function in OSCC., Results: CES2 was downregulated in OSCC patients, especially those with recurrence or metastasis. CES2 high OSCC patients showed better overall survival than CES2 low OSCC patients. Restoring CES2 expression reduced OSCC cell viability and suppressed their migration and invasion in vitro, and it inhibited OSCC tumor growth in vivo. CES2 reprogrammed lipid metabolism in OSCC cells by hydrolyzing neutral lipid diacylglycerols (DGs) to release free fatty acids and reduce the membrane structure lipid phospholipids (PLs) synthesis. Free FAs were converted to acyl-carnitines (CARs) and transferred to mitochondria for oxidation, which induced reactive oxygen species (ROS) accumulation, mitochondrial damage, and apoptosis activation. Furthermore, the reduction in signaling lipids, e.g., DGs, PLs and substrates, suppressed PI3K/AKT/MYC signaling pathways. Restoring MYC rescued the diminished cell viability, suppressed migratory and invasive abilities, damaged mitochondria and reduced apoptosis rate induced by CES2., Conclusions: We demonstrated that CES2 downregulation plays an important role in OSCC by maintaining lipid homeostasis and reducing lipotoxicity during tumor progression and may provide a potential therapeutic target for OSCC., Competing Interests: Conflict of Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Vascular smooth muscle cell (VSMC) phenotypic modulation regulates the initiation and progression of intracranial aneurysm (IA). Dexmedetomidine (DEX) is suggested to play neuroprotective roles in patients with craniocerebral injury. Therefore, we investigated the biological functions of DEX and its mechanisms against IA formation and progression in the current study. The rat primary VSMCs were isolated from Sprague-Dawley rats. IA and superficial temporal artery (STA) tissue samples were obtained from patients with IA. Flow cytometry was conducted to identify the characteristics of isolated VSMCs. Hydrogen peroxide (H 2 O 2 ) was used to mimic IA-like conditions in vitro. Cell viability was detected using CCK-8 assays. Wound healing and Transwell assays were performed to detect cell motility. ROS production was determined by immunofluorescence using DCFH-DA probes. Western blotting and RT-qPCR were carried out to measure gene expression levels. Inflammation responses were determined by measuring inflammatory cytokines. Immunohistochemistry staining was conducted to measure α 2 -adrenergic receptor levels in tissue samples. DEX alleviated the H 2 O 2 -induced cytotoxicity, attenuated the promoting effects of H 2 O 2 on cell malignancy, and protected VSMCs against H 2 O 2 -induced oxidative damage and inflammation response. DEX regulated the GSK-3β/MKP-1/NRF2 pathway via the α2AR. DEX alleviates the inflammatory responses and oxidative damage of VSMCs by regulating the GSK-3β/MKP-1/NRF2 pathway via the α2AR in IA., (© 2022. The Author(s).)

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is the ninth member of the proprotein convertase family that regulates lipoprotein homeostasis and altered PCSK9 expression was reportedly associated with tumor development and progression. This study assessed PCSK9 expression and functions in human colon cancer and then explored the underlying molecular events., Methods: Colon cancer tissues were utilized for analysis of PCSK9 expression for association with clinicopathological factors from patients by immunohistochemistry assay. Manipulation of PCSK9 expression was assessed in vitro and in vivo for colon cancer cell proliferation, migration, and invasion using cell viability CCK-8, Transwell tumor cell migration and invasion, and wound-healing assays. Next, proteomic analysis, Western blot, qRT-PCR and Flow cytometry were conducted to assess downstream targets and tumor cell-derived PCSK9 action on macrophage polarization., Results: PCSK9 expression was upregulated in colon cancer tissues versus the normal tissues, and associated with advanced tumor pathological grade. Knockdown of PCSK9 expression reduced colon cancer cell proliferation, migration, and invasion and suppressed tumor metastasis in vivo. PCSK9 directly or indirectly upregulated Snail 1 and in turn to downregulate E-cadherin expression, but upregulate N-cadherin and MMP9 levels and thereafter, to induce colon cancer cell epithelial-mesenchymal transition (EMT) process and activated PI3K/AKT signaling. However, PCSK9 overexpression showed the inverse effects on colon cancer cells. Knockdown of PCSK9 expression inhibited M2 macrophage polarization, but also promoted M1 macrophage polarization by reduction of lactate, protein lactylation and macrophage migration inhibitory factor (MIF) levels., Conclusion: PCSK9 played an important role in the progression and metastasis of colon cancer by regulation of tumor cell EMT and PI3K/AKT signaling and in the phenotypic polarization of macrophages by mediating MIF and lactate levels. Targeting PCSK9 expression or activity could be used to effectively control colon cancer., (© 2022. The Author(s).)

This study investigated the possibility of exosomes loaded with si-PDGFR β ability to suppress the progression of glioma. Common gliomas develop from neuroglial progenitor cells. Many variables affect the survival rate and occurrence of gliomas. Understanding oxidative stress processes and creating new, efficient treatments are crucial because oxidative stress is linked to the development of brain tumors. For this purpose, selected clinical samples were subjected to various tests like quantitative real-time PCR, Cignal Finder RTK signaling 7-pathway reporter array analysis, CCK-8 analysis, flow cytometry, and immunoblotting. Here, we demonstrated that PDGFR β expression was increased in glioma patients. Following that, cell-derived exosomes were extracted and collected and traced in vivo, and selected tissue samples were subjected to immunohistochemical analysis. The results indicated that the knockdown of PDGFR β (si-PDGFR β ) inhibited the proliferation of glioma cells. Besides this, si-PDGFR β -loaded exosomes induced a similar antitumor effect in glioma cells. The anticancer effect of si-PDGFR β -loaded exosomes was mediated by the inactivation of the PI3K/Akt/EZH2 pathway. Finally, we verified that this exosome delivery system, si-PDGFR β -loaded exosomes, had robust targeting and no associated toxicity. In conclusion, the study confirmed that si-PDGFR β -loaded exosomes inhibit glioma progression via inactivating the PI3K/Akt/EZH2 signaling pathway., Competing Interests: The authors state that there is no conflict of interest., (Copyright © 2022 Yuping Li et al.)

