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Hyperglycemia induces chronic stresses, such as oxidative stress and endoplasmic reticulum (ER) stress, which can result in β -cell dysfunction and development of Type 2 Diabetes Mellitus (T2DM). Ginsenoside Rk1 is a minor ginsenoside isolated from Ginseng. It has been shown to exert anti-cancer, anti-inflammatory, anti-oxidant, and neuroprotective effects; however, its effects on pancreatic cells in T2DM have never been studied. This study aims to examine the novel effects of Ginsenoside Rk1 on ER stress-induced apoptosis in a pancreatic β -cell line MIN6 and HFD-induced diabetic pancreas, and their underlying mechanisms. We demonstrated that Ginsenoside Rk1 alleviated ER stress-induced apoptosis in MIN6 cells, which was accomplished by directly targeting and activating insulin-like growth factor 1 receptor (IGF-1R), thus activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Bcl-2-associated agonist of cell death (Bad)-B-cell lymphoma-2 (Bcl-2) pathway. This pathway was also confirmed in an HFD-induced diabetic pancreas. Meanwhile, the use of the IGF-1R inhibitor PQ401 abolished this anti-apoptotic effect, confirming the role of IGF-1R in mediating anti-apoptosis effects exerted by Ginsenoside Rk1. Besides, Ginsenoside Rk1 reduced pancreas weights and increased pancreatic insulin contents, suggesting that it could protect the pancreas from HFD-induced diabetes. Taken together, our study provided novel protective effects of Ginsenoside Rk1 on ER stress-induced β -cell apoptosis and HFD-induced diabetic pancreases, as well as its direct target with IGF-1R, indicating that Ginsenoside Rk1 could be a potential drug for the treatment of T2DM. [ABSTRACT FROM AUTHOR]

Simple Summary: Targeted therapies are typically well tolerated and frequently lead to rapid tumor regression. However, the re-emergence of a drug-resistant tumors poses a significant challenge to their long-term effectiveness. A small subset of drug-tolerant "persister" cells have been identified as crucial precursors to full-fledged resistance. While various studies have pinpointed the activation of alternative signaling pathways as a key mechanism for evading therapy effects, targeting these secondary pathways alone has not yet reached the clinic. Currently, there is a pressing need for a more comprehensive profiling of the early drug-tolerant cellular landscape. This profiling is essential for understanding the underlying mechanisms driving resistance emergence and for devising more impactful therapeutic strategies. Drug resistance can evolve from a subpopulation of cancer cells that initially survive drug treatment and then gradually form a pool of drug-tolerant cells. Several studies have pinpointed the activation of a specific bypass pathway that appears to provide the critical therapeutic target for preventing drug tolerance. Here, we take a systems-biology approach, using proteomics and genomics to examine the development of drug tolerance to EGFR inhibitors in EGFR-mutant lung adenocarcinoma cells and BRAF inhibitors in BRAF-mutant melanoma cells. We found that there are numerous alternative mitogenic pathways that become activated in both cases, including YAP, STAT3, IGFR1, and phospholipase C (PLC)/protein kinase C (PKC) pathways. Our results suggest that an effective therapeutic strategy to prevent drug tolerance will need to take multiple alternative mitogenic pathways into account rather than focusing on one specific pathway. [ABSTRACT FROM AUTHOR]

Background: Prognosis is critically important in stroke cases, with angiogenesis playing a key role in determining outcomes. This study aimed to investigate the potential protective effects of Atractylenolide I (Atr I), Atractylenolide III (Atr III), and Paeoniflorin (Pae) in promoting angiogenesis following cerebral ischemia. Methods: The bEnd.3 cell line was used to evaluate the effects of these three compounds on vascular endothelial cell proliferation, migration, and tube formation. Male C57BL/6 mice underwent transient middle cerebral artery occlusion (MCAO), followed by daily intragastric administration of the Chinese medicine compounds to assess their impact on brain protection and angiogenesis. In vivo experiments included measuring infarct size and assessing neurological function. Immunofluorescence staining and an angiogenesis antibody array were used to evaluate angiogenesis in ischemic brain tissue. Functional enrichment analysis was performed to further investigate the pathways involved in the protective effects of the compounds. Molecular docking analysis explored the potential binding affinity of the compounds to insulin-like growth factor 2 (IGF-2), and Western blotting was used to measure levels of angiogenesis-related proteins. Results: In vitro, the combination of Atr I, Atr III, and Pae enhanced cell proliferation, promoted migration, and stimulated tube formation. In vivo, the combined treatment significantly facilitated neurological function recovery and angiogenesis by day 14. The treatment also increased levels of angiogenesis-related proteins, including IGF-2. Pearson correlation analysis revealed a strong positive association between IGF-2 levels in ischemic brain tissue and angiogenesis, suggesting a good affinity of the compounds for the IGF-2 binding site, as supported by molecular docking analysis. Conclusion: The administration of Atr I, Atr III, and Pae has shown significant enhancements in long-term stroke recovery in mice, likely due to the promotion of angiogenesis via increased activation of the IGF-2 pathway in ischemic brain tissue. [ABSTRACT FROM AUTHOR]

Background: Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that play an important role in cancer progression. Although the mechanism by which CAFs promote tumorigenesis has been well investigated, the underlying mechanism of CAFs activation by neighboring cancer cells remains elusive. In this study, we aim to investigate the signaling pathways involved in CAFs activation by gastric cancer cells (GC) and to provide insights into the therapeutic targeting of CAFs for overcoming GC. Methods: Alteration of receptor tyrosine kinase (RTK) activity in CAFs was analyzed using phospho-RTK array. The expression of CAFs effector genes was determined by RT-qPCR or ELISA. The migration and invasion of GC cells co-cultured with CAFs were examined by transwell migration/invasion assay. Results: We found that conditioned media (CM) from GC cells could activate multiple receptor tyrosine kinase signaling pathways, including ERK, AKT, and STAT3. Phospho-RTK array analysis showed that CM from GC cells activated PDGFR tyrosine phosphorylation, but only AKT activation was PDGFR-dependent. Furthermore, we found that connective tissue growth factor (CTGF), a member of the CCN family, was the most pronouncedly induced CAFs effector gene by GC cells. Knockdown of CTGF impaired the ability of CAFs to promote GC cell migration and invasion. Although the PDGFR-AKT pathway was pronouncedly activated in CAFs stimulated by GC cells, its pharmacological inhibition affected neither CTGF induction nor CAFs-induced GC cell migration. Unexpectedly, the knockdown of SRC and SRC-family kinase inhibitors, dasatinib and saracatinib, significantly impaired CTGF induction in activated CAFs and the migration of GC cells co-cultured with CAFs. SRC inhibitors restored the reduced expression of epithelial markers, E-cadherin and Zonula Occludens-1 (ZO-1), in GC cells co-cultured with CAFs, as well as CAFs-induced aggregate formation in a 3D tumor spheroid model. Conclusions: This study provides a characterization of the signaling pathways and effector genes involved in CAFs activation, and strategies that could effectively inhibit it in the context of GC. DC5ErEG73tsyg3F8WfE1xi Video Abstract [ABSTRACT FROM AUTHOR]

