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Background and aim: Achyranthis Bidentatae Radix plus Semen Vaccariae are traditional Chinese medicines, which have been widely applied in the treatment of migraine and Erectile Dysfunction (ED) for many years. This study verified the effect of Achyranthis Bidentatae Radix plus Semen Vaccariae in improving migraine-induced ED and explored its potential mechanism. Methods: Key targets and signaling pathways of Achyranthis Bidentatae Radix plus Semen Vaccariae in migraine-induced erectile dysfunction treatment were predicted by network pharmacology. A rat model of migraine was established by nitroglycerin injection. Apomorphine was injected into rats to screen the migraine-induced erectile dysfunction model, Achyranthis Bidentatae Radix-Semen Vaccariae granule suspension administered, and erectile function evaluated. Hematoxylin and eosin staining was used to compare the histological structure of the penile tissue, while RT-qPCR and Western blotting were used to determine mRNA and protein levels, respectively. Results: Screening allowed us to identify common targets for migraine and ED; the signaling pathway exhibiting the greatest change was the Myosin light chain kinase- Calcium (MLCK-CaM) signal pathway. From Western blotting and RT-qPCR, we found that the levels of MLCK mRNA and protein in rats from Group B rats were significantly higher (P Conclusion: Data indicate that the regulatory effects of Achyranthis Bidentatae Radix plus Semen Vaccariae on migraine-induced ED in a rat model are mediated by the MLCK-CaM signaling pathway.

Aim and Objective: We investigated the inhibitory effects of fractions from Lycopus lucidus Turcz. leaves on genomic DNA oxidation, Nitric Oxide (NO) production, and Matrix Metalloproteinase (MMP) activity. Material and Methods: Oxidative damage of genomic DNA was detected after Fenton reaction with H2O2 using DNA electrophoresis. Western blotting was performed to compare the expression levels of MMP-2 in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Lipopolysacchride (LPS)-induced NO production in RAW 264.7 cells was measured using Griess reagent. Results: All fractions (n-Hexane, 85% aq. MeOH, n-BuOH, and water fractions) from the leaves of L. lucidus Turcz. significantly inhibited intracellular production of reactive oxygen species (ROS) (p Conclusion: Overall, these results indicated that L. lucidus Turcz. leaves can be exploited as plant based sources of antioxidants in the pharmaceutical, cosmetic, nutraceutical, and food industries.

Background Di-(2-ethylhexyl) phthalate (DEHP) is the most common endocrine disrupting chemical used as a plasticizer. DEHP is associated with the development of endometrium-related diseases through the induction of inflammation. The major therapeutic approaches against endometrial cancer and endometriosis involve the suppression of inflammatory response. Korean red ginseng (KRG) is a natural product with anti-inflammatory and anti-carcinogenic properties. Thus, the purpose of this study is to investigate the effects of KRG on DEHP-induced inflammatory response in endometrial cancer Ishikawa cells and a mouse model of endometriosis. Methods RNA-sequencing was performed and analyzed on DEHP-treated Ishikawa cells in the presence and absence of KRG. The effects of KRG on DEHP-induced cyclooxygenase-2 (COX-2) mRNA levels in Ishikawa cells were determined by RT-qPCR. Furthermore, the effects of KRG on the extracellular signal-regulated kinases (ERKs) pathway, COX-2, and nuclear factor-kappa B (NF-κB) p65 after DEHP treatment of Ishikawa cells were evaluated by western blotting. In the mouse model, the severity of endometriosis induced by DEHP and changes in immunohistochemistry were used to assess the protective effect of KRG. Results According to the RNA-sequencing data, DEHP-induced inflammatory response-related gene expression was downregulated by KRG. Moreover, KRG significantly inhibited DEHP-induced ERK1/2/NF-κB/COX-2 levels in Ishikawa cells. In the mouse model, KRG administration significantly inhibited ectopic endometriosis growth after DEHP-induced endometriosis. Conclusions Overall, these results suggest that KRG may be a promising lead for the treatment of endometrial cancer and endometriosis via suppression of the inflammatory response.

Background The paper is aimed at uncovering the mechanism of miR-204-5p in regulating inflammatory responses of human osteoarthritic synovial fibroblasts (SFs). Methods IL-1β-induced osteoarthritic SFs were established as an osteoarthritis (OA) cell model. The osteoarthritic SFs were accordingly transfected with mimics-miR-204-5p, inhibitors-miR-204-5 or FOXC1 siRNA. MTT tested the vitality of osteoarthritic SFs by analyzing the cell optical density. The expressions of miR-204-5p, FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 in osteoarthritic SFs were measured by qRT-PCR, Western blotting and/or ELISA. The binding of miR-204-5p to FOXC1 was verified through luciferase reporter assay. The regulatory effect of miR-204-5p on FOXC1 was also tested in normal SFs. Results miR-204-5p was under-expressed and FOXC1 was over-expressed in osteoarthritic SFs. The expressions of FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 were up-regulated in IL-1β-treated SFs. Up-regulation of miR-204-5p or down-regulation of FOXC1 suppressed the inflammatory responses of osteoarthritic SFs. miR-204-5p negatively regulated FOXC1 by being a sponge in osteoarthritic SFs as well as in normal SFs. Conclusion miR-204-5p down-regulates FOXC1 to ameliorate inflammation of SFs in OA.

Background: Triple Negative Breast Cancer (TNBC) commonly displays Epidermal growth factor receptor (EGFR). Effective EGFR degradation results in the suppression of tumor in various models. Studies have addressed the relevance of this strategy in the treatment of TNBC. In the present study, we examined the effect of 17 β- estradiol on EGFR expression in MDA-MB-231 (TNBC) cell line and assessed whether 17 β-estradiol degrades EGFR by ubiquitination pathway. Objectives: The objectives of this study are to treat MDA-MB-231 cell lines with Cycloheximide with or without 17β-estrdiol to observe whether 17β-estradiol leads to EGFR degradation and to treat with MG-132 to assess whether degradation occurs through ubiquitination pathway. Methods: MDA-MB-231 cells were treated with 17β-estradiol (E2) and EGFR expression was studied by western blotting at different intervals by using Cycloheximide chase. To assess ubiquitination pathway of degradation of EGFR in MDA-MB-231 cell line, MG-132 was used. Results: EGFR expression was reduced with β-estradiol treatment in MDA-MB-231 cell line with Cycloheximide chase. Upon Treatment with MG-132 and E2, EGFR expression did not reduce, suggesting that Estrogen degrades EGFR by ubiquitination pathway. Conclusion: Estrogen degrades EGFR in MDA-MB-231 cells and this degradation occurs by ubiquitination.

Objective: Psoriasis is a chronic inflammatory skin disease highly depending on angiogenesis. Our prior results showed that the mRNA and protein of Del-1 in dermal mesenchymal stem cells (dMSCs) was up-regulated from psoriasis. Our aim was further to investigate the role of Del-1 from dMSCs in the pathogenesis of psoriasis and confirm the effect of Del-1 on the pathogenesis of psoriasis. Methods: We conducted an immunohistochemistry experiment to further investigate the expression of Del-1in psoriatic lesions. In addition, dMSCs with over-expressed Del-1 via the lentiviral vector of Del-1 were co-cultured with ECs, and the protein expression of integrins (αvβ3, αvβ5 ,and α5β1) of ECs were detected by western blotting. Results: This research showed that Del-1 was significantly increased in lesions of patients with psoriasis (p< .05, 9.96 vs. 2.18), and Del-1 from dMSCs successfully induced up-regulation of integrins α5β1 and αvβ3 (all p < .05). Conclusion: This study demonstrated that Del-1 from dMSCs was involved in the pathogenesis of psoriasis through induced angiogenesis. And Del-1, αvβ3 and α5β1 may be potential new targets for inhibiting angiogenesis in psoriasis.