Aim: Thin endometrium remains a severe clinical challenge with no effective therapy to date. We aimed at exploring the role and molecular mechanism of human umbilical cord mesenchymal stem cell- (hucMSC-) derived exosomes (hucMSC-Ex) in repairing hypoxic injury of endometrial epithelial cells (EECs)., Methods: Exosomes were harvested from the conditioned medium of hucMSC and characterized using western blot, transmission electron microscopy (TEM), flow cytometry, and nanoparticle tracking analysis (NTA). EECs were subjected to hypoxic conditions before cocultured with hucMSC-Ex. Cell viability, apoptosis, and migration were determined with CCK-8, flow cytometry, and wound healing assay, respectively. Apoptosis/EMT-related proteins were detected by western blot. The miRNA profiling was determined by RNA sequencing. The expression of miR-663a and CDKN2A was measured by qRT-PCR. MiR-663a in EECs was overexpressed by transfecting with miR-663a mimics., Results: Mesenchymal stem cells (MSCs) markers CD73, CD90, and CD106 were positively expressed in hucMSCs. Exosome isolated from hucMSC expressed CD63 and TSG101, and were 100-150 nm in diameter. HucMSC-Ex promoted cell proliferation inhibited by hypoxia. And hucMSC-Ex also inhibited hypoxia-induced apoptosis, migration, and EMT of EECs by upregulating the expression of Bcl-2 and E-cadherin and downregulating Bax and N-cadherin levels. Further, bioinformatics research found that hucMSC-Ex coculture can significantly upregulate the expression of miR-663a and decrease the expression of CDKN2A in hypoxia-induced EECs. Furthermore, miR-663a overexpression inhibited CDKN2A expression and increased the expression of Bcl-2 and E-cadherin in hypoxia-induced EECs., Conclusions: HucMSC-Ex promoted cell proliferation, inhibited cell apoptosis, migration, and EMT in hypoxia-induced EECs, thereby alleviating hypoxia-induced EECs injury, which may be related to its regulation of miR-663a/CDKN2A expression. Our study indicated that hucMSC-Ex might benefit for repairing thin endometrium., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Hanbi Wang et al.)

Pancreatic stellate cells (PSCs) constitute the fibrotic tumor microenvironment composed of the stroma matrix, which blocks the penetration of gemcitabine (GEM) in pancreatic adenocarcinoma (PDAC) and results in chemoresistance. We analyzed the expression of α-SMA, collagen type I, and fibronectin by immunohistochemistry of pancreatic cancer tissues and demonstrated that the abundant interstitial stroma is associated with dismal survival. Two desmoplastic pancreatic tumor models are treated with arsenic trioxide (ATO) and GEM to confirm the sensitizing effect of ATO on GEM. RNA-seq was performed to analyze the potential fibrotic genes regulated by ATO. Western blotting, CCK-8 methods, colony formation, and wound healing and transwell assays were utilized to verify that ATO attenuates the tumor-promoting ability of PSCs by inhibiting its activation and decreasing matrix secretion via the PI3K/AKT/AP4/galectin-1 pathway. Furthermore, we developed targeted ATO-loaded nanoparticles self-assembled by poly (D,L-lactide) and poly(ethylene glycol) (PEG-PDLLA) and modified by the single-chain antibody of FAP-α (scAb FAP-α ) (scAb-ATO-NPs) to promote the delivery efficiency of ATO to PSCs and enhance anti-tumor effects with gemcitabine. Herein, we elucidate the mechanism of how ATO inhibits the activation of PSCs and enhances the therapeutic effect of GEM. We propose a novel cocktail therapy including scAb-ATO-NPs and GEM, indicating a new perspective in the treatment of PDAC.