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Background: Swertiamarin is the main hepatoprotective component of Swertiapatens and has anti-inflammatory and antioxidation effects. Our previous study showed that it was a potent inhibitor of idiopathic pulmonary fibrosis (IPF) and can regulate the expressions of α-smooth muscle actin (α-SMA) and epithelial cadherin (E-cadherin), two markers of the TGF-β/Smad (transforming growth factor beta/suppressor of mothers against decapentaplegic family) signaling pathway. But its targets still need to be investigated. The main purpose of this study is to identify the targets of swertiamarin. Methods: GEO2R was used to analyze the differentially expressed genes (DEGs) of GSE10667, GSE110147, and GSE71351 datasets from the Gene Expression Omnibus (GEO) database. The DEGs were then enriched with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for their biological functions and annotated terms. The protein-protein interaction (PPI) network was constructed to identify hub genes. The identified hub genes were predicted for their bindings to swertiamarin by molecular docking (MD) and validated by experiments. Results: 76 upregulated and 27 downregulated DEGs were screened out. The DEGs were enriched in the biological function of cellular component (CC) and 7 cancer-related signaling pathways. Three hub genes, i.e., LOX (lysyl oxidase), COL5A2 (collagen type V alpha 2 chain), and CTGF (connective tissue growth factor) were selected, virtually tested for the interactions with swertiamarin by MD, and validated by in vitro experiments. Conclusion: LOX, COL5A2, and CTGF were identified as the targets of swertiamarin on IPF. [ABSTRACT FROM AUTHOR]

Background & Aims: Idiopathic pulmonary fibrosis (IPF) is a devastating chronic form of interstitial lung disease, characterized by an inflammatory infiltrate, deposition of extracellular matrix (ECM) components like collagen, and change in the architectural lung parenchyma. IPF has a poor prognosis and is lethal, with a median survival of 2 to 3 years. The etiology of the disease is obscure; however, several studies indicated that the disease is initiated by inflammation and the release of profibrotic growth factor, namely transforming growth factor β (TGF-β). The definitive treatment for patients has not yet been approved. Although. However, evidence suggests that inflammation plays an essential role in the development and pathogenesis of IPF as a stimulus. However, inflammation is sometimes referred to as a secondary event in fibrosis, partly due to the failure of anti-inflammatory drugs in clinical trials. Therefore, more studies are needed to evaluate the effectiveness of anti-inflammatory therapies. In primary injury or inflammation, TGF-β, a critical cytokine and regulator of fibrosis, promotes inflammation and increases the activity and proliferation of fibroblasts at the site of inflammation, differentiation into myofibroblasts, and production of extracellular matrix, leading to severe pulmonary fibrosis. As a downstream mediator, the connective tissue growth factor (CTGF) is induced by TGF-β and promotes its fibrotic effects, enhancing lung fibrosis through fibroblast proliferation and collagen deposition. Evidence has shown that CTGF expression is increased in fibroblasts of IPF patients. In addition, Endothelin-1 (ET-1) is another downstream mediator of TGF-β fibrogenic responses in fibroblasts, which produces an extracellular matrix and differentiates fibroblasts into myofibroblasts. Also, the expression of tumor necrosis factoralpha (TNF-α), which in addition to its inflammatory properties, also has fibrogenic properties, is significantly increased in the lungs of IPF patients. This expression is associated with increased fibroblasts and the deposition of extracellular matrix proteins in the interstitial region of the lung. In general, these findings indicate that these factors are reasonable targets for designing treatment strategies or evaluating the effect of proposed drugs in the treatment of IPF. Broad-acting anti-inflammatory molecules, including glucocorticoids like Dexamethasone, were considered potential therapy. Glucocorticoids are anti-inflammatory agents that can reduce proinflammatory molecules by suppressing cellular and humoral immunity. Dexamethasone is one of the most potent glucocorticoid drugs; however, despite the various investigations on the inhibition of pulmonary fibrosis, its anti-fibrosis effects are still debated, and there are contradictory findings in this regard. Also, the underlying mechanism of its impact on IPF is not clear. Therefore, in this study, the therapeutic effect of Dexamethasone on pulmonary fibrosis was scrutinized, based on the impact of various players of this scenario, namely the expression of TGF-β, TNF-α, CTGF, ET-1and hydroxyproline. Methods: 6 to 8 week-old male mice were used in this study. All animal care conditions, including temperature, light, and humidity, were observed. Mice aged 8-10 weeks were randomly divided into the following groups, and ten mice were placed in each group. 1. Control group treated with normal saline (Control) 2. Bleomycin inoculated group on day one (BLM) 3. Bleomycin inoculated on day one and dexamethasone-treated for 14 days (BLM+DEXA) The experiments were performed according to the approved guideline from the Faculty Ethics Committee (IR.IUMS.FMD.REC1396.9511127007). Lung fibrosis was induced intratracheal in anesthetized mice using 50 µL bleomycin (5mg/kg) by a single dose. The intervention with Dexamethasone (1mg/Kg/day) was done by 14 days intraperitoneal injection under sterile conditions. The mice were euthanized on day 21 under deep anesthesia, and their lungs were extracted. For evaluation, architectural changes occurred in the lungs based on bleomycin administration and dexamethasone intervention; the left lungs were fixed in formalin. The right lungs were frozen in liquid nitrogen for RNA extraction, gene expression analysis, ELISA, and hydroxyproline assay and were stored at -80 ºC. After 24 hours of immersion in formalin, paraffin blocks were prepared from the left lungs, and tissue incisions were made and transferred to the slide. After paraffin removal and leaching of slides, pathological examinations were performed using Masson Trichrome staining. The amount of hydroxyproline was measured using a hydroxyproline kit. Quantitative measurement of mRNA from CTGF and ET-1 genes in the lungs of mice was performed using real-time PCR. Data analysis of different groups was performed using Prism software and one-way ANOVA and Tukey multiple comparison test. In order to evaluate the anti-fibrotic effect of Dexamethasone in IPF, the bleomycin-induced mouse model was treated with Dexamethasone. After fibrosis was induced by intratracheal BLM administration, histopathological evaluation and hydroxyproline assay, ELISA for measurement of TGF-β and TNF-α, and RT-PCR were performed to evaluate the expression CTGF and ET-1 genes. Results: Histological examination indicated the deposition of collagen after administration of one dose of Bleomycin; an enzymatic analysis of hydroxyproline showed that administration of a single dose of BLM intratracheally leads to extensive fibrosis in the lungs of C57BL/6 mice on day 21. In comparison, 14 days of intraperitoneal treatment with Dexamethasone reduced the severity of fibrosis. Histological examination of Mason trichrome-stained tissue sections from lungs of control mice (receiving saline intratracheally) showed normal lung structure including no extracellular matrix deposition (based on the absence of blue color in the interstitium) and the alveolar space was distinct. While the lung tissue sections of BLM mice showed histopathological changes, including increased fibrotic areas and increased collagen deposition (based on increased blue color), and decreased alveolar spaces compared to the control group. However, lung tissue sections from dexamethasone-treated mice (BLM+DEXA group) showed only mild fibrosis. Dexamethasone treatment reduced the amount of hydroxyproline compared to the Bleomycin treated group; however, this reduction was not statistically significant (P> 0.05). Dexamethasone treatment significantly reduced TGF-β levels in the lungs of the BLM + DEXA group compared with the BLM group (P <0.001). Further, treatment of received Bleomycin (BLM) received mice with Dexamethasone (BLM + DEXA) reduced the amount of TNF-α when compared with Bleomycin received only group nevertheless, this reduction was not statistically significant to mice but statistically significant (P <0.05). The levels of TNF-α and TGF-β in the lungs of Bleomycin treatment mice (BLM) were higher when compared with Normal saline-treated mice (Control) (P <0.001). Dexamethasone treatment can induce its effect by inhibiting a prominent signaling pathway, namely TGF-β. Further, Dexamethasone treatment of Bleomycine received mice significantly reduced the expression of CTGF gene (P <0.001). CTGF expression occurs before the TGF- β cytokine is expressed during the phenomenon of fibrosis. It seems that CTGF is responsible for extracellular accumulation in the fibrosis pathway. In addition, 14 days of treatment of Bleomycin received mice with Dexamethason reduced ET-1 genes in the lungs of the BLM+DEXA group compared with the BLM group (P <0.001). ET1 gene is responsible for the differentiation of fibroblasts to myofibroblasts. It is known that Dexamethasone can ameliorate fibrosis; however, it is not known how this steroid can induce its effect or whether its impact is based on its role in the inhibition of inflammation Conclusion: Our findings showed that intratracheal inoculation of Bleomycin in mice resulted in extensive accumulation of extracellular matrix in the lungs of C57BL/6 mice. Our study demonstrated that administration of Dexamethasone attenuated Bleomycin-induced pulmonary fibrosis in C57BL/6 mice by reducing hydroxyproline, production of two critical cytokines, the TGF-β and TNF-α, also by reduction of CTGF and ET-1 gene expression. Still, further investigations are required to understand how this compound can reduce fibrosis. [ABSTRACT FROM AUTHOR]