Background: Neurofibrillary Tangles (NFTs), formed by hyperphosphorylation of Tau protein in Alzheimer's Disease (AD), arethe main pathomechanisms of neuronal degeneration, which indicate a sign of brain disorder. NFTs are positively correlated with the degree of cognitive impairment in AD. Objective: The objective of this study isto investigate the effect of flavonoids from the stems and leaves of Scutellaria baicalensis Georgi (SSF) on the hyperphosphorylated expression levels at multiple sites of Tau protein induced by β-amyloid protein 25-35 (Aβ25-35) in combination with aluminum trichloride (AlCl3) and recombinant human transforming growth factor-β1(RHTGF-β1) (composited Aβ) in rats. Methods: The AD rat models were established by intracerebroventricular injection of Aβ25-35 and AlCl3 combined with RHTGF-β1. On day 45, after the operation, the Morris water maze test was conducted to screen the memory impairment of AD models. The successful model rats were randomly divided into the model group and the three-dose drug group. The drug group rats were orally administered SSF daily for 38 days. Western blotting was performed to detect the protein expression of P-Tau (Thr 181), P-Tau (Thr 217), P-Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-- Tau (Ser 396), and P-Tau (Ser 404) in the hippocampus and cerebral cortex of rats. Results: Compared with the sham group, the expression of P-Tau (Thr 181), P-Tau (Thr 217), P-- Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-Tau (Ser 396), and P-Tau (Ser 404)was significantly increased in the hippocampus and cerebral cortex of the model group (P < 0.01). However, the three doses of SSF, i.e., 35, 70, and 140 mg/kg, regulated the expression of phosphorylated Tau proteinto varying degrees in the hippocampus and cerebral cortex of AD model rats (P < 0.01). Conclusion: SSF could significantly reduce the protein expression levels of P-Tau (Thr 181), PTau (Thr 217), P-Tau (Thr 231), P-Tau (Ser 199), P-Tau (Ser 235), P-Tau (Ser 396), and P-Tau (Ser 404), induced by the intracerebroventricular injection of composited Aβ, in rats’ brain. These results indicated that the neuro-protection and the improvement in the impaired memory of rats by SSF were due to the inhibition of hyperphosphorylation of Tau protein at multiple sites in rats' brain.

Introduction The transplantation of mesenchymal stem cells (MSCs) in patients with premature ovarian failure (POF) could lead to clinical improvement. The transplantation to the ovaries among other transplantation methods have been reported in various animal models, however, there is little evidence regarding the optimal method, including the clinical safety and the efficiency for the treatment of age associated ovarian hypofunction. Objectives To establish the most effective transplantation route of MSCs, explore the resistance to therapy, its safety and role in the natural aging process of the ovaries. Methods Highly purified MSCs were injected intraperitoneally, directly into the ovaries or tail-intravenously in mice animal model. The ovarian function, quantity and quality of oocytes, cell viability/apoptosis, were evaluated, applying chemiluminescence analysis (CLIA), western blotting, immunofluorescence staining, transmission electron microscope (TEM), TdT mediated dUTP Nick End Labeling (TUNEL) assay and other techniques. The organ tumorigenicity was also evaluated by long-term observation and histopathological examination. The efficiency of MSCs was further verified in non-human primates by the most effective transplantation route. Results The 32nd week was ultimately determined as the time point of MSCs transplantation. Our results showed that the intra-ovarian injection was the best transplantation method with a more conspicuous effect. With deeper investigations, we found that the transplanted MSCs showed an effective influence on the follicular number, promoted follicle maturation and inhibited cell apoptosis, which was further verified in non-human primates. In addition, the long-term observation and the histopathological examinations ruled out neoplasms or obvious prosoplasia after MSCs transplantation. Conclusion MSCs transplantation by intra-ovarian injection could within a month exert the most conspicuous anti-age-associated ovarian hypofunction effects, which may improve the quantity and quality of oocytes by changing the mitochondrial structure, regulating mitochondrial function and attenuating cell apoptosis to increase the storage of the follicle pool without a remarkable potential of tumorigenicity.

Pomegranate peel extract (PPE), a polyphenolic compound derived from pomegranate, has been widely concerned for its anti-oxidant, anti-inflammatory, and bacteriostatic effects. The potential therapeutic effect of PPE on burn injury was investigated, and its possible mechanisms were explored. Minipigs with second-degree burn were treated with PPE, Jing Wan Hong, and silver sulfadiazine. Hematoxylin-eosin (HE) staining was performed to detect burn severity, and then biological tissues were biopsied on days 0, 7, 14, 21, and 28 after administration. Immunohistochemistry, western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect the protein and mRNA expression levels of VEGF-A and TGF-β1 in skin tissues after treatment with PPE. Furthermore, the skin wound healing at different time points was monitored by macroscopic observation. HE showed that after 28-day PPE treatment, the morphology of the skin tissue showed a significant improvement. Macroscopic data monitoring indicated that the decrustation and fur growing time was shortened. Meanwhile, the rate of wound healing increased after PPE treatment. The combination of immunohistochemistry, western blotting, and RT-PCR showed that after PPE treatment, expression of VEGF-A and TGF-β1 increased sharply on day 7, maintaining a high level until day 14, showing a downward trend on day 21, and approaching normal levels on day 28. However, in the model group, the protein and mRNA expression levels of VEGF-A and TGF-β1 increased on day 28 after burn injury, which was a slow process. Results indicated that compared with the model group, the peak expression level of VEGF-A and TGF-β1 was earlier, which was consistent with decrustation, shortening of fur growing time, and improvement of wound healing rate in minipig second-degree burn model. PPE showed a significant promoting effect on minipig second-degree burn model, which might be associated with the upregulation of the protein and gene expression levels of VEGF-A and TGF-β1.

Background: High methionine-diet (HMD) causes Alzheimer's disease (AD)-like symptoms. Previous studies have shown that Dendrobium nobile Lindle. alkaloids (DNLA) had potential benefits for AD. Object: Whether DNLA can improve AD-like symptoms induced by HMD is to be explored. Method: Mice were fed with 2% HMD diet for 11 weeks, the DNLA20 control group (20 mg/kg), DNLA10 group (10 mg/kg), and DNLA20 group (20 mg/kg) were administrated with DNLA for 3 months. Morris water maze test was used to detect learning and memory ability. Neuron damage was evaluated by HE and Nissl stainings. Levels of homocysteine (Hcy), beta-amyloid 1-42 (Aβ1-42), S-adenosine methionine (SAM), and S-adenosine homocysteine (SAH) were detected by ELISA. Immunofluorescence and western blotting (WB) were used to determine the expression of proteins. CPG island methylation. Results: Morris water maze test revealed that DNLA improved learning and memory dysfunction. HE, Nissl, and immunofluorescence stainings showed that DNLA alleviated neuron damage and reduced the 5-methylcytosine (5-mC), Aβ1-40, and Aβ1-42 levels. DNLA also decreased the levels of Hcy and Aβ1-42 in the serum, along with decreased SAM/SAH levels in the liver tissue. WB results showed that DNLA down-regulated the expression of the amyloid-precursor protein (APP), presenilin-1 (PS1), beta-secretase-1 (BACE1), DNA methyltransferase1 (DNMT1), Aβ1-40, and Aβ1-42 proteins. DNLA also up-regulated the expression of the protein of insulin-degrading enzyme (IDE), neprilysin (NEP), DNMT3a, and DNMT3b. Meanwhile, DNLA increased CPG island methylation levels of APP and BACE1 genes. Conclusions: DNLA alleviated AD-like symptoms induced by HMD via the DNA methylation pathway.