Background: Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on the function of bovine mammary epithelial cells (BMECs) and milk synthesis under heat stress., Methods and Results: We cultured the BMECs in fetal bovine serum (FBS) or exosome-free FBS medium and examined, their viability using CCK-8 kit. The results showed that culturing the cells in an exosome-free medium decreased viability and increased the levels of reactive oxygen species. The BMECs cultured in the exosome-free medium had reduced mitochondrial membrane potential, decreased manganese superoxide dismutase activity, and disrupted mitochondrial dynamics. They exhibited apoptosis due to upregulated Drp1, Fis1, Bax and HSP70. Lastly, we observed downregulation of milk fat and lactoprotein-related genes: mTOR, PPARγ, p-mTOR and ADD1 and SREBP1, ELF5, and CSN2, respectively, after culturing the cells in an exosome-free medium. These negative effects of the exosome-free medium on the BMECs could be further reinforced under heat stress., Conclusion: Our results demonstrated that exosomes from serum are critical for maintaining the normal function of BMECs., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Human chorionic villous mesenchymal stem cells (CV-MSCs) are a promising and effective therapeutic option for tissue injury. Vascular dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work aims to investigate how CV-MSCs regulate the function of vascular endothelial cells. In this study, RNA-seq analysis was used to examine the changes in HUVECs treated with CV-MSC conditioned medium (CM). We examined the levels of ABCA9 and AKT signaling in human umbilical vein endothelial cells (HUVECs) by immunohistochemistry, western blotting, and qRT-PCR assays. CCK-8, colony formation, and tube formation assays were used to understand the role of ABCA9 in HUVEC proliferation and angiogenesis mediated by CV-MSCs. The CV-MSC treatment significantly enhanced the HUVEC proliferation and angiogenesis. Furthermore, a significant increase in the ABCA9 expression and AKT pathway activation was observed in CV-MSCs -treated HUVECs. Consistent with these findings, ABCA9 overexpression exhibited the same proliferation-and angiogenesis-promoting effect in HUVECs as induced by CV-MSC CM, also accompanied the AKT signaling activation. In addition, inhibition of ABCA9 inactivated the AKT signaling in HUVECs and reduced the HUVEC proliferation and angiogenesis. Importantly, the elevation of proliferation and angiogenesis induced by ABCA9 overexpression in HUVECs could be reversed by AKT pathway inhibition. Our results suggest that ABCA9-dependent AKT signaling activation mediated by CV-MSCs could promote HUVEC proliferation and angiogenesis., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Objective: To investigate the potential antitumor effect and its mechanism of dihydroartemisinin (DHA) on diffuse large B-cell lymphoma (DLBCL)., Methods: OCI-Ly7 cells were respectively treated with different concentrations of DHA (0, 12.5, 25, 50 and 100 μmol/L) , CCK-8 was used to detect the cells viability. Subsequently, OCI-Ly7 cells were divided into 5 groups : DHA 0,25,50,100 μmol / L and DHA (100 μmol / L) + Colivelin (STAT3 activator). Aldehyde dehydrogenase (ALDH) positive cells were sorted by flow cytometry, the sphere-forming ability of stem cells was detected. Transwell assay and scratch test were used to analyze the invasion and migration of cells. Western blot was used to detect the expression of migration and invasion-related proteins, as well as the phosphorylation levels of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3(STAT3)., Results: DHA induced obvious cytotoxicity to OCI-Ly7 cells. Compared with the control group, the stem cell-like properties, invasion and migration of OCI-Ly7 were significantly inhibited in DHA 50 μmol/L group and 100 μmol/L group, while the phosphorylation levels of JAK2 and STAT3 were significantly reduced. There was no significant difference in DHA 25 μmol/L group compared with the control group. Treated with Colivelin, the inhibition of DHA on OCI-Ly7 stem cell-like properties, invasion and migration was significantly reversed, and the expression of p-STAT3 was significantly up-regulated., Conclusion: DHA has antitumor effect on DLBCL, and its mechanism may be through inhibiting the activation of JAK2/STAT3 pathway to inhibit the stem cell-like properties, invasion and migration of DLBCL cells.

Context: The high expression of long non-coding RNA deleted in lymphocytic leukaemia 2 (lncRNA DLEU2) has been confirmed in gastric cancer (GC). Objective: However, the detailed mechanism concerning its involvement in GC remained unclear, which we aimed to explore in this study. Materials and methods: LncRNA DLEU2 expression in GC was estimated by bioinformatic analysis, and the relationship between the expression of DLEU2 and the clinicopathological characteristics of patients with GC was performed. qRT-PCR was employed to detect the expression of lncRNA DLEU2 and confirm the transfection efficiency following the knockdown or overexpression of DLEU2. Functional assays, including CCK-8, flow cytometry, scratching test and Transwell assays, were used to determine the role of DLEU2 in tumor phenotypes. The effects of DLEU2 on the PI3K/Akt pathway were detected by western blot. For elucidating the functions of DLEU2/PI3K/Akt axis in GC, we inhibited the PI3K/Akt pathway in rescue experiments, and evaluated the expression levels of epithelial-mesenchymal transition (EMT)-related proteins by western blot. Results: The expression of DLEU2 was aberrantly up-regulated in GC tissues and cells, which was correlated with the degree of tumor differentiation, cancer antigen 19-9 (CA19-9) and Lauren histologic classification of patients with GC. Silencing of DLEU2 induced apoptosis, attenuated viability, migration and invasion as well as inhibited the PI3K/Akt signaling pathway in GC cells. Mechanistically, the DLEU2/PI3K/Akt axis promoted the progression of GC and the EMT by down-regulating the expression of E-Cadherin and up-regulating those of N-Cadherin and Vimentin. Discussion and conclusions: LncRNA DLEU2 promoted the migration, invasion and EMT in GC by activating the PI3K/Akt pathway.