Fibrosis is characterized by excessive accumulation of extracellular matrix, which is a key feature of uterine fibroids. Our prior research supports the tenet that inhibition of fibrotic processes may restrict fibroid growth. Epigallocatechin gallate (EGCG), a green tea compound with powerful antioxidant properties, is an investigational drug for uterine fibroids. An early phase clinical trial showed that EGCG was effective in reducing fibroid size and its associated symptoms; however, its mechanism of action(s) has not been completely elucidated. Here, we probed effects of EGCG on key signaling pathways involved in fibroid cell fibrosis. Viability of myometrial and fibroid cells was not greatly affected by EGCG treatment (1–200 µM). Cyclin D1, a protein involved in cell cycle progression, was increased in fibroid cells and was significantly reduced by EGCG. EGCG treatment significantly reduced mRNA or protein levels of key fibrotic proteins, including fibronectin (FN1), collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and actin alpha 2, smooth muscle (ACTA2) in fibroid cells, suggesting antifibrotic effects. EGCG treatment altered the activation of YAP, β-catenin, JNK and AKT, but not Smad 2/3 signaling pathways involved in mediating fibrotic process. Finally, we conducted a comparative study to evaluate the ability of EGCG to regulate fibrosis with synthetic inhibitors. We observed that EGCG displayed greater efficacy than ICG-001 (β-catenin), SP600125 (JNK) and MK-2206 (AKT) inhibitors, and its effects were equivalent to verteporfin (YAP) or SB525334 (Smad) for regulating expression of key fibrotic mediators. These data indicate that EGCG exhibits anti-fibrotic effects in fibroid cells. These results provide insight into mechanisms behind the observed clinical efficacy of EGCG against uterine fibroids. [ABSTRACT FROM AUTHOR]

To investigate the role of a disintegrin and metalloprotease protein 17 (ADAM17) in regulating the proliferation and extracellular matrix (ECM) expression of keloid fibroblasts (KFs) via the epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway. ADAM17 expression in keloid tissues was detected by western blotting. KFs were isolated, cultured and divided into the control, shNC (negative control), shADAM17, transforming growth factor-β1 (TGF-β1), TGF-β1 + shNC and TGF-β1 + shADAM17 groups. The expression of ECM was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Western blotting was performed to detect the expression of proteins. Cell proliferation was detected by a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, while cell invasion and migration were examined by Transwell and wound healing assays. The expression of ADAM17 was increased in keloid tissues and KFs. Compared with the control group, the expression of p-EGFR and p-ERK/1/2/ERK1/2, as well as the expression of collagen I, collagen III, connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA), were decreased in KFs from the shADAM17 group, with decreased cell proliferation, invasion and migration. In contrast, the TGF-β1 group presented the opposite trend in these aspects. In addition, compared with the TGF-β1 group, KFs from the TGF-β1 + shADAM17 group had decreased ECM expression, proliferation, invasion and migration. ADAM17 expression was upregulated in keloid tissues. Silencing ADAM17 may inhibit the activity of the EGFR/ERK pathway to limit the deposition of ECM in KFs with reduced proliferation, invasion and migration. [ABSTRACT FROM AUTHOR]

Renal fibrosis is the final pathway for chronic kidney disease to end-stage renal failure. Noncoding RNAs have been reported to play a crucial role in renal fibrosis. Here, the effects of long noncoding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) and miR-31 on renal fibrosis and their regulatory mechanism were evaluated. RT-qPCR was used to assess NEAT1, miR-31, and RhoA levels. Western blot was performed to analyze the expression of fibrosis markers, RhoA, rho-related kinase (ROCK1), and connective tissue growth factor (CTGF). RNA immunoprecipitation (RIP), fluorescence in situ hybridization (FISH), and luciferase reporter assays verified the interaction between miR-31 and NEAT1 or RhoA. Renal fibrosis and injury were observed by Masson and hematoxylin and eosin (H&E) staining. The expression level of inflammatory cytokines was detected by ELISA. Immunohistochemistry (IHC) was performed to examine the expression levels of α-smooth muscle actin (a-SMA) and RhoA in renal tissues. We showed that NEAT1 was highly expressed, whereas miR-31 was decreased in renal fibrosis. NEAT1 was found to directly bind miR-31 to positively regulate RhoA expression. Furthermore, NEAT1 silencing inhibited renal fibrosis and inflammation and suppressed the RhoA/ROCK1 signaling pathway. However, knockdown of miR-31 could reverse these effects. NEAT1 silencing or overexpression of miR-31 alleviated renal fibrosis in vivo. In conclusion, NEAT1 accelerates renal fibrosis progression via negative regulation of miR-31 and the activation of RhoA/ROCK1 pathway, thereby upregulating the expression level of CTGF, providing a theoretical basis for treatment and prognostic evaluation of renal fibrosis. [ABSTRACT FROM AUTHOR]