Background: Bevacizumab (Bev) resistance is hypothesized to be overcome by combining inhibitors of other signalling pathways. Objective: We aimed to study the effect of combining Bev with knocked down β-catenin (Bev-β-cat-siRNA) on the expression of VEGF-A, Slug, NFкB, and its two target genes, c-Flip and FasR, in HepG2. Expression of VEGF-A and Slug was also studied in Caco-2 cells. Methods: Cultured cells were divided into six groups 1) cells treated with Bev, 2) cells treated with β-catenin-siRNA, 3) cells treated with Bev-β-cat-siRNA, 4) cells treated with negative control, 5) cells treated with Bev-negative control, and 6) untreated cells. Expressions were assessed using qPCR and western blotting. Results: Bev-β-cat-siRNA significantly reduced the mRNA level of VEGF-A, which was initially increased in response to Bev alone in HepG2 but not in Caco-2. Additionally, Bev-β-cat-siRNA significantly decreased Slug mRNA level compared to Bev treated HepG2 cells. In contrast, VEGF-A and Slug mRNA levels in Bev group were remarkably lower than Bev-β-cat-siRNA in Caco-2 cells. Distinct β-catenin and Slug protein expressions were noticed in HepG2 and Caco-2 cells. On the other hand, Bev-β-catsiRNA remarkably reduced the level of NFкB, FasR, and c-Flip compared to Bev treated HepG2 cells, although the difference was not statistically significant. Conclusion: We conclude that combining Bevacizumab with knocked down β-catenin reduces the expression of VEGF-A and Slug in HepG2 but not in Caco-2 cells.

Study Design: An experimental study with extracellular vesicles (EVs) from mesenchymal stem cell (MSC) of the epidural fat (EF) of the spine.Purpose: This study aims to isolate the exosomes from epidural fat-derived mesenchymal stem cells (EF-MSCs) and fully characterize the EF-MSC-EVs.Overview of Literature: EF-MSCs were reported in 2019, and a few studies have shown the positive outcomes of using EF-MSCs to treat specific spine pathologies. However, MSCs have significant limitations for conducting basic studies or developing therapeutic agents. Although EVs are an emerging research topic, no studies have focused on EVs, especially exosomes, from EF and EF-MSCs.Methods: In this study, we isolated the exosomes using the tangential flow filtration (TFF) system with exosome-depleted fetal bovine serum and performed the characterization tests via western blotting, reverse transcription–polymerase chain reaction, nanoparticle tracking analysis (NTA), and transmission electron microscopy.Results: In transmission electron microscopy, the exosome had a diameter of approximately 100–200 nm and had a spherical shape, whereas in the NTA, the exosome had an average diameter of 142.8 nm with a concentration of 1.27×1010 particles/mL. The flow cytometry analysis showed the expression of CD63 and CD81. The western blotting analysis showed the positive markers.Conclusions: These findings showed that isolating the exosomes via TFF resulted in high-quality EF-MSC exosome yield. Further studies with exosomes from EF-MSC are needed to evaluate the function and role of the EF tissue.

Background: Macrophage migration inhibitory factor (MIF), originally reported as an inflammation regulating molecule, is elevated in various cancer cells, which may promote carcinogenesis. Meanwhile, ISO-1 is a potent small molecular inhibitor of MIF, which has not been investigated in nasopharyngeal carcinoma (NPC), hence the impact of ISO-1 on NPC cells remains to be illustrated. Objective: This study intended to explore the biological function of ISO-1 in NPC cells in vitro and prove a possibility of ISO-1 being a novel agent in NPC treatments. Methods: Gene expression of MIF in Head and Neck squamous cell carcinoma was obtained from The Cancer Genome Atlas (TCGA) database. Nasal pharyngeal tissues were collected from adult patients undergoing nasopharyngeal biopsy for MIF level detection. Proliferation of NPC cell lines 5-8B and 6-10B was studied using Cell Counting Kit-8 (CCK-8) assay and plate-colony-formation assay, apoptosis was determined by flow cytometry and TUNEL staining, migration and invasion capacities were measured by wound-healing assay and transwell assay, all to explore the function of ISO-1 in NPC cells in vitro. Epithelial-to-mesenchymal transition (EMT) level of NPC cells was determined by Western blot analysis and immunofluorescence assay. Results: Transcript level of MIF was significantly higher in head and neck squamous cell carcinoma. Protein MIF was overexpressed in human NPC tissues compared to non-cancerous ones, and its expression could be compromised by ISO-1 in vitro. 100μM ISO-1 significantly hindered NPC cells' migration and invasion capacities in vitro but acted relatively poorly on proliferation and apoptosis. Immunofluorescence assay and Western blotting implied a downregulated EMT level through TGF-β/Smad4 axis in ISO-1 treated NPC cells compared to the vehicle Conclusion: This study indicated that MIF antagonist ISO-1 holds an impact on NPC progression by influencing the migration and invasion of NPC cells ISO-1 inhibits the EMT process of NPC cells through TGF-β/Smad4 axis, supporting that prudent application of ISO-1 may be a potential adjuvant treatment for NPC.

For patients at risk for multidrug-resistant organisms, IDSA/ATS guidelines recommend empiric therapy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas. Following negative cultures, the guidelines recommend antimicrobial de-escalation. We assessed antibiotic de-escalation practices across hospitals and their associations with outcomes in hospitalized patients with pneumonia with negative cultures.We included adults admitted with pneumonia in 2010-2015 to 164 US hospitals if they had negative blood and/or respiratory cultures and received both anti-MRSA and antipseudomonal agents other than quinolones. De-escalation was defined as stopping both empiric drugs on day 4 while continuing another antibiotic. Patients were propensity adjusted for de-escalation and compared on in-hospital 14-day mortality, late deterioration (ICU transfer), length-of-stay (LOS), and costs. We also compared adjusted outcomes across hospital de-escalation rate quartiles.Of 14 170 patients, 1924 (13%) had both initial empiric drugs stopped by hospital day 4. Hospital de-escalation rates ranged from 2-35% and hospital de-escalation rate quartile was not significantly associated with outcomes. At hospitals in the top quartile of de-escalation, even among patients at lowest risk for mortality, the de-escalation rates were50%. In propensity-adjusted analysis, patients with de-escalation had lower odds of subsequent transfer to ICU (adjusted odds ratio, .38; 95% CI, .18-.79), LOS (adjusted ratio of means, .76; .75-.78), and costs (.74; .72-.76).A minority of eligible patients with pneumonia had antibiotics de-escalated by hospital day 4 following negative cultures and de-escalation rates varied widely between hospitals. To adhere to recent guidelines will require substantial changes in practice.