Rheumatoid arthritis (RA) is a systemic autoimmune disease, which has been reported closely associated with the dysfunction of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. This study aims to explore the potential therapeutic effect of Tofacitinib, a putative JAK/STAT inhibitor, in RA. Tofacitinib suppressed proliferation and accelerated apoptosis of rheumatoid arthritis synovial fibroblasts (RA-FLS) as confirmed by CCK-8, EdU and Western blot assays. Tofacitinib significantly inhibited expression of pro-inflammatory factors including tumor necrosis factor-α (TNF-α), vascular endothelial growth factor A, matrix metalloproteinase 1, matrix metalloproteinase 3, interleukin-6 and interferon gamma in RA-FLS cells. mechanistically, tofacitinib decreased signal transducer and activator of transcription 6 (STAT6), which transcriptionally activates miR-425-5p, and thus increased insulin like growth factor 1 (IGF1) expression, a target of miR-425-5p in RA-FLS. Overexpression of STAT6 restored the expression of pro-inflammatory factors and proliferation inhibited by Tofacitinib in RA-FLS. Overall, Tofacitinib exerted inhibitory effect on proliferation and inflammation of RA-FLS through modulating STAT6/miR-425-5p/IGF1 signal axis. These findings shed light on the novel strategies for improving RA., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: The main target of current drugs for alleviating bone loss is osteoclasts. However, the long-term application of such drugs will also cause side effects. Therefore, it is of great need to develop new and safer therapeutics for osteoporosis. In recent years, drug development based on gut microbiota has gradually attracted attention. This manuscript investigates the inhibitory effect of urolithin B (UB) on osteoclastogenesis and differentiation in vitro and in ovariectomized (OVX) mice., Materials and Methods: CCK-8 was used to analyse the cytotoxicity of UB; BMMs cells were differentiated into osteoclasts by RANKL, and respectively treated with 1, 5, and 25 μmol/L UB during this process. After one week of intervention, tartrate-resistant acid phosphatase (TRAP) staining was used to analyse the number and average area of osteoclasts. F-actin staining and immunofluorescence staining were conducted to evaluate the morphology and function of osteoclasts. Bone resorption function of osteoclasts was detected by Pit Formation Assay. The expression of osteoclast-related protein genes in RAW264.7 cells were investigated via western blot and RT-PCR assays. Western blot analysis of RANKL-mediated activation of MAPK/NF-κB pathway after 0, 5, 15, 30, 60 min of intervention. For in vivo experiments, OVX mice received intraperitoneal injection of 10, 50 mg/kg every two days, 8 weeks later, the femurs of mice were taken for morphological analysis, and the serum content of CTX-1, a bone metabolism index, was analysed., Results: UB could inhibit the osteoclast differentiation of rankl-induced bone marrow macrophages (BMMs) and RAW264.7 cells in vitro, suppress the uptake activity of hydroxyapatite and expression of osteoclast-related gene MMP9, CTSK, NFATc1 and c-fos. Furthermore, UB repressed the rankl-induced phosphorylation and degradation of IκB and the phosphorylation of P65 in the NF-κB pathway of RAW264.7 cells, and also down-regulated the phosphorylation level of ERK in the MAPK pathway. For in vivo studies, UB-treated OVX mice showed more significant improved various parameters of distal femur compared with the control group, with fewer NFATc1, MMP9 and TRAP-positive osteoclasts in bone tissues, and less serum content of CTX-1., Conclusion: Urolithin B attenuated bone loss in OVX mice by inhibiting the formation and activation of osteoclasts via down-regulation of the ERK/NF-κB signalling pathway., (© 2022 The Authors. Cell Proliferation published by European Cell Proliferation Society and John Wiley & Sons Ltd.)

1. Chicken primary myoblasts (CPMs) are precursors that form muscle fibres. The proliferation and differentiation of CPMs is an essential stage in muscle development. Previous RNA-seq analysis showed that phosphoglycerate dehydrogenase ( PHGDH ) is a differentially expressed gene in chicken muscle tissue at different growth stages. Therefore, the following study explored the effect of PHGDH on the proliferation and differentiation of CPMs.2. The effect on the proliferation of CPMs by RT-qPCR, CCK-8, and EdU assays after the overexpression and knockdown of PHGDH was evaluated. RT-qPCR, western blotting, and indirect immunofluorescence were used to detect the effect of PHGDH on the differentiation of the CPMs. The expression was observed at different time points for differentiation induced by the CPMs.3. The results showed that PHGDH significantly promoted proliferation and differentiation in CPMs. The results showed that overexpression of PHGDH significantly upregulated CPM proliferation, while knockdown had the opposite effect. Marker genes showed that overexpression of PHGDH significantly upregulated the expression of P21, MYOG and MYOD genes, significantly downregulated the expression of the MSTN gene and promoted the expression of the MYHC protein. In contrast, PHGDH knockdown had the opposite effect.4. Desmin immunofluorescence analysis of myotube differentiation in primary myoblasts showed that overexpression of PHGDH significantly increased the area of myotube differentiation and promoted the proliferation and differentiation of myoblasts. Knockdown of PHGDH had the opposite effect.5. In summary, PHGDH was shown to play a positive role in regulating myoblast proliferation and differentiation. This provides a theoretical basis for further analysis of the regulatory mechanism of the PHGDH gene in chicken muscle development and for improving poultry production.