Aim. Pirfenidone (PFD), an antifibrotic drug, has various beneficial functions such as antioxidant, antifibrotic, and anti-inflammatory effects. This study aimed to explore the molecular mechanisms underlying how PFD modulates retinal pigment epithelial (RPE) cells involved in neovascularization and subretinal fibrosis. Methods. ARPE-19 cell lines were treated with transforming growth factor-beta 2 (TGF-β2) alone or in combination with PFD. RPE cell viability, as a consequence of PFD use, was determined by the CCK-8 assay. Cell migration was assessed by the wound closure assay and quantified by the Image J software. Protein expression of the following markers was measured by the western blot analysis: an epithelial cell marker and E-cadherin; mesenchymal cell markers, fibronectin, matrix metalloprotein-9 (MMP-9), and alpha-smooth muscle actin (α-SMA); a fibrotic marker and connective tissue growth factor (CTGF); an angiogenesis marker and vascular endothelial growth factor (VEGF); NF-κB/Snail. The mRNA levels of fibronectin and α-SMA were determined by quantitative real-time PCR. VEGF was quantitatively measured by the enzyme-linked immunosorbent assay. Results. The cell viability assay revealed that PFD had no significant cytotoxic effect on RPE cells at concentrations of less than 1 mg/mL. The cell scratch assay showed that TGF-β2 stimulation significantly improved the migration of RPE cells and that PFD attenuated this effect. PFD significantly inhibited the TGF-β2-induced protein expression of E-cadherin and increased the TGF-β2-induced protein expression of fibronectin, MMP-9, α-SMA, CTGF, and VEGF in ARPE-19 cells. The mRNA expression of fibronectin and α-SMA was inhibited by PFD in TGF-β2-inducedARPE-19 cells. Additionally, the increased intracellular and supernatant expression of VEGF protein was suppressed by PFD. Mechanistically, RPE cells treated with PFD + TGF-β2 exhibited a decrease in phosphorylation of the NF-κB P65 subunit and activation of Snail, compared with the RPE cells treated with TGF-β2 alone. Conclusion. PFD ameliorated TGF-β2-induced neovascularization and fibrosis by suppressing the NF-κB/Snail signaling pathway. Therefore, PFD may be a potential drug in the treatment of age-related macular degeneration. [ABSTRACT FROM AUTHOR]

Background. Myocardial fibrosis (MF) is an essential pathological factor for heart failure. Previous studies have shown that the combination of Carthamus tinctorius L. and Lepidium apetalum Willd. (C-L), two types of Chinese herbal medicine, can ameliorate MF after myocardial infarction (MI) in rats and inhibit the activation of myocardial fibroblasts. However, the mechanism of C-L in the treatment of MF remains unclear. Methods. A rat model of MF with left anterior descending coronary ligation-induced MI was first established. Then, the effects of C-L on cardiac function, MF, and endothelial-to-mesenchymal transition (EndMT) were evaluated by the left ventricular ejection fraction (LVEF), serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels, Masson's trichrome staining, and immunohistochemical and immunofluorescence staining. Next, a hypoxia-induced cardiac microvascular endothelial cell (CMEC) model was established to observe the effects of C-L on EndMT. The supernatant of CMECs was collected and used to culture cardiac fibroblasts (CFs) and observe the effects of CMEC paracrine factors on CFs. Results. Animal experiments indicated that C-L improves the cardiac function of rats after MI, inhibits the progression of EndMT and MF, and downregulates TGFβ1, Snail, and CTGF expression. Cell experiments showed that drug-loaded serum containing C-L inhibits the EndMT of CMECs under hypoxic conditions. The culture supernatant of CMECs grown under hypoxic conditions significantly activated CFs. After treatment with C-L, the activating factor for CFs in hypoxic CMEC culture supernatant was substantially downregulated, and the effect of the culture supernatant on CF activation was also reduced. However, TGFβ1 agonists inhibited the effects of C-L on CMECs and CFs. Conclusion. Our data demonstrated that by regulating the TGFβ1/Snail pathway, C-L inhibits EndMT of CMECs and reduces the release of CF-activating factors in cells undergoing EndMT. [ABSTRACT FROM AUTHOR]

Objectives This study aimed to investigate the high-resolution CT (HRCT) characteristics of anti-melanoma differentiation-associated gene 5 (MDA5) antibody positive dermatomyositis-associated interstitial lung disease (anti-MDA5 DM-ILD), and to clarify the underlying mechanisms of the clinical phenomenon. Methods Clinical data and HRCT patterns were compared between anti-MDA5 DM-ILD (n  = 32) and antisynthetase syndrome-associated ILD (ASS-ILD) (n  = 29). RNA sequencing of whole-blood samples from the two groups, and in vitro experiments using human embryonic lung fibroblasts (HELFs) were conducted to explore the potential mechanisms of the clinical findings. Results The anti-MDA5 DM-ILD subset had a significantly higher incidence of rapidly progressive ILD (RPILD) than ASS-ILD (65.6% vs 37.9%; P  = 0.031). The relative percentage of the lung fibrosis HRCT pattern was significantly lower in the anti-MDA5 DM-ILD group, especially the RPILD subgroup (P  = 0.013 and 0.003, respectively). RNA sequencing detected the upregulated genes including interferon-induced helicase C domain 1 (encoding MDA5), and a trend towards downregulated expression of TGF-β signalling components in anti-MDA5 DM-ILD. In vitro culture of HELFs revealed that upregulated expression of MDA5 in HELFs was correlated with the downregulated expression of alpha smooth muscle actin, connective tissue growth factor, collagen I and collagen III by suppressing the TGF-β signalling pathway. Conclusions Anti-MDA5 DM-ILD patients have significantly less lung fibrosis and elevated MDA5 expression. The upregulated expression of MDA5 has relations with the suppression of the pro-fibrotic function of fibroblasts via the TGF-β signalling pathway, which may partially explain the mechanism of the clinical phenomenon. [ABSTRACT FROM AUTHOR]

Dysregulation of long intergenic non-protein coding RNA 00,641 (LINC00641) is associated with the malignancy progression of multiple cancers, including thyroid carcinoma. The current study aimed to determine the role of LINC00641 in papillary thyroid carcinoma (PTC) and the underlying mechanism. We found that LINC00641 was downregulated in PTC tissues and cells(p < 0.05), and overexpression of LINC00641 inhibited PTC cell proliferation and invasion, and induced apoptosis(p < 0.05), while silencing LINC00641 promoted the proliferation and invasion in PTC cells, and inhibited cell apoptosis(p < 0.05). Furthermore, we found that Glioma-associated oncogene homolog 1 (GLI1) expression was negatively correlated with LINC00641 expression in PTC tissues (r 2 = 0.7649, p < 0.0001), and silencing GLI1 inhibited PTC cell proliferation and invasion, and induced apoptosis(p < 0.05). Meanwhile, RNA immunoprecipitation (RIP) and RNA pull-down assays confirmed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) bound to LINC00641 as an RNA binding protein, and overexpression of LINC00641 destabilized GLI1 mRNA by competitively binding to IGF2BP1. Rescue experiments revealed that overexpression of GLI1 restored the inhibitory effect of LINC00641 overexpression on activation of the AKT pathway, as well as PTC cell proliferation and invasion, and counteracted the induction of cell apoptosis by LINC00641 overexpression. Finally, in vivo experimental results showed that overexpression of LINC00641 markedly suppressed tumor growth and reduced expression of GLI1 and p-AKT in xenograft tumor mice(p < 0.05). In summary, this study highlighted that LINC00641 plays a critical role in the malignant biological progression of PTC by regulating the LINC00641/IGF2BP1/GLI1/AKT signaling pathway, which may serve as a potential therapeutic target for PTC. [ABSTRACT FROM AUTHOR]