ZFP91 positively regulates IL-1β production in macrophages and may be a potential therapeutic target to treat inflammatory-related diseases. We investigated whether this process is modulated by convallatoxin, which is a cardiac glycoside isolated from the traditional Chinese medicinal plant Adonis amurensis Regel et Radde.In vitro, the mechanisms by which convallatoxin inhibits ZFP91-regulated IL-1β expression were investigated using molecular docking, western blotting, RT-PCR, ELISA, immunofluorescence and immunoprecipitation assays.In vivo, mice liver injury was induced by an intraperitoneal injection of D-GalN and LPS, colitis was induced by oral administration of dextran sulfate sodium (DSS) in drinking water and peritonitis was induced by an intraperitoneal injection of alum.We confirmed that convallatoxin inhibited the release of IL-1β by down-regulating ZFP91. Importantly, we found that convallatoxin significantly reduced K63-linked polyubiquitination of pro-IL-1β regulated by ZFP91 and decreased the efficacy of pro-IL-1β cleavage. Moreover, convallatoxin suppressed ZFP91-mediated activation of the non-canonical cysteine-requiring aspartate protease-8 (caspase-8) inflammasome and MAPK signalling pathways in macrophages. Furthermore, we showed that ZFP91 promoted the assembly of the caspase-8 inflammasome complex, whereas convallatoxin treatment reversed this result. Mice in vivo studies further demonstrated that convallatoxin ameliorated D-GalN/LPS-induced liver injury, DSS-induced colitis and alum-induced peritonitis by down-regulating ZFP91.We show for the first time that convallatoxin-mediated inhibition of ZFP91 is an important regulatory event that prevents inappropriate inflammatory responses to maintain immune homeostasis. This mechanism provides new insight for the development of convallatoxin as a novel anti-inflammatory drug targeting ZFP91.This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

Graphical abstract, Highlights • PLA2G16 is up-regulated in CRC, and high expression of PLA2G16 is associated with the advanced stages. • PLA2G16 promotes the malignant progression of CRC through the Hippo signaling pathway. • GCK exerts its anti-CRC effects by inhibiting the protein expression of PLA2G16. • Provide a new insights towards the development of effective therapeutic strategies for CRC treatment by targeting PLA2G16., Introduction Colorectal cancer (CRC) is a common malignant tumor with a high global incidence, metastasis rate and low cure rate. Changes in lipid metabolism-related genes can affect the occurrence and development of CRC, and may be a potential therapeutic target for CRC. Therefore, starting from lipid metabolism-related genes to find natural medicines for tumor treatment may become a new direction in CRC research. Objectives This study aimed to investigate the effect of PLA2G16, a key gene involved in lipid metabolism, on the biological function of CRC, and whether the anti-CRC effect of GCK is related to PLA2G16. Methods To explore the role of PLA2G16 in CRC in vitro and in vivo, we performed cell proliferation, migration, invasion and nude mice tumorigenesis assays. As for the mechanism, we designed RNA-seq analysis and verified by western blotting and immunofluorescence experiments. Subsequently, we found the anti-CRC effect of GCK is related to PLA2G16 through western blotting and rescue experiments. Results We showed that PLA2G16 was significantly higher in CRC tissues than the adjacent normal appearing tissues, and high PLA2G16 expression correlates with unfavorable prognosis of CRC patients. Further, PLA2G16 promoted the malignant progression of CRC by inhibiting the Hippo signaling pathway determined by RNA-seq analysis, and GCK exerted anti-CRC effects by inhibiting the protein expression of PLA2G16 in vitro and in vivo. Conclusion Our results suggested that PLA2G16 promote the malignant progression of CRC by inhibiting the Hippo signaling pathway and the anti-CRC effect of GCK is through inhibiting the protein expression of PLA2G16.

Neuroinflammation is an important secondary aggravating factor in spinal cord injury (SCI). Inhibition of the inflammatory response is critical for SCI treatment. Glycyrrhizic acid (GA) is an anti-inflammatory drug, but its utility for SCI is unclear. This study aimed to evaluate the effects of GA on inflammation after SCI and the underlying mechanism.Cell counting kit-8 assays were performed to assess the viability of highly aggressively proliferating immortalized cells that had been treated with lipopolysaccharide (LPS) and/or GA. Reverse transcription quantitative polymerase chain reaction and Western blotting were performed to assess expression of high mobility group box-1 protein (HMGB1), ionized calcium binding adaptor molecule 1, and inflammatory factors in vitro and in vivo. GA (100 mg/kg) was intraperitoneally injected into rats. Anti-inflammatory effects of GA were analyzed in SCI tissues. p38/Jun N-terminal kinase signaling pathway proteins were analyzed by Western blotting.Cell counting kit-8 assay results showed that treatment with 100 ng/mL LPS for 12 hours was optimal. After LPS treatment, highly aggressively proliferating immortalized cells were activated; messenger RNA expression levels of HMGB1 and inflammatory factors were increased. GA significantly inhibited LPS-induced HMGB1 expression and inflammatory responses, as determined by reverse transcription quantitative polymerase chain reaction and Western blotting. Transfection with an HMGB1-overexpression plasmid reversed the anti-inflammatory effects of GA. In addition, intraperitoneal injection of GA (100 mg/kg) into rats for 3 days significantly reduced expression levels of HMGB1 and inflammatory factors after SCI in vivo. GA reduced phosphorylation, but not levels, of p38 and Jun N-terminal kinase proteins.GA attenuates the inflammatory response after SCI by inhibiting HMGB1 through the p38/JNK signaling pathway and thus has therapeutic potential for SCI.

Background Due to the inconsistent results of anti-treponema pallidum (TP) specific antibodies by enzyme-linked immunosorbent assay (ELISA) and Treponema pallidum granule agglutination assay (TPPA) in clinical work, there will be a certain proportion of false-positives and false-negatives depending on TPPA as confirmation results. This study aimed to evaluate the necessity of Western blotting (WB) in samples with inconsistent results in detecting anti-TP antibodies by ELISA and TPPA. Methods Specific anti-TP test results in our clinical laboratory were retrospectively analyzed. The specimens with a positive or a negative result, but with colored ELISA plates, were retested by TPPA. WB was used to confirm the suspicious results between ELISA and TPPA. The Chi-square test was used to analyze whether the difference was statistically significant. Results A total of 106,757 anti-TP specimens were screened by ELISA from August 2018 to December 2019; 3972 were retested by TPPA, and 3809 were positive by TPPA. ELISA and TPPA showed different results in 163 specimens. Among them, 29 specimens were negative and 134 were positive by ELISA; 76 were negative, 23 were positive, and 64 were “reserve” by TPPA; 93 were negative, 31 were positive, and 39 were suspicious by the WB confirmation test. Compared with WB, the difference in the results of ELISA and TPPA was statistically significant. Conclusions TPPA is an effective retest method for anti-TP antibody detection. If the results of anti-TP antibodies by ELISA and TPPA are inconsistent, it is necessary to use WB for confirmation. Trial registration This retrospective analysis is in accordance with the ethical guidelines of China and approved by the second hospital of Jiaxing (jxey-2018048).