Abstract: Sepsis leads to the damage of multiple organs, and thereby adversely affects the cardiovascular system. At present, no effective method has been found to treat myocardial injury caused by sepsis. Although Puerarin was reported to attenuate lipopolysaccharide (LPS)-induced mitochondrial injury in H9C2 cells, the effects of Puerarin in sepsis-induced myocardial injury remain unclear. In this study, H9C2 cells were stimulated with LPS, CCK-8 assays were performed to assess cell viability, and flow cytometry and TUNEL staining were used to assess cell apoptosis. Levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and enzyme activity were investigated using commercial kits. Reactive oxygen species (ROS) levels in H9C2 cells were detected by flow cytometry. Autophagosomes in the mitochondria of H9C2 cells were observed by transmission electron microscope, and protein expression was assessed by western blotting. Furthermore, in vivo experiments were applied to test the function of Puerarin in sepsis. We found that Puerarin significantly reversed LPS-induced decreases in H9C2 cell viability by inhibiting apoptosis. The ROS levels in H9C2 cells were significantly upregulated by LPS, but that effect was markedly reduced by Puerarin. In addition, Puerarin attenuated LPS-induced mitochondrial injury in H9C2 cells by regulating dynamin-related protein 1 (Drp1) and mitofusin 1 (MFN1). LPS decreased enzyme activity and reduced the levels of ADP, ALP, and AMP in mitochondria; however, those effects were reversed by Puerarin. Puerarin and Torin1 reversed LPS-induced inhibition of autophagy in the mitochondria of H9C2 cells via mediation of p62, LC3B, Pink1, and Parkin. Puerarin notably inhibited the progression of sepsis in vivo . Puerarin inhibited LPS-induced H9C2 cell injury by inducing mitochondrial autophagy, which acts as a mechanism for preventing myocardial injury caused by sepsis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Cholecystokinin (CCK) is a peptide hormone mainly secreted by small intestinal endocrine I-cells and functions as a regulator of gallbladder contraction, gastric emptying, gastrointestinal (GI) motility, and satiety. The cellular effects of CCK in these peripheral tissues are predominantly mediated via CCK-A receptors which are found in smooth muscles, enteric neurons, and vagal afferent neurons in humans and animal models. Although various functions of CCK have been reported to be neurally mediated, it can also stimulate contraction via the CCK receptor on the smooth muscle. However, the entire underlying neural and cellular mechanisms involved in CCK-induced GI contractions are not clearly understood. Here, we first determined the cDNA and amino acid sequences of CCK and CCK-A receptor along with the distributions of cck mRNA and CCK-producing cells in house musk shrew (Suncus murinus, the laboratory strain named as suncus) and examined the mechanism of CCK-induced contraction in the GI tract. Mature suncus CCK-8 was identical to other mammalian species tested here, and suncus CCK-A receptor presented high nucleotide and amino acid homology with that of human, dog, mouse, and rat, respectively. Suncus CCK mRNA and CCK-producing cells were found mainly in small intestine and colon. In the organ bath study, CCK-8 induced dose-dependent contractions in the suncus stomach, duodenum, and jejunum, and these contractions were inhibited by atropine and CCK-A receptor antagonist. These results suggest that CCK-8-induced contraction is mediated in the myenteric cholinergic neural network and that CCK-A receptor is partly responsible for CCK-8-induced contractions. This study indicates that suncus is a useful animal model to study the functions of CCK involved in GI motility., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The study aimed to explore tumor suppressor mechanism of ARNTL from the perspective of autophagy in oral cancer. Human oral squamous carcinoma HN6 cells stably overexpressing ARNTL were established, cell viability and apoptosis were detected by CCK-8 and TUNEL assays, and intracellular autophagosomes were observed under electron microscopy. Western Blot detected expressions of Beclin1, LC3 II/I, ATG-12, P62, BAX and BCL-2. Bafilomycin A1 was used to detect autophagic flux, and Western Blot was used to detect changes of LC3II and P62 proteins. Autophinib was added to cells with ARNTL overexpression for recovery experiments, and cell proliferation and apoptosis were detected by flow cytometry. In vivo tumorigenesis experiment was used to evaluate the in vivo anti-tumor efficacy of ARNTL, and Western blot simultaneously detected ARNTL, LC3 II/I, Beclin1, P62 and ATG-12 expressions. ARNTL overexpression promoted apoptosis and autophagy and inhibited cell viability. In ARNTL-overexpressing cells, expressions of Beclin1, LC3 II/I, and BAX were significantly up-regulated, while P62 and BCL-2 expressions were decreased, and ATG-12 expression wasn't significantly changed. When the autophagy inhibitor Autophinib was used, expressions of elevated BAX and decreased BCL-2 were reversed effectively, as were decreased cell proliferation index and increased apoptosis index. An in vivo tumorigenesis assay also showed ARNTL overexpression inhibited tumor growth, and autophagy-related protein expressions were consistent with the in vitro data. The research demonstrated for the first time that ARNTL induced apoptosis and inhibited cell proliferation dependent on autophagy in oral cancer, which provides theoretical basis for potential therapeutic targets., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Respiratory syncytial virus (RSV) infection can deteriorate asthma by inducing persistent airway inflammation. Increasing evidence elucidated that pyroptosis plays a pivotal role in asthma. Conciliatory anti-allergic decoction (CAD) exhibits an anti-inflammatory effect in ovalbumin (OVA)-induced asthma; however, the effects and mechanisms of CAD in RSV-infected asthmatic mice have not yet been elucidated. The RSV-infected asthmatic mice model and lipopolysaccharide (LPS)-induced 16HBE cell pyroptosis model were established, respectively. Pulmonary function, ELISA, and histopathologic analysis were performed to assess the airway inflammation and remodeling in mice with CAD treatment. Furthermore, ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) was conducted to identify the chemical compounds of high-dose CAD (30 g/kg). Cell viability and apoptosis of 16HBE cells were assessed by CCK-8 and flow cytometry assays, respectively. Finally, the expression levels of apoptosis-, pyroptosis-, and TLR3/NLRP3/NF- κ B/IRF3 signaling-related genes were measured with qRT-PCR or western blotting, respectively. Pulmonary function tests showed that CAD significantly ameliorated respiratory dysfunction, airway hyperresponsiveness, inflammation cell recruitment in BALF, pulmonary inflammation, collagen deposition, and cell death in lung tissues. CAD significantly decreased the content of TNF- α , IL-13, IL-4, IL-1 β and IL-5 in the bronchoalveolar lavage fluid (BALF), IL-17, IL-6, and OVA-specific IgE in serum and increased serum IFN- γ in asthma mice. The results of UPLC-Q-TOF/MS showed that high-dose CAD had 88 kinds of chemical components. In vitro, CAD-contained serum significantly suppressed LPS-induced 16HBE cell apoptosis. Additionally, CAD and CAD-contained serum attenuated the up-regulated expressions of Bax, Cleaved caspase-3, NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-18, IL-1 β , TLR3, p-P65, p-I κ B α , and IRF3 but increased Bcl-1 and GSDMD levels in the asthma mice and LPS-induced 16HBE cells, respectively. These results illustrated that CAD may have a potential role in improving airway inflammation and pyroptosis through inhibition of the TLR3/NLRP3/NF- κ B/IRF3 signaling pathway., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Ya-qin Chen et al.)

Blood brain barrier (BBB) dysfunction is a critical complication of diabetes mellitus type 2 (T2DM), and the oxidative stress-induced apoptosis of human brain microvascular endothelial cells (HBMECs) is a main cause of BBB dysfunction. In this study, oxygen and glucose deprivation/reoxygenation (OGD/R) models were established with HBMECs to analyze the effects of miR-602 on the apoptosis of HMBECs. Western Blot, qRT-PCR, CCK-8, flow cytometry assay, ROS detection assay, and dual-luciferase reporter gene assay were used to measure the expression levels of corresponding factors and changes in intracellular environment. The results showed that miR-602 was overexpressed in HBMECs after OGD/R treatment, and miR-602 could reduce ROS level of OGD/R-induced HBMECs and promote cells survival via increasing the expression level of NRF2 and the transcription activity of NRF2/ARE. Besides, it was found that KEAP1 and HRD1 were downstream factors of miR-602, and the increase of both KEAP1 and HRD1 could reverse the effects of miR-602 on the OGD/R-induced HMBECs. Therefore, miR-602 may be a potential target for research and treatment of the oxidative stress injury induced by apoptosis in HMBECs., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 Chunli Ma et al.)