Purpose: Agaro-oligosaccharides (AGO), hydrolysis products of agarose, is known to have antioxidant and anti-inflammatory properties. Speculating that AGO is effective for preventing aging, we investigated the longevity-supporting effects of AGO and their mechanisms using Caenorhabditis elegans. Methods: Caenorhabditis elegans were fed AGO from young adulthood. The lifespan, locomotory activity, lipofuscin accumulation, and heat stress resistance of the worms were examined. To elucidate mechanisms of AGO-mediated longevity, we conducted comprehensive expression analysis using microarrays. Moreover, we used quantitative real-time PCR (qRT-PCR) to verify the genes showing differential expression levels. Furthermore, we measured the lifespan of loss-of-function mutants to determine the genes related to AGO-mediated longevity. Results: AGO extended the lifespan of C. elegans, reduced lipofuscin accumulation, and maintained vigorous locomotion. The microarray analysis revealed that the endoplasmic reticulum-unfolded protein response (ER-UPR) and insulin/insulin-like growth factor-1-mediated signaling (IIS) pathway were activated in AGO-fed worms. The qRT-PCR analysis showed that AGO treatment suppressed sir-2.1 expression, which is a negative regulator of ER-UPR. In loss-of-function mutant of sir-2.1, AGO-induced longevity and heat stress resistance were decreased or cancelled completely. Furthermore, the pro-longevity effect of AGO was decreased in loss-of-function mutants of abnormal Dauer formation (daf) -2 and daf-16, which are IIS pathway-related genes. Conclusion: AGO delays the C. elegans aging process and extends their lifespan through the activations of ER-UPR and the IIS pathway. [ABSTRACT FROM AUTHOR]

Objective and Background: Gingival overgrowth (GO) is a common side effect of some drugs such as anticonvulsants, immunosuppressant, and calcium channel blockers. Among them, the antiepileptic agent phenytoin is the most common agent related to this condition due to its high incidence. Transforming growth factor β (TGFβ) importantly contributes to the pathogenesis of GO. Connective tissue growth factor (CTGF or CCN2) is a key mediator of tissue fibrosis and is positively associated with the degree of fibrosis in GO. We previously showed that Src, c‐jun N‐terminal kinase, and Smad3 mediate TGFβ1‐induced CCN2 protein expression in human gingival fibroblasts (HGFs). This study investigates whether phenytoin can induce CCN2 synthesis through activated latent TGFβ in HGFs and its mechanisms. Methods: CCN2 synthesis, latent TGFβ1 activation, and cellular reactive oxygen species (ROS) generation in HGFs were studied using western blot analysis, a TGFβ1 Emax® ImmunoAssay System, and 2′,7′‐dichlorodihydrofluorescein diacetate (an oxidation‐sensitive fluorescent probe), respectively. Results: Phenytoin significantly stimulated CCN2 synthesis, latent TGFβ1 activation, and ROS generation in HGFs. Addition of an TGFβ‐neutralizing antibody, TGFβ receptor kinase inhibitor SB431542, and Smad3 inhibitor SIS3 completely inhibited phenytoin‐induced CCN2 synthesis. General antioxidant N‐acetylcysteine, NADPH oxidase (NOX) inhibitor diphenylene iodonium, and specific NOX4 inhibitor plumbagin almost completely suppressed phenytoin‐induced total cellular ROS and latent TGFβ1 activation. Curcumin dose‐dependently decreased phenytoin‐induced TGFβ1 activation and CCN2 synthesis in HGFs. Conclusions: Our findings indicated that NOX4‐derived ROS play pivotal roles in phenytoin‐induced latent TGFβ1 activation. Molecular targeting the phenytoin/NOX4/ROS/TGFβ1 pathway may provide promising strategies for the prevention and treatment of GO. Curcumin‐inhibited phenytoin‐induced CCN2 synthesis is caused by the suppression of latent TGFβ1 activation. [ABSTRACT FROM AUTHOR]

Simple Summary: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy arising from the aberrant proliferation of immature T-cell progenitors. Despite improved insights in genetic and biological characteristics of T-ALL, clinical therapy has remained largely similar. Recent studies have shown that leukemia-initiating cells (LICs) and leukemic niches play major roles in the initiation and progression of T-ALL, thus, facilitating the development of targeted therapies. This review provides a broad overview of the recent discoveries on LICs and leukemic niches in the context of T-ALL, with a particular focus on the current precision medicine. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of hematological malignancy characterized by its high heterogeneity and potentially life-threatening clinical features. Despite the advances in risk stratification and therapeutic management of T-ALL, patients often suffer from treatment failure and chemotherapy-induced toxicity, calling for greater efforts to improve therapeutic efficacy and safety in the treatment of T-ALL. During the past decades, increasing evidence has shown the indispensable effects of leukemia-initiating cells (LICs) and leukemic niches on T-ALL initiation and progression. These milestones greatly facilitate precision medicine by interfering with the pathways that are associated with LICs and leukemic niches or by targeting themselves directly. Most of these novel agents, either alone or in combination with conventional chemotherapy, have shown promising preclinical results, facilitating them to be further evaluated under clinical trials. In this review, we summarize the latest discoveries in LICs and leukemic niches in terms of T-ALL, with a particular highlight on the current precision medicine. The challenges and future prospects are also discussed. [ABSTRACT FROM AUTHOR]

Objective: Acteoside (ACT) has been reported to regulate the inflammation and immune response. The study aims to explore the effect of ACT on renal fibrosis in unilateral ureteral obstruction (UUO) rats. Methods: Eighteen Sprague-Dawley rats were randomly divided into 3 groups: sham group, opened the abdominal cavity and sutured abdominal; UUO group, performed UUO surgery; and ACT + UUO group, ACT (40 mg/kg) was given by gavage every day after UUO surgery. After 2 weeks of rat model construction, urine and blood samples were collected for biochemical analysis, while kidney tissues were harvested for hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemistry staining. The expression of connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA), collagen III, heat shock protein 47 (HSP47), and β-catenin in the renal tissue was detected and the correlation between these proteins was analyzed. Results: ACT improved the parameters of renal function in UUO rats, including decreased creatinine and urea nitrogen, and declined urinary protein. Pathological analysis suggested that ACT improved the conditions of renal tubule lesion (including structure destruction, atrophy and lumen obstruction), renal interstitial fibrosis and inflammatory cell infiltration in UUO rats. It also down-regulated the expressions of fibrin-related proteins β-catenin, CTGF, α-SMA, collagen III, and HSP47. Correlation analysis found that β-catenin and CTGF were correlated with the expressions of α-SMA, collagen III, and HSP47. Conclusions: ACT could alleviate renal fibrosis in UUO rats probably via inhibiting β-catenin/CTGF signaling pathway. [ABSTRACT FROM AUTHOR]

The WNT1 inducible signaling pathway protein 1 (WISP1), a member of the connective tissue growth factor family, plays a crucial role in several important cellular functions in a highly tissue-specific manner. Results of a RT-qPCR indicated that WISP1 expressed only in cells of the human prostate fibroblasts, HPrF and WPMY-1, but not the prostate carcinoma cells in vitro. Two major isoforms (WISP1v1 and WISP1v2) were identified in the HPrF cells determined by RT-PCR and immunoblot assays. The knock-down of a WISP1 blocked cell proliferation and contraction, while treating respectively with the conditioned medium from the ectopic WISP1v1- and WISPv2-overexpressed 293T cells enhanced the migration of HPrF cells. The TNFα induced WISP1 secretion and cell contraction while the knock-down of WISP1 attenuated these effects, although TNFα did not affect the proliferation of the HPrF cells. The ectopic overexpression of WISP1v1 but not WISP1v2 downregulated the N-myc downstream regulated 1 (NDRG1) while upregulating N-cadherin, slug, snail, and vimentin gene expressions which induced not only the cell proliferation and invasion in vitro but also tumor growth of prostate carcinoma cells in vivo. The results confirmed that WISP1 is a stroma-specific secreting protein, enhancing the cell migration and contraction of prostate fibroblasts, as well as the proliferation, invasion, and tumor growth of prostate carcinoma cells. [ABSTRACT FROM AUTHOR]