Objectives The dysregulation of circular RNAs (circRNAs) is implicated in the progression of various cancers. This study was aimed at expounding the role and mechanism of hsa_circ_0002141 in the OSCC progression. Materials and methods Circ_0002141 expression was examined in 52 pairs of OSCC tissues and matched adjacent tissue samples by quantitative real-time polymerase chain reaction (qRT-PCR) assay. After circ_0002141 was overexpressed or knocked down in OSCC cell lines, cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry, and Western blotting were conducted to detect the changes in the growth, migration, invasion and apoptosis of OSCC cells. Western blot assay, qRT-PCR and dual luciferase reporter assay were performed to clarify the interplay among circ_0002141, miR-1231, and epidermal growth factor receptor (EGFR). Results Circ_0002141 expression was significantly up-regulated in OSCC tissues and cell lines. Circ_0002141 overexpression markedly promoted the proliferation, migration, and invasion of OSCC cells whereas reduced the apoptotic of OSCC cells. Also, circ_0002141 knockdown suppressed the malignant characteristics of OSCC cells. EGFR was validated as the target of miR-1231. Besides, circ_0002141 could sponge miR-1231 and up-regulate EGFR expression in OSCC cells. Conclusion Circ_0002141/miR-1231/EGFR axis is involved in the progression of OSCC.

Astaxanthine (AST) has important biological activities including antioxidant and anti-inflammatory effects that could alleviate neurological and heart diseases, but its role in the prevention of cisplatin-induced hearing loss (CIHL) is not yet well understood. In our study, a steady interaction between AST and the E3 ligase adapter Kelch-like ECH-associated protein 1, a predominant repressor of nuclear factor erythroid 2-related factor 2 (NRF2), was performed and tested via computer molecular docking and dynamics. AST protected against cisplatin-induced ototoxicity via NRF2 mediated pathway using quantitative PCR and Western blotting. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential revealed that AST reduced ROS overexpression and mitochondrial dysfunction. Moreover, AST exerted anti-apoptosis effects in mouse cochlear explants using immunofluorescence staining and HEI-OC1 cell lines using quantitative PCR and Western blotting. Finally, AST combined with poloxamer was injected into the middle ear through the tympanum, and the protection against CIHL was evaluated using the acoustic brain stem test and immunofluorescent staining in adult mice. Our results suggest that AST reduced ROS overexpression, mitochondrial dysfunction, and apoptosis via NRF2-mediated pathway in cisplatin-exposed HEI-OC1 cell lines and mouse cochlear explants, finally promoting cell survival. Our study demonstrates that AST is a candidate therapeutic agent for CIHL.

Introduction Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. Methods We established the nitro- l -arginine methyl ester ( l -NAME)-induced preeclamptic mice model and a hypoxia‐reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence‐associated β‐galactosidase (SA‐β‐gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). Results CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l -NAME. CsA ameliorated placental SASP and SAMD level induced by l -NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l -NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. Discussion CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Made available in DSpace on 2022-05-01T09:47:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 This study aimed to evaluate the immunomodulatory effect of vitamin D (VD) on the NLRP1 and NLRP3 inflammasomes in placental explants from preeclamptic (PE) and normotensive (NT) pregnant women. Placental explants from eight PE and eight NT pregnant women were cultured with or without hydrogen peroxide (H2O2), VD or H2O2 + VD. Gene and protein expression of NLRP1, NLRP3, HMGB1, caspase-1, IL-1β, TNF-α and IL-18 were determined by qPCR and Western blotting/ELISA. Compared to NT pregnant women, the endogenous gene expression of NLRP1, NLRP3, HMGB1, IL-1β, TNF-α and IL-18 was significantly higher in explants from PE and became decreased after VD treatment. Similarly, VD decreased the protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE. Placental explants from NT cultured with H2O2 showed increased gene and protein expression of NLRP1, NLRP3, caspase-1, IL-1β, TNF-α and HMGB1, while H2O2 was also able to increase TNF-α and caspase-1 gene expression in PE. Treatment with H2O2 + VD decreased gene/protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE and NT explants with H2O2. NLRP1 and NLRP3 are upregulated in the PE. VD may play an immunomodulatory role in the placental inflammation and downregulates oxidative stress induced in vitro by H2O2. Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp) Botucatu Medical School Sao Paulo State University (Unesp) Institute of Biosciences Sao Paulo State University (Unesp)

Pregnancy causes changes in the uterus, such as increased cell volume and altered water content. However, the mechanisms that protect the structure and maintain the function of uterine smooth muscle cells against these changes during pregnancy have not been clarified. This study focused on the volume-regulated anion channel (VRAC), which opens with cell swelling under low osmotic pressure and releases Cl- ions and various organic osmolytes to resist cell swelling and regulates a wide range of biological processes such as cell death. In this study, myometrial smooth muscle (MSM) tissues and cells (MSMCs) were collected from non-pregnant and pregnant mice. Using western blotting and immunocytochemistry, leucine-rich repeat containing protein 8A (LRRC8A), an essential membrane protein that constitutes part of the VRAC, was determined to be diffused throughout MSMCs including in the cell membrane. Patch-clamp experiments were performed to investigate the electrophysiology of swelling-induced Cl- currents (ICl, swell) mediated by the VRAC. No significant changes between non-pregnancy and pregnancy groups were observed in either the expression density of LRRC8A or the current density of ICl, swell, however the presence of LRRC8A on the cell membrane was significantly increased in the third trimester of pregnancy compared to the non-pregnancy. This study suggests that the VRAC may play a role, such as maintaining cellular homeostasis in the pregnant MSM.

Background Circular RNAs (circRNAs) participate in the genesis and progression of tumors, including non‐small cell lung cancer (NSCLC). At present, the role and regulatory mechanisms of circRNAs in NSCLC have not been fully elucidated. The aim of this study was to explore the role and regulatory mechanism of circRNA hsa_circ_0008037 (circ_0008037) in NSCLC. Methods Expression of circ_0008037 in NSCLC tissues and cells was detected by quantitative real‐time polymerase chain reaction (RT‐qPCR). Loss‐of‐function experiments were performed to analyze the influence of circ_0008037 knockdown on proliferation, migration, invasion, and the Warburg effect of NSCLC cells. Western blotting was utilized for protein analysis. The regulatory mechanism of circ_0008037 was surveyed by bioinformatics analysis, RNA pulldown assay, and dual‐luciferase reporter assay. Xenograft assay was used to validate the oncogenicity of circ_0008037 in NSCLC in vivo. Results Circ_0008037 was upregulated in NSCLC tissues and cells. Circ_0008037 downregulation reduced tumor growth in vivo and repressed proliferation, migration, invasion, and decreased the Warburg effect of NSCLC cells in vitro. Mechanically, circ_0008037 regulated nuclear ubiquitous casein kinase and cyclin‐dependent kinase substrate 1 (NUCKS1) expression via sponging miR‐433‐3p. Furthermore, MiR‐433‐3p inhibitor reversed the inhibiting influence of circ_0008037 silencing on proliferation, migration, invasion, and the Warburg effect of NSCLC cells. Also, NUCKS1 elevation overturned the repressive influence of miR‐433‐3p mimic on proliferation, migration, invasion, and the Warburg effect of NSCLC cells. Conclusion Circ_0008037 accelerated tumor growth and elevated the Warburg effect via regulating NUCKS1 expression by adsorbing miR‐433‐3p, providing an underlying target for NSCLC treatment., A model depicting the regulatory mechanism of circ_0008037 in NSCLC. Circ_0008037 elevates NUCKS1 expression through binding to miR‐433‐3p and inhibiting miR‐433‐3p activity, promoting NSCLC tumorigenesis.