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik . Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly ( P <0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P -Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P <0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences ( P <0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference ( P =0.024) , but there was no significant difference in the expression level of Bik mRNA ( P =0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups ( P =0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups ( P =0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups ( P =0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Lumbar spinal stenosis (LSS) is a common degenerative spinal condition in older individuals that causes impaired walking and other disabilities due to severe lower back and leg pain. Ligamentum flavum hypertrophy is a major LSS cause that may result from oxidative stress caused by degenerative cascades, including imbalanced iron homeostasis that leads to excessive reactive oxygen species production. We investigated the effects of Harpagophytum procumbens (HP) on iron-induced oxidative stress associated with LSS pathophysiology. Primary spinal cord neuron cultures were incubated in FeSO 4 -containing medium, followed by addition of 50, 100, or 200  μ g/mL HP. Cell viability was assessed by CCK-8 and live/dead cell assays and by propidium iodide-live imaging. In an in vivo rat model of LSS, HP were administered at 100, 200, and 400 mg/kg, and disease progression was monitored for up to 3 weeks. We investigated the in vitro and in vivo effects of HP on iron-induced neurotoxicity by immunochemistry, real-time PCR, and flow cytometry. HP exerted neuroprotective effects and enhanced neurite outgrowths of iron-injured rat primary spinal cord neurons in vitro . HP treatment significantly reduced necrotic cell death and improved cells' antioxidative capacity via the NRF2 signaling pathway in iron-treated neurons. At 1 week after HP administration in LSS rats, the inflammatory response and oxidative stress markers were substantially reduced through regulation of excess iron accumulation. Iron that accumulated in the spinal cord underneath the implanted silicone was also regulated by HP administration via NRF2 signaling pathway activation. HP-treated LSS rats showed gradually reduced mechanical allodynia and amelioration of impaired behavior for 3 weeks. We demonstrated that HP administration can maintain iron homeostasis within neurons via activation of NRF2 signaling and can consequently facilitate functional recovery by regulating iron-induced oxidative stress. This fundamentally new strategy holds promise for LSS treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Jin Young Hong et al.)

Alzheimer's disease (AD) is the most commonly seen neurodegenerative brain disorder. The paracrine effects of mesenchymal stem cells (MSCs) signify to trigger immunomodulation and neural regeneration. However, the role and mechanism of bone marrow MSC- (BMSC-) derived CX3CL1 in AD remains elusive. In this study, A β 1-42 -intervened SH-SY5Y cells were used for AD cell model construction. pcDNA-ligated CX3CL1 overexpression plasmids were transfected into BMSCs. The levels of soluble and membrane-bound CX3CL1 were detected by ELISA and Western blotting (WB), respectively. The growth, apoptosis, and pathology of AD model cells were evaluated by CCK-8, flow cytometry, immunofluorescence, morphology observation, biochemical examination, and WB. It was found that A β 1-42 significantly reduced CX3CL1 expression either in soluble or membrane-bound form, cell viability, relative protein expression of synaptic markers, SOD, CAT, and GSH-Px contents, as well as Trx protein expression; in addition, it enhanced the apoptosis rate, the relative expression of cleaved caspase-3, A β , tau, p-Tau, Iba1, MDA, TXNIP, and NLRP3 in SH-SY5Y cells; however, the above effects were prominently reversed by the coculture of BMSCs. Moreover, overexpression of CX3CL1 in BMSCs observably strengthened the corresponding tendency caused by BMSCs. In conclusion, through the TXNIP/NLRP3 pathway, CX3CL1 derived from BMSCs inhibited pathological damage in A β 1-42 -induced SH-SY5Y., Competing Interests: The authors report that there are no competing interests to declare., (Copyright © 2022 Chuan Guo et al.)

Objective: Gastric cancer is one of the most common and deadly cancers worldwide. Basic leucine zipper transcription factor ATF-like 3 (BATF3) plays a key role in tumor immunity. However, the function of BATF3 in gastric cancer remains unclear. Here, we demonstrated BATF3 positively regulated proliferation and radioresistance of gastric cancer cells by regulating S1PR1/STAT3 pathway., Methods: The RNA-seq analyzed the gene expression by UALCAN web portal and Tumor Immune Estimation Resource. RT-qPCR and western blot was performed to verify BATF3 expression in gastric cancer cells. The assays of CCK-8, EdU incorporation and colony formation were used to analyze cell proliferation, and radioresistance in AGS and MKN45 cells. Flow cytometry was used to detect the cell apoptosis of AGS and MKN45 in treatment with si-BATF3 or radiation. Finally, western blot was performed to measure the expression of cell apoptosis-related modules including Bax, cleaved-caspase3, cleaved-PARP and assess the regulation of S1PR1/STAT3 pathway., Results: BATF3 expression was upregulated in gastric cancer cells. Knockdown of BATF3 suppressed proliferation, radioresistance but promoted the radiation-induced apoptosis of gastric cancer cells through positively regulating S1PR1 expression and STAT3 phosphorylation., Conclusions: Knockdown of BATF3 inhibits gastric cancer cell growth and radioresistance via S1PR1/STAT3 pathway. BATF3 would become a potential diagnostic indicator for gastric cancer and target of therapeutic treatment., (© 2022 by the Association of Clinical Scientists, Inc.)