Objective. This work is aimed at revealing the role and the molecular mechanism of connective tissue growth factor 2 (CTGF) in the osteoblast differentiation of periodontal ligament stem cells (PDLSCs). Methods. The osteogenic differentiation of PDLSCs was induced by osteogenic induction medium (OM), and the expression level of osteogenic related proteins ALP, RUNX2, OCN, and CTGF was estimated using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis. We constructed cell lines with CTGF overexpression or knockdown to verify the role of CTGF in the osteoblast differentiation of PDLSCs. Alkaline phosphatase (ALP) staining was introduced to measure the osteoblasts activity, and alizarin red S (ARS) staining was employed to test matrix mineralization. The interaction between CTGF and bone morphogenetic protein-2 (BMP-2) was determined by endogenous coimmunoprecipitation (Co-IP). Results. The expression level of CTGF was increased during the osteogenic induction of PDLSCs. Additionally, CTGF overexpression effectively maintained the stemness and facilitated the osteoblast differentiation in PDLSCs, and CTGF knockdown exerted opposite effects. Moreover, at molecular mechanism, CTGF increased the activity of BMP-2/Smad signaling pathway. Conclusion. This investigation verified that CTGF promotes the osteoblast differentiation in PDLSCs at least partly by activating BMP-2/Smad cascade signal. [ABSTRACT FROM AUTHOR]

Muscle atrophy (MA) is a case in which protein degeneration occurs excessively due to an imbalance between protein synthesis and breakdown, and is characterized by decreased muscle mass and weakened muscle strength. Despite mounting concern about MA, the number of patients with MA is increasing every year. The aim of the present study was to assess the impact of Gardeniae Fructus (GF) hot water extract on dexamethasone (DEX)-induced MA in mice. C57BL/6N mice were grouped (n = 8) as follows: Normal mice (Normal), MA mice were treated with distilled water (Control), MA mice were treated with GF 100 mg/kg (GF100), MA mice were treated with GF 200 mg/kg (GF200). For 10 days, DEX (25 mg/kg body weight, i.p.) injection was used to induce MA, and GF was administered. GF treatment restored the muscle weight decreased due to MA, and in particular, the weights of EDL+TA and Sol were significantly increased in the GF200 group. Also, it was confirmed that the swimming time was improved in the GF200 group. In addition, the expression of NADPH oxidase related to oxidative stress was significantly reduced, and protective (insulin-like growth factor I/phosphoinositide 3-kinase/protein kinase B pathway) and catabolic (AMP-activated kinase [AMPK]/sirtuin 1 [SIRT1]/proliferator-activated receptor-gamma coactivator-1α (PGC-1α)-forkhead box O (FOXO) pathway) pathways were significantly modulated. These results demonstrate that GF regulates muscle protein synthesis and catabolic pathways, and in particular, it is judged to improve MA by regulating the proteolytic AMPK/SIRT1/PGC-1α-FOXO pathway. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is a common complication of diabetes and it is urgent to develop effective therapies for DN. In this study, high‐sucrose and high‐fat diet combined with streptozotocin was used to induce DN in rats to observe the effects of natural flavonoid ombuin on renal function, inflammation, and interstitial fibrosis. Immunohistochemistry and western blotting analysis were used to detect protein expression levels. Results showed that ombuin significantly improved renal function and pathological injury, inhibited accumulation of advanced glycation end‐products, suppressed the release of inflammatory cytokines, and improved renal interstitial fibrosis in DN rats. Ombuin also significantly downregulated the expressions of transforming growth factor beta1 (TGF‐β1), connective tissue growth factor (CTGF), fibronectin (FN), p65, phosphorylated (p)‐p65, Cleaved‐Notch 1, and hairy and enhancer of split 1 (Hes 1), and upregulated the expression of peroxisome proliferator‐activated receptor γ (PPAR γ). When PPAR γ activity was inhibited by T0070907, the effects of ombuin on improving DN were significantly reversed, and the expressions of TGF‐β1, FN, CTGF, p‐p65, and p65 increased, while the expressions of Cleaved‐Notch 1 and Hes 1 were not significantly affected. These results suggest that ombuin may activate PPAR γ to exert anti‐inflammatory and antifibrotic effects by inhibiting Notch 1 activity in DN. It is also possible that ombuin acts on these two independent signal pathways synchronously. [ABSTRACT FROM AUTHOR]