MoS2 nanosheets (NSs) are novel 2D nanomaterials (NMs) with potential uses in many areas, and therefore oral exposure route to MoS2 NSs is plausible. Currently, MoS2 NSs are considered as biocompatible NMs, but there is lacking of systemic investigations to study the interactions of MoS2 NSs with intestinal cells. In this study, we exposed the 3D Caco-2 spheroids to MoS2 NSs or MoS2 powders (denoted as MoS2-bulk), and investigated the potential adverse effects of MoS2-materials based on transcriptomics and lipidomics analysis. As expected, both MoS2 NSs and MoS2-bulk were dose-dependently internalized into 3D Caco-2 spheroids but did not induce cytotoxicity, membrane disruption or decrease of thiols. However, the Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis indicated that nutrient absorption and metabolism was decreased. One of the most significantly decreased KEGG pathways is fat digestion and absorption (map04975), and Western blotting analysis further showed that fatty acid binding protein 1 and apolipoprotein A1, key proteins involved in fat digestion and absorption, were down-regulated by MoS2 NSs or MoS2-bulk. In addition, BODIPY 493/503 staining suggested that exposure to MoS2 NSs and MoS2-bulk decreased lipid levels in the spheroids. However, lipidomics data indicated that MoS2 materials only decreased 8 lipid classes, including lysophosphatidylcholine, lysodimethylphosphatidylethanolamine, N-acylethanolamine, ceramide phosphoethanolamines, gangliosides, lysosphingomyelin and sulfatide, whereas most of the lipid classes were indeed increased. In addition, MoS2 NSs was more potent to decrease the lipid classes compared with MoS2-bulk. Combined, the results from this study showed that MoS2 NSs and bulk materials were non-cytotoxic but altered lipid profiles in 3D Caco-2 spheroids.

Purpose Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. Methods Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70–80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. Results Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a −16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. Conclusions AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, which is not sensitive to radiotherapy and chemotherapy and very often experiences postoperative relapse. In this regard, effective screening of liver cancer is considered as the most important and urgent task. The aim of our study was to determine whether N-methyl-D-aspartate receptor (NMDAR) and, in particular, its subunits, can serve as biomarkers to distinguish the precancerous liver at early stages of liver fibrosis. We assessed the development of HCC after 10, 15 and 22 weeks using a HCC rat model. The expression of NMDAR subunits was monitored at different stages of HCC by means of immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting and direct bisulfite sequencing. NMDAR subunits were not found in healthy liver tissues. In contrast, NMDAR subunits, in particular NR1 and NR2B, appeared at the stage of severe liver fibrosis (precancerous liver disease) in rats and were expressed during the development of HCC in rats and mice. Using the direct bisulfite sequencing, we detected that increased expression of NMDAR directly correlated with the demethylation of CpG islands in the promoter region of genes encoding receptor subunits. The obtained results confirmed that NMDAR subunits can serve as new biomarkers of precancerous liver disease, severe fibrosis, and its progression towards HCC.

Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.

S4A ((1R, 2R, 3S)-1, 2-propanediol acetal- zeylenone) is one of the derivatives of Zeylenone, which exhibits superior cytotoxicity against canine breast cancer cell line CIPp. However, its poor aqueous solubility and toxicity to normal tissue limit its clinical application. Therefore, in order to enhance the anticancer effect of S4A, in this article, BSA/BSA-Au-nanocluster-aggregated core/shell nanoparticles (B-BANC-NPs) were prepared by using bovine serum albumin (BSA) and HAuCl4, and then we further synthesized S4A-BSA-Au NPs which were spherical, with a diameter of about 60 nm. In vitro cytotoxicity assessed by using CCK-8 assay demonstrated that the IC50 value of the S4A-BSA-Au NPs was 10.39 μg/mL, which was not significantly different from that of S4A (10.45 μg/mL). In vitro apoptosis assay showed that the apoptosis rate of cells treated with S4A-BSA-Au NPs was 20.12%, which was significantly higher than that of the control group treated with S4A (11.3%). Notably, S4A-BSA-Au NPs were shown to effectively accumulate at tumor sites with fluorescence tracing. Besides, the effect of S4A-BSA-Au NPs on SPARC expression was determined by western blotting, and the result showed that at 24 h after applying S4A-BSA-Au NPs, SPARC expression in low, middle and high dosage groups was all lower than that of the control group, and the tendency showed dose dependence. The results revealed that S4A-BSA-Au NPs could effectively improve the anti-tumor activity of S4A on canine breast cancer, which may be associated with their abilities to effectively accumulate within tumor and to reduce the expression of SPARC.

Background: Demethoxycurcumin (DMC), a natural derivative of curcumin, has anti-inflammatory activities. However, the mechanism has not been fully elucidated. Objective: The aim of the current study was to investigate the role of DMC on NLRP3 inflammasome priming. Methods: Protein expression was quantified by western blotting. Inflammatory cytokines were measured by ELISA. Autophagosomes were evaluated by transmission electron microscopy. Results: DMC inhibited LPS-stimulated NLRP3, pro-caspase-1, and pro-IL-1β expression. Meanwhile, DMC diminished NLRP3-dependent IL-1β maturation, caspase-1 activation, IL-1β, and IL-18 production caused by LPS plus ATP. Moreover, DMC induced autophagy and autophagy inhibitor 3-MA abrogated the role of DMC on NLRP3 inflammasome priming and subsequent activation. DMC also inhibited LPS-stimulated phosphorylation and nuclear translocation of p65 NF-κB. Additionally, DMC significantly increased the PPARγ expression, and the effects of DMC in NF- κB inhibition, autophagy, and NLRP3 inflammasome priming were abrogated by specific PPARγ antagonist T0070907. Conclusion: The evidence presented here has confirmed that DMC increases PPARγ expression, resulting in autophagy and NF-κB inhibition, and subsequently inhibits LPS-induced NLRP3 inflammasome priming and subsequent activation.

PURPOSE Breast cancer (BC) is one of the most common malignant tumors for women. The role and potential mechanisms of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) were explored in BC cell migration and invasion. METHODS PVT1, miR-148a-3p and Rho‑associated, coiled‑coil containing protein kinase 1 (ROCK1) mRNA expressions were detected using real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROCK1 protein expression was detected by Western blotting. The relationship of PVT1, miR-148a-3p and ROCK1 was analyzed by Dual Luciferase activity, RNA immunoprecipitation (RIP) and Spearman correlation analysis. Cell invasion and migration were detected by Transwell assay. RESULTS Upregulation of PVT1 and ROCK1, and downregulation of miR-148a-3p were observed in BC tissues and cell lines. According to the analysis of Dual Luciferase activity, RIP and Spearman correlation analysis, miR-148a-3p directly binds to PVT1, and ROCK1 is a target of miR-148a-3p. In addition, PVT1 regulated the cells migration and invasion by regulating miR-148a-3p and ROCK1 expression. CONCLUSION These data demonstrated that PVT1 was upregulated and facilitated to the cell migration and invasion of BC by the regulation of miR-148a-3p and ROCK1, indicating that PVT1 may be a potential biomarker of BC diagnosis and treatment.