Background and Objective: Periodontitis is a chronic progressive inflammation that invades periodontal supporting tissues, in which periodontal tissue regeneration engineering offers new hope for prevention and treatment, including seed cells, scaffolds, and growth factors. In recent years, scholars have shown that autologous teeth can be used as new bone tissue repair materials for periodontal regeneration and bone tissue repair. The aim of this study was to establish a human periodontal ligament cell line that expresses the human bone morphogenetic protein 2 gene (BMP2) in a stable manner using lentiviral mediation in order to explore the effect of BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells (hPDLCs)., Materials and Methods: Human periodontal ligament cells were cultured, subcultured, and identified, and then homologous recombinant lentivirus plasmid plv-BMP2 was constructed and transfected into the third passage (P 3 ) hPDLCs. After that, the effect of BMP2 on its proliferation was detected by CCK-8, at the same time, the osteogenic induction of hPDLCs was carried out at 7, 14, and 21 days, and then the effect of BMP2 on its osteogenic ability was detected by alizarin red staining, alkaline phosphatase activity determination, and the mRNA expression levels of osteogenic-related genes using real-time fluorescence quantitative PCR, including alkaline phosphatase, runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen I. Finally, spss26.0 software was used for statistical processing., Results: The results showed that cells transfected with the homologous recombinant lentiviral plasmid pLV-BMP2 had a similar morphology to normal hPDLCs, showing a typical radial arrangement; the cell proliferative capacity of the pLV-BMP2 group as measured by CCK-8 was enhanced compared with the control group and the pLV-puro group (p < .05); alizarin red staining and alkaline phosphatase activity assay showed that the osteogenic ability of pLV-BMP2 was significantly enhanced compared with the control and pLV-puro groups (p < .01), and the findings of real-time fluorescence-based quantitative PCR showed high expression of osteogenic-related genes in pLV-BMP2 group (p < .01)., Conclusion: In conclusion, a stable periodontal ligament cell line overexpressing BMP2 was successfully established by a lentivirus-mediated method, which proved that BMP2 has a strong ability to promote the proliferation and osteogenesis of hPDLCs, thereby providing an opportunity for the study of periodontal tissue regeneration as well as providing an experimental basis for the application of autologous teeth as a new type of bone repair material for periodontal therapy and even for maxillofacial bone tissue repair., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: Non-small cell lung carcinoma (NSCLC) is a subtype of lung cancer with unfavorable outcome. Autophagy, a mechanism responsible for cellular component degradation, has been recorded to play either a positive or negative regulatory role in apoptosis. Tectonin Beta-Propeller Repeat Containing 1 (TECPR1) is recognized relevant to autophagy. This study aimed to investigate the molecular mechanisms through which TECPR1 regulates NSCLC cell apoptosis., Methods: Analysis of TECPR1 expression in the subcategories of NSCLC was conducted using GEPIA. Survival analysis for NSCLC patients was performed with Kaplan-Meier's plotter. The interaction between ATG5 and TECPR1 was predicted by STRING and validated through co-immunoprecipitation. NSCLC cells were transfected with short hairpin RNA against ATG5 and/or ATG5/TECPR1 overexpression plasmids, followed by viability and apoptosis assay using CCK-8 and flow cytometry. Expressions of TECPR1, ATG5, LC3-II/LC3-I, P62, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) in NSCLC cells with or without transfection were assessed by qRT-PCR and/or Western blot., Results: TECPR1 was low-expressed in LUAD and LUSC samples as well as NSCLC cells. Higher TECPR1 expression was associated with better outcomes. TECPR1 overexpression and ATG5 overexpression both decreased viability, promoted apoptosis, upregulated Bax and LC3-II/LC3-I, and downregulated P62 and Bcl-2. TECPR1 could form a complex with ATG5 in NSCLC cells. ATG5 was upregulated by TECPR1 overexpression and could positively modulate TECPR1 expression. ATG5 knockdown induced effect oppositely to TECPR1 overexpression, and this effect reversed the TECPR1 overexpression-induced effect and vice versa., Conclusion: TECPR1 induces NSCLC cell apoptosis via ATG5 upregulation-induced autophagy promotion., (© 2022 by the Association of Clinical Scientists, Inc.)

Objective: Activation of PI3K pathway has been reported to promote survival, tumorigenicity and metastasis in the esophageal cancer (EC), and the PI3Kp110β (one of PI3K family) contributed to tumorigenesis in types of cancers. However, it is still unclear whether PI3Kp110β effects the progression of EC., Methods: RT-qPCR and western blot were used to detect the expression of PI3Kp110β in the patients with EC and ECA-109 cells transfected with si-PI3Kp110β. CCK-8 and clonogenic assays were performed to analyze the proliferation of ECA-109 cells. Flow cytometry was used to investigate cell cycle and apoptosis of ECA-109 cells., Results: The expression of PI3Kp110β was up-regulated in the patients with EC by online RNA Sep data and RT-qPCR assay. Compared with the ECA-109 cells with si-control, the proliferation was suppressed in the ECA-109 cells with si-PI3Kp110β. The G1 phase of the cells with si-PI3Kp110β was significantly higher than that of cells with si-control. The apoptosis of cells with si-PI3Kp110β was accelerated, compared with that of cells with si-control. The expression of cyclinD1 and Bcl-2 was decreased, while the expression of Bax was increased in the ECA-109 cells with si-PI3Kp110β., Conclusion: Inhibition of si-PI3Kp110β attenuates cell viability and enhances apoptosis in esophageal cancer., (© 2022 by the Association of Clinical Scientists, Inc.)