Objective. Microglial BV-2 cells are activated in the brain following insomnia. Naringin (NAR) is a polymethoxylated flavonoid that is also commonly found in citrus fruits and is known for its antioxidant potential. However, the effect of NAR on microglial cells has rarely been studied in the brain of an organism after insomnia. This study aimed to investigate the effects and potential mechanisms of action of NAR on microglial cell activation and inflammation. Methods. BV-2 cells were obtained from the China Center for Type Culture Collection and randomly divided into five treatment groups: control, model, NAR (10 μM), WP1066 (5 μM), and NAR + WP1066. With the exception of the control group, all groups were stimulated with LPS (1 μg/mL) for 6 h. CCK8 was used to quantify cell viability and a scratch test was performed to detect cell migration. The expression levels of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), nterleukin 10 (IL-10), and insulin like growth factor (1IGF-1) were measured by ELISA. Western blotting was performed to determine the levels of p-STAT3 and p-JAK. The Focalcheck™ Thin-Ring Fluorescent Microspheres kit was used to detect cell phagocytosis. Immunofluorescence was used to observe the expression of iNOS and arginase1 in BV-2 cells. Results. Compared with the control group, cell migration, cell viability, and the expression of IL-1β, IL-6, TNF-α, and iNOS were significantly increased in the model group, whereas the expression levels of IL-10, IGF-1, and arginase 1, as well as cell phagocytosis were reduced. With the increase in NAR concentration, cell migration, cell viability, the expression levels of IL-1β, IL-6, TNF-α, and iNOS decreased, while the expression of IL-10, IGF-1, and arginase 1 increased. Compared with the control group, p-STAT3, and p-JAK expression in the model group were significantly increased (P<0.05). Compared with the model group, the expression of p-STAT3 and p-JAK in the NAR, NAR + WP1066, and WP1066 groups was significantly decreased (P < 0.05). Conclusion. NAR treatment inhibited the proliferation, migration, and inflammation of BV-2 cells as well as the activation of microglia to the M1 phenotype. Conversely, NAR treatment promoted the activation of microglia to the M2 phenotype and enhanced the phagocytic function of BV-2 cells by regulating the activity of the JAK/STAT3 pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Hepatocellular carcinoma (HCC) progression depends on two major processes, tumor growth and invasion. The present study investigated how these events are linked. A panel of HCC cell lines were stimulated with insulin-like growth factor-1 (IGF1) and the biological behavior was evaluated. IGF1 activated the proliferation and invasion cascade by altering the expression level of integrin α subtypes, which were associated with the AKT-mTOR pathway and the CDK-Cyclin axis. We assume that HCC progression is controlled by a fine-tuned network between IGF1 driven integrin signaling, the Akt-mTOR pathway, and the CDK-Cyclin axis. Concerted targeting of these pathways may, therefore, become an innovative option to prevent cancer dissemination. Integrin receptors contribute to hepatocellular carcinoma (HCC) invasion, while AKT-mTOR signaling controls mitosis. The present study was designed to explore the links between integrins and the AKT-mTOR pathway and the CDK-Cyclin axis. HCC cell lines (HepG2, Huh7, Hep3B) were stimulated with soluble collagen or Matrigel to activate integrins, or with insulin-like growth factor 1 (IGF1) to activate AKT-mTOR. HCC growth, proliferation, adhesion, and chemotaxis were evaluated. AKT/mTOR-related proteins, proteins of the CDK-Cyclin axis, focal adhesion kinase (FAK), and integrin-linked kinase (ILK) were determined following IGF1-stimulation or integrin knockdown. Stimulation with collagen or Matrigel increased tumor cell growth and proliferation. This was associated with significant alteration of the integrins α2, αV, and β1. Blockade of these integrins led to cell cycle arrest in G2/M and diminished the number of tumor cell clones. Knocking down the integrins α2 or β1 suppressed ILK, reduced FAK-phosphorylation and diminished AKT/mTOR, as well as the proteins of the CDK-Cyclin axis. Activating the cells with IGF1 enhanced the expression of the integrins α2, αV, β1, activated FAK, and increased tumor cell adhesion and chemotaxis. Blocking the AKT pathway canceled the enhancing effect of IGF on the integrins α2 and β1. These findings reveal that HCC growth, proliferation, and invasion are controlled by a fine-tuned network between α2/β1-FAK signaling, the AKT-mTOR pathway, and the CDK–Cyclin axis. Concerted blockade of the integrin α2/β1 complex along with AKT-mTOR signaling could, therefore, provide an option to prevent progressive dissemination of HCC. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the present study was to investigate the efficacy of recombinant human endostatin (ES) (rh-ES) combined with radiation on rat cardiomyocyte apoptosis and the regulatory mechanism of transforming growth factor beta1 (TGF-β1)/Sma and Mad-related protein 3 (Smad3)/connective tissue growth factor (CTGF) signaling.Method: The primary cardiomyocytes were isolated from neonatal Sprague-Dawley rats for culture in vitro and divided into blank control group (without treatment), 10 Gy radiation + siTGF-β1 siRNA (gene silencing) group, ES + siTGF-β1 siRNA group, and 10 Gy radiation + ES + siTGF-β1 siRNA group. Methyl thiazolyl tetrazolium assay was used to calculate the half-maximal inhibitory concentration (IC50) of rh-ES on cardiomyocytes. Adenoviral vector was constructed for virus packaging to silence TGF-β1 expression in cardiomyocytes. Quantitative real-time polymerase chain reaction and Western blot were carried out to analyze TGF-β1, Smad2, Smad3 and CTGF expression at both gene and protein levels. Flow cytometry and electron microscope were used to examine cell apoptosis.Results: ES had a dose-dependent inhibitory effect on the proliferation of primary rat cardiomyocytes. ES combined with radiotherapy significantly inhibited cardiomyocyte proliferation and promoted cell apoptosis (P < 0.01). The gene and protein expression of TGF-β1, Smad2, Smad3 and CTGF were significantly up-regulated in primary cardiomyocytes transfected with TGF-β1 gene (P < 0.05).Conclusion: The combination therapy with rh-ES and radiation can promote cardiomyocyte apoptosis and aggravate myocardial cell damage via TGF-β1/Smad3/CTGF signaling pathway. [ABSTRACT FROM AUTHOR]

Impaired keratinocyte functions are major factors that are responsible for delayed diabetic wound healing. In addition to its antimicrobial activity, the antimicrobial peptide derived from insulin‐like growth factor‐binding protein 5 (AMP‐IBP5) activates mast cells and promotes keratinocyte and fibroblast proliferation and migration. However, its effects on diabetic wound healing remain unclear. Human keratinocytes were cultured in normal or high glucose milieus. The production of angiogenic growth factor and cell proliferation and migration were evaluated. Wounds in normal and streptozotocin‐induced diabetic mice were monitored and histologically examined. We found that AMP‐IBP5 rescued the high glucose‐induced attenuation of proliferation and migration as well as the production of angiogenin and vascular endothelial growth factors in keratinocytes. The AMP‐IBP5‐induced activity was mediated by the epidermal growth factor receptor, signal transducer and activator of transcription 1 and 3, and mitogen‐activated protein kinase pathways, as indicated by the inhibitory effects of pathway‐specific inhibitors. In vivo, AMP‐IBP5 markedly accelerated wound healing, increased the expression of angiogenic factors and promoted vessel formation in both normal and diabetic mice. Overall, the finding that AMP‐IBP5 accelerated diabetic wound healing by protecting against glucotoxicity and promoting angiogenesis suggests that AMP‐IBP5 might be a potential therapeutic target for treating chronic diabetic wounds. [ABSTRACT FROM AUTHOR]

Introduction and objectives: Caffeine consumption is associated with beneficial effects on hepatic disorders. The objectives of this study were to evaluate the antifibrotic effects of caffeine on experimental nonalcoholic steatohepatitis (NASH) induced with a high-fat, high-sucrose, high-cholesterol diet (HFSCD), as well as to evaluate the ability of caffeine to prevent the progression of experimental liver fibrosis induced by the administration of thioacetamide (TAA) in rats and explore the mechanisms of action. Methods: NASH and fibrosis were induced in rats by the administration of an HFSCD for 15 weeks, and liver fibrosis was induced by intraperitoneal administration of 200 mg/kg TAA 3 times per week, for 6 weeks. Caffeine was administered at a dose of 50 mg/kg body weight. The effects of diet, TAA, and caffeine on fibrosis were evaluated by biochemical and histological examinations. The profibrotic pathways were analyzed by western blotting and immunohistochemistry. Results: Rats exhibited liver fibrosis after HFSCD feeding and the administration of TAA. Caffeine could reduce the hepatic level of collagen and the fibrotic area in the liver. Caffeine prevented the progression of liver fibrosis by decreasing transforming growth factor-beta (TGF-b), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (a-SMA) expression and by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and Smad3 phosphorylation. Conclusions: Caffeine attenuates NASH and the progression of liver fibrosis due to its antifibrotic effects and modulating the MAPK and TGF-b pathways. Therefore, caffeine could be a suitable candidate for treating liver diseases associated with fibrosis. [ABSTRACT FROM AUTHOR]