PURPOSE: To identify the genetic causes of multiple morphological anomalies of the flagella (MMAF) and oligoasthenoteratozoospermia (OAT). METHODS: Whole-exome sequencing (WES) was performed on the proband to identify pathogenic mutation for infertility. Western blotting and immunofluorescence analysis detected the expression level and localization of adenylate kinase 7 (AK7). RESULTS: We identified a novel homozygous missense mutation (NM_152327: c.1846G > A; p.E616K) in AK7 in two brothers with MMAF and OAT from a consanguineous family by WES. Western blotting and immunofluorescence experiments determined that the expression level of AK7 decreased in the sperm from the proband. The proband and his wife underwent two cycles of intracytoplasmic sperm injection (ICSI) treatment but got unfavorable outcomes. CONCLUSION: This study could provide precise genetic diagnosis for the patient and expand the spectrum of AK7 mutations.

Myocardial ischemia/reperfusion (I/R) injury is a serious issue during the therapy of myocardial infarction. Herein, we explored the beneficial influence of Epigallocatechin-3-gallate (EGCG) on hypoxia/reoxygenation (H/R)-stimulated cardiomyocyte H9c2 cells damage, along with possible internal molecular mechanism related autophagy related 4C (ATG4C). H9c2 cells were subjected to H/R stimulation and/or EGCG treatment. ATG4C mRNA expression was measured via q-PCR assay. ATG4C overexpression plasmid (OE-ATG4C) was transfected to arise ATG4C level. Cell viability, apoptosis, reactive oxygen species (ROS) production, ATP level were tested via CCK-8 assay, Annexin V-FITC/PI staining, DCFH-DA staining and ATP Assay Kit, respectively. Western blotting was performed to test Cleaved-caspase 3, Cleaved-caspase 9, cytochrome C, and LC3B protein levels. H/R stimulation resulted in H9c2 cell viability loss, promoted cell apoptosis, and ROS overproduction, as well as lowered ATP level in cells. EGCG treatment alleviated H/R-resulted H9c2 cell viability loss, cell apoptosis, ROS overproduction, and reduction of ATP level. Moreover, H/R stimulation reduced the ATG4C expression in H9c2 cells, while EGCG raised the ATG4C expression. Overexpression of ATG4C strengthened the beneficial influence of EGCG on H/R-stimulated H9c2 cell viability, apoptosis and ROS production. Besides, ATG4C overexpression weakened the H/R-stimulated H9c2 cell autophagy via reducing LC3B II/I expression. EGCG exerted beneficial influence on H/R-stimulated cardiomyocytes, which protected cardiomyocytes from H/R-stimulated viability loss, apoptosis, and ROS overproduction via enhancing ATG4C expression.

Breast cancer, with high morbidity worldwide, is a threat to the life of women. MiR-543 was identified as playing an active part in the development of breast cancer involving multiple molecules. The goal of this study was to explore the molecular mechanisms of the involvement of miR-543 in the development of breast cancer. Quantitative real-time PCR (qRT-PCR) or Western blotting was used to detect mRNA or protein expression. Cell counting kit-8 (CCK-8), and the 5-bromo-2ʹ-deoxyuridine (BrdU), wound healing, and Transwell assays were the main experimental procedures. Furthermore, subcutaneous tumor formation experiments were conducted to detect the function of miR-543 in breast cancer development in vivo. The match of miR-543 and ubiquitin-conjugating enzyme E2T (UBE2T) was detected through a dual-luciferase reporter experiment and RNA pull-down assay. Based on these results, miR-543 exhibited reduced expression in breast cancer tissues and cell lines, whereas UBE2T exhibited high levels. Furthermore, miR-543 directly targeted UBE2T, and a negative correlation between miR-543 and UBE2T was also observed in breast cancer tissues. Moreover, miR-543 overexpression led to inhibition of viability, proliferation, migration, and invasion of breast cancer. Furthermore, miR-543 overexpression undermined the UBE2T promotional effect by inhibiting ERK/MAPK pathway activity in breast cancer cells. Our study revealed that miR-543 impaired breast cancer progression by targeting UBE2T and downregulating UBE2T expression through the ERK/MAPK pathway, which suggested that miR-543 and UBE2T might serve as promising therapeutic gene targets for breast cancer in clinical application.

Objectives Danzhi Jiangtang capsule (DJC) is widely used for preventing and treating diabetic cardiomyopathy (DCM). However, the underlying mechanisms of the anti-inflammatory and antiapoptotic activities are unclear. Methods In the in vivo diabetic cardiomyopathy rat model, cardiac function was measured through echocardiography, histological changes in the myocardium were visualized using HE staining, and cardiomyocyte apoptosis was detected using TUNEL. The serum levels of anti-inflammatory cytokines were detected using ELISA. Finally, TLR4, MyD88, and NF-κB mRNA expressions were analyzed using RT-qPCR. In the in vitro experiments, the apoptosis rate of the H9c2 cells was detected using FCM; moreover, TLR4, MyD88 and NF-κB mRNA expressions were measured using RT-qPCR and related protein levels were investigated using Western blotting. Results In vivo, DJC effectively improved cardiac function, alleviated the pathological changes, and reduced the apoptosis rate. Moreover, DJC reduced TNF-α, IL-1β, and IL-6 activities, with significant inhibition of the TLR4, MyD88 and NF-κB p65 mRNA expression. Moreover, in vitro, DJC effectively inhibited high-glucose-induced H9c2 apoptosis-an effect similar to that for TAK242. Finally, both the DJC and TAK242 considerably reduced TLR4, MyD88, NF-κB, Bax, and caspase-3 protein expression but increased that of BCL-2. Conclusions DJC prevented the overactivation of the TLR4/MyD88/NF-κB signaling pathway and regulate cardiomyocyte apoptosis against DCM.

Linc00467 is a vital regulator in tumor progression. This study explores the molecular mechanisms of linc00467 in gastric cancer (GC). Linc00467 expression was obtained and analyzed in GC tissue through exploration in the cancer genome atlas database. Then, real-time quantitative polymerase chain reaction was used to detect linc000467 expression in GC cells. Cell functions were observed using cell counting Kit-8, Transwell assay, Western blotting to testify the proliferation, migration, invasion, and the relative expression of epidermal growth factor receptor (EGFR) in GC cells. Moreover, a dual-luciferase reporter gene assay was used to verify the relationship between linc00467 and miR-7-5p. Results showed that the expression of linc00467 was overexpressed in GC. Linc00467 silencing decreased the GC cell proliferation, migration, and invasion. With mRNA verification and combined previous research, linc00467 directly regulated the miR-7-5p expression and downstream EGFR expression. Inhibited miR-7-5p could restore cell function, EGFR expression of GC cells when linc00467 knockdown occurs. Altogether, linc00467 directly regulates the miR-7-5p and EGFR signaling pathway to promote GC proliferation, migration, and invasion.