Atrial natriuretic peptide (ANP), a canonical cardiac hormone, is mainly secreted from atrial myocytes and is involved in the regulation of body fluid, blood pressure homeostasis, and antioxidants. Cholecystokinin (CCK) is also found in cardiomyocytes as a novel cardiac hormone and induces multiple cardiovascular regulations. However, the direct role of CCK on the atrial mechanical dynamics and ANP secretion is unclear. The current study was to investigate the effect of CCK octapeptide (CCK-8) on the regulation of atrial dynamics and ANP secretion. Experiments were performed in isolated perfused beating rat atria. ANP was measured using radioimmunoassay. The levels of hydrogen peroxide (H 2 O 2 ) and arachidonic acid (AA) were determined using ELISA Kits. The levels of relative proteins and mRNA were detected by Western blot and RT-qPCR. The results showed that sulfated CCK-8 (CCK-8s) rather than desulfated CCK-8 increased the levels of phosphorylated cytosolic phospholipase A2 and AA release through activation of CCK receptors. This led to the upregulation of NADPH oxidase 4 (NOX4) expression levels and H 2 O 2 production and played a negative inotropic effect on atrial mechanical dynamics via activation of ATP-sensitive potassium channels and large-conductance calcium-activated potassium channels. In addition, CCK-8s-induced NOX4 subsequently upregulated peroxisome proliferator-activated receptor γ (PPAR γ ) coactivator-1 α (PGC-1 α ) expression levels through activation of p38 mitogen-activated protein kinase as well as the serine/threonine kinase signaling, ultimately promoting the secretion of ANP via activation of PPAR α and PPAR γ . In the presence of the ANP receptor inhibitor, the CCK-8-induced increase of AA release, H 2 O 2 production, and the upregulation of NOX4 and CAT expressions was augmented but the SOD expression induced by CCK-8s was repealed. These findings indicate that CCK-8s promotes the secretion of ANP through activation of NOX4-PGC-1 α -PPAR α /PPAR γ signaling, in which ANP is involved in resistance for NOX4 expression and ROS production and regulation of SOD expression., Competing Interests: The authors declare that there is no conflict of interest in this article., (Copyright © 2022 Zhuo-na Han et al.)

Objective: To explore the effects of fibroblast growth factor 3 (FGF3) on the proliferation, cell cycle, and apoptosis of the tongue squamous cell carcinoma SCC-9 cell line (SCC-9)., Methods: We measured the proliferation of SCC-9 cells in a control group, an FGF3 intervention group, and a fibroblast growth factor (FGFR) inhibitor intervention group in cholecystokinin octapeptide (CCK-8) experiments. We studied effects of FGF3 on the cell cycle and apoptosis of tongue cancer cells using flow cytometry. We further explored the IRS1/PI3K/AKT signaling pathway by measuring BCL-2 and Bcl-2 Associated X-protein (BAX) mRNA and protein levels with RT-PCR and western blot, respectively., Results: Results from the CCK-8 experiment showed that survival rates of cells in the control group, FGF3 intervention group, and FGFR inhibitor intervention group were 100.000% ± 4.026%, 136.330% ± 9.779%, and 83.199% ± 4.954%, respectively; survival rates of SCC-9 cells in all three groups were statistically significant ( P < 0.05). Compared with that in the control group, the ratio of cells in G0/G1 phase in the FGFR inhibitor intervention group was higher ( P < 0.05) and that in G2/M phase was lower, while the FGF3 intervention group showed opposite results ( P < 0.05). The apoptosis rate of tongue cancer cells differed significantly between the FGFR inhibitor intervention and the control groups ( P < 0.05). The mRNA and protein expression levels of IRS1, PI3K, and BCL-2 were all increased in the FGF3 intervention group ( P < 0.05), while BAX mRNA and protein expression levels were decreased ( P < 0.05). The mRNA expression levels of protein kinase B (AKT) showed no differences between groups. The p-AKT protein was overexpressed, while the total amount of AKT protein remained stable ( P < 0.05)., Conclusion: FGF3 contributes to the proliferation of SCC-9 cells by increasing the proportion of cells in G2/M phase. Therefore, appropriately timed inhibition of FGF3 can potentially promote tumor apoptosis through the IRS1/PI3K/AKT signaling pathway. Our results demonstrate the role of FGF3 in the tumor microenvironment in tongue squamous cell carcinoma SCC-9 cells and suggest new therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any conflicts of interest., (Copyright © 2022 Yang Zhang et al.)

Background and Purpose: Osteosarcoma is the most commonly seen type of primary malignant bone tumors in children and adolescents. Partial patients with osteosarcoma cannot tolerate the side effects of chemotherapy drugs. Hence, it is urgent to find anti-osteosarcoma drugs with low side effects. Melittin is an anti-tumor Traditional Chinese Medicine with low side effects. The purpose of this study was to explore the anti-osteosarcoma effect of melittin and its possible molecular mechanisms., Methods: The effects of melittin on cell growth were detected by CCK-8, clonal formation, and flow cytometry. The related molecules were also investigated by Real-time PCR and Western blot. A xenograft model in nude mice was established to observe the effects of melittin on tumor growth and the related molecular expression was detected by immunohistochemistry., Results: Melittin can inhibit the proliferation of osteosarcoma 143B cells, reduce colony formation, and induce apoptosis while significantly up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 proteins. Moreover, treatment with melittin significantly reduced the mRNA and protein levels of β-catenin and Wnt/β- catenin related genes (LRP5, c-Myc, and Survivin) in osteosarcoma 143B cells in vitro. The xenograft model found that melittin significantly inhibited tumor growth and decreased the protein expression levels of β-catenin and Wnt/β- catenin related genes in vivo., Conclusion: These findings show that melittin could inhibit the growth of osteosarcoma 143B cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway activity and induce apoptosis by up-regulating the ratio of Bax/Bcl-2 in osteosarcoma 143B cells. Therefore, melittin is a promising anti-tumor drug for the treatment of osteosarcoma., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)