A variety of studies indicate that microRNAs (miRNAs) are involved in diabetes. However, the direct role of miR-320a in the pathophysiology of pancreatic β cells under diabetes mellitus remains unclear. In the current study, islet transplantation and hyperglycemic clamp assays were performed in miR-320a transgenic mice to explore the effects of miR-320a on pancreatic β cells in vivo. Meanwhile, β cell-specific overexpression or inhibition of miR-320a was delivered by adeno-associated virus (AAV8). In vitro, overexpression or downregulation of miR-320a was introduced in cultured rat islet tumor cells (INS1). RNA immunoprecipitation sequencing (RIP-Seq), luciferase reporter assay, and western blotting were performed to identify the target genes. Results showed that miR-320a was increased in the pancreatic β cells from high-fat-diet (HFD)-treated mice. Overexpression of miR-320a could not only deteriorate the HFD-induced pancreatic islet dysfunction, but also initiate pancreatic islet dysfunction spontaneously in vivo. Meanwhile, miR-320a increased the ROS level, inhibited proliferation, and induced apoptosis of cultured β cells in vitro. Finally, we identified that MafF was the target of miR-320a that responsible for the dysfunction of pancreatic β cells. Our data suggested that miR-320a could damage the pancreatic β cells directly and might be a potential therapeutic target of diabetes., Graphical abstract, Overexpression of miR-320a spontaneously initiated pancreatic islet dysfunction in vivo. Meanwhile, miR-320a suppressed the insulin secretion and induced apoptosis in pancreatic β cells via targeting MafF. These findings suggested that miR-320a was a key regulator in diabetes which could potentially serve as a new therapeutic target.

Background: LncRNA homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism is not fully understood.Methods: HOXA11-AS, miR-148b-3p and IGFBP5 expression were detected by RT-qPCR or western blots. CCK8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p HOXA11-AS and IGFBP5 was confirmed by luciferase reporter assays or RNA immunoprecipitation.Results: The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis.Conclusion: These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid.

Background Echinococcus multilocularis is the causative agent of human hepatic alveolar echinococcosis (AE). AE can cause damage to several organs, primarily the liver, and have severe outcomes, such as hepatic failure and encephalopathy. The main purpose of this study was to explore the interactions between hepatic stellate cells (HSCs) and E. multilocularis protoscoleces (PSCs). The results of this study provide an experimental basis for further examination of the pathogenesis of hepatic fibrosis due to AE infection. Methods We investigated the role of Echinococcus multilocularis (Echinococcus genus) PSCs in hepatic fibrosis by examining structural changes and measuring hepatic fibrosis-related protein levels in cocultures of PSCs and human HSCs. Structural changes were detected by transmission electron microscopy (TEM), and levels of the hepatic fibrosis-related proteins collagen I (Col-I), alpha-smooth muscle actin (α-SMA) and osteopontin (OPN) were measured by western blotting and enzyme-linked immunosorbent assay (ELISA). Results Under coculture (1) both PSCs and HSCs exhibited morphological changes, as observed by TEM; (2) Col-I, α-SMA, and OPN expression levels, which were determined by western blotting and ELISA, significantly increased after 3 days of incubation. Conclusions The results of this study provide insights into the molecular mechanisms of AE-induced hepatic fibrosis. Graphical abstract

Vasorin (VASN) is an important transmembrane protein associated with development and disease. However, it is not clear whether the death of mice with VASN deficiency (VASN −/−) is related to cardiac dysfunction. The aim of this research was to ascertain whether VASN induces pathological cardiac hypertrophy by targeting myosin light chain 7 (MYL7). VASN −/− mice were produced by CRISPR/Cas9 technology and inbreeding. PCR amplification, electrophoresis, real‐time PCR and Western blotting were used to confirm VASN deficiency. Cardiac hypertrophy was examined by blood tests, histological analysis and real‐time PCR, and key downstream factors were identified by RNA sequencing and real‐time PCR. Western blotting, immunohistochemistry and electron microscopy analysis were used to confirm the downregulation of MYL7 production and cardiac structural changes. Our results showed that sudden death of VASN −/− mice occurred 21–28 days after birth. The obvious increases in cardiovascular risk, heart weight and myocardial volume and the upregulation of hypertrophy marker gene expression indicated that cardiac hypertrophy may be the cause of death in young VASN −/− mice. Transcriptome analysis revealed that VASN deficiency led to MYL7 downregulation, which induced myocardial structure abnormalities and disorders. Our results revealed a pathological phenomenon in which VASN deficiency may lead to cardiac hypertrophy by downregulating MYL7 production. However, more research is necessary to elucidate the underlying mechanism.

The aim of this study was to explore the neurocognitive effects of dexmedetomidine-loaded gold nanoparticles (AuNPs-dexmedetomidine) on anesthetized rats. Sixty Sprague Dawley rats (age, 2–3 weeks; weight, 250–280 g) were randomly divided into three groups (n = 20): the control group and two groups that received intraperitoneal injection of AuNPs-dexmedetomidine at 50 and 100 μg/kg each. Western blotting and RT-PCR were used to determine the protein and mRNA expression of GSK-3β, respectively. Compared with that in the control group, GSK-3β expression in AuNP-dexmedetomidine groups increased (P < 0.05). The protein expression of GSK-3β was higher and mRNA expression was significantly lower in the 100 μg/kg AuNP-dexmedetomidine group (P < 0.05). AuNPs-dexmedetomidine reduced the neurocognitive effect on anesthetized rats through the regulation of the GSK-3β signaling pathway.

Introduction Osteoporosis, a common skeletal disorder mainly affecting postmenopausal women, is characterized by the imbalance between osteogenesis and osteoclastogenesis. Circ_0134944 has been recently found to be upregulated in postmenopausal osteoporosis (PMOP) patients. However, its role in osteogenesis remains unknown. Here we aimed to explore the role of circ_0134944 in osteogenesis and reveal the underlying mechanism. Methods qRT-PCR was used to determine the expression of circ_0134944, miR-127-5p, PDX1 and SPHK1 in the blood mononuclear cells (BMCs) of PMOP patients. Bone marrow mesenchymal stem cells (BMSCs) were used as the cellular model. Western blotting and qRT-PCR were used to determine the expression of osteogenesis-related genes (Runx2, OPN, OCN). ALP and Alizarin Red S staining were performed to evaluate osteogenic differentiation. The interactions between circ_0134944 and miR-127-5p, miR-127-5p and PDX1, PDX1 and SPHK1 were determined by dual-luciferase reporter and ChIP assay. Results Circ_0134944, PDX1 and SPHK1 were upregulated while miR-127-5p was downregulated in PMOP patients. Enhanced expression of circ_0134944 suppressed osteogenesis, which was then reversed by miR-127-5p overexpression. The binding between circ_0134944 and miR-127-5p, PDX1 and miR-127-5p were confirmed by dual-luciferase reporter assay. Moreover, PDX1 was enriched in the promoter region of SPHK1, and SPHK1 overexpression prevented the promotion of osteogenesis induced by miR-127-5p overexpression. Conclusions Taken together, these results demonstrate that circ_0134944 inhibit osteogenesis via miR-127-5p/PDX1/SPHK1 axis. Thus, the present study offered evidence that circ_0134944/miR-127-5p/PDX1/SPHK1 axis could be a potential therapeutic target for PMOP.