Background: Our previous study indicated that Ginkgo biloba leaf extract (EGb) could protect against cisplatin-induced acute kidney injury in rabbits. The present study aimed to determine the effects and potential molecular mechanisms of EGb on chronic renal interstitial fibrosis induced by cisplatin using in vivo and in vitro models. Methods: Rats received a single dose of cisplatin on Day 1, and a subset of rats was intraperitoneally injected with EGb daily between Days 22–40. In vitro, HK-2 cells were treated with cisplatin, and a subset of cells was cultivated with EGb or SIS3 (Smad3 inhibitor) for 48 h. Renal function of rats was assessed by detecting the levels of serum creatinine (Scr), blood urea nitrogen (BUN) and urinary N-acetyl-β-D-glucosaminidase (NAG). Hematoxylin and eosin staining and Masson's trichrome staining were used to evaluate the damage and fibrosis of renal tissue. Western blotting, immunohistochemistry and immunofluorescence were used to detect the protein levels of fibrosis-associated proteins and signaling pathway-related proteins. RT–qPCR analysis was used to examine the mRNA levels of related indicators. Results: EGb significantly decreased the increased levels of Scr, BUN and urinary NAG and attenuated renal damage and the relative area of renal interstitial fibrosis induced by cisplatin. Additionally, EGb decreased the protein levels of α-SMA, Col I, TGF-β1, smad2/3, phosphorylated (p)-smad2/3, p38 MAPK, and p-p38 MAPK; the ratio of p-p38 MAPK/p38 MAPK; and the mRNA level of p38 MAPK in renal tissues induced by cisplatin. In agreement with in vivo studies, EGb significantly reduced the increased protein levels of these indicators. Additionally, EGb significantly reduced the increased protein levels of vimentin, TIMP-1, and CTGF, as well as the mRNA levels of α-SMA, vimentin, and TGF-β1, while it significantly increased the reduced E-cadherin protein level and the MMP-1/TIMP-1 ratio in HK-2 cells induced by cisplatin. It's worth noting that the effects of SIS3 in changing the above indicators were similar to those of EGb. Conclusion: Our study demonstrated that EGb improved cisplatin-induced chronic renal interstitial fibrosis, and its mechanisms were associated with inhibiting the epithelial-mesenchymal transition of renal tubular epithelial cells via the Smad3/TGF-β1 and Smad3/p38 MAPK pathways. [ABSTRACT FROM AUTHOR]

The role of PI3K and Hippo signaling in chronic pancreatitis (CP) pathogenesis is unclear. Therefore, we assessed the involvement of these pathways in CP by examining the PI3K and Hippo signaling components PTEN and SAV1, respectively. We observed significant decreases in pancreatic PTEN and SAV1 levels in 2 murine CP models: repeated cerulein injection and pancreatic ductal ligation. Additionally, pancreas-specific deletion of Pten and Sav1 (DKO) induced CP in mice. Pancreatic connective tissue growth factor (CTGF) was markedly upregulated in both CP models and DKO mice, and pancreatic CCAAT/enhancer-binding protein-α (CEBPA) expression was downregulated in the CP models. Interestingly, in pancreatic acinar cells (PACs), CEBPA knockdown reduced PTEN and SAV1 and increased CTGF levels in vitro. Furthermore, CEBPA knockdown in PACs induced acinar-to-ductal metaplasia and activation of cocultured macrophages and pancreatic stellate cells. These results were mitigated by CTGF inhibition. CP in DKO mice was also ameliorated by Ctgf gene deletion, and cerulein-induced CP was alleviated by antibody-mediated CTGF neutralization. Finally, we observed significantly decreased PTEN, SAV1, and CEBPA and increased CTGF levels in human CP tissues compared with nonpancreatitis tissues. Taken together, our results indicate that dysregulation of PI3K and Hippo signaling induces CP via CTGF upregulation. [ABSTRACT FROM AUTHOR]

As a well-known traditional Chinese medicine, Amomi fructus (A. fructus) (Sharen) has been used therapeutically to treat gastrointestinal illnesses, including gastric ulcer (GU). The mechanism underlying this impact is still not fully known, though. To investigate the hidden mechanism by which A. fructus influences the pathogenesis of GU, we employed network pharmacology approaches and in vivo validated studies. Multiple public databases were used to compile information on bioactive compounds, potential targets of A. fructus , and associated genes of GU. Then, the STRING database's protein-protein interaction (PPI) data of the drug-disease overlapping gene targets was obtained, and the core targets for A. fructus against GU were discovered. Additionally, molecular docking was done to examine the binding capabilities of the active substances and core targets. Then, the pathways of A. fructus that target GU were examined using the Annotation, Visualization and Integrated Discovery (DAVID)'s Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway studies. In a mouse model of acute stomach mucosal damage brought on by absolute ethanol, the findings of network pharmacology were finally validated. In total, 610 targets derived from the 196 bioactive compounds in A. fructus , were discovered, and along with 115 A. fructus target genes for therapy of GU. Then, ten core targets associated with apoptosis and inflammation were determined based on node degree, and ALB, AKT1, TNF, EGFR, MAPK3, CASP3, MMP9, STAT3, SRC, and HRAS were identified as promising therapeutic targets of A. fructus against GU. The results of molecular docking also revealed that 65 active compounds had strong binding activity with the core targets, with volatile chemicals being the most significant active ingredients. So, for following in vivo tests, A. fructus volatile oils (AVO) were used. The KEGG analysis showed that the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) signaling pathway may be crucial for the therapeutic mechanism of GU. In experiments that were validated in vivo, AVO considerably decreased the ulcer area and enhanced the histological appearance of the gastric tissues. In addition, compared to the model group, up-regulated the expression of IGF-1, p-PI3K, and p-AKT and down-regulated the protein levels of TNF-α and Caspase 3 in the stomach tissues. According to preliminary findings from this work, A. fructus may influence inflammatory response and apoptosis via regulating the PI3K/AKT signaling pathway and associated gene targets. Importantly, our research might offer a theoretical foundation for future research into the intricate anti-GU mechanism of A. fructus. [Display omitted] [ABSTRACT FROM AUTHOR]

Optimized New Shengmai Powder (ONSMP) is a traditional Chinese medicine (TCM) formula for heart failure treatment. MAPK signaling pathway is the key driver of myocardial fibrosis in heart failure. However, the mechanism of ONSMP on myocardial fibrosis and MAPK signaling pathway remains unclear. To evaluate the effect of ONSMP against myocardial fibrosis in heart failure and the underlying mechanisms. Firstly, UHPLC-Q-Exactive-MS/MS was used to identify the active components in ONSMP. Secondly, a rat model of heart failure was established by ligating the left anterior descending branch of the coronary artery. After four weeks of intragastric administration of ONSMP, we used various classic tests, including echocardiography, exhaustive swimming, cardiopulmonary coefficient, heart failure markers, and cardiac pathological section, to assess the prescription's anti-myocardial fibrosis in heart failure properties. AGEs, Ang Ⅱ, VEGF, CTGF, and TGFβ levels in rat serum were quantified using ELISA. The positive expression of p-ERK1/2 and p-JNK1/2 of rat myocardium was determined immunohistochemical. The protein and mRNA levels of genes involved in the MAPK signaling pathway and myocardial fibrosis were measured using western blotting or real-time PCR. The main components of ONSMP that regulate the MAPK signaling pathway are isorhamnetin, kaempferol, quercetin, and tanshinone ⅡA. ONSMP ameliorated cardiac function and exercise tolerance and reduced cardiopulmonary coefficient, heart failure marker levels, and myocardial fibrosis in the heart failure rats. In addition, ONSMP diminished the serum MAPK pathway activator levels, positive expression level of p-ERK1/2 and p-JNK1/2, protein and mRNA levels of components of the MAPK signaling pathway in the myocardial tissue of heart failure rat, indicating that it inhibits MAPK signaling pathway. ONSMP delayed heart failure by inhibiting myocardial fibrosis via the MAPK signaling pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

Jiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. This study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. 30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients' decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. The decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). Abnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA. [Display omitted] • Established that impaired decidualization contributes to the occurrence of recurrent spontaneous abortion. • Verified the involvement of the SGK1/ENaC pathway in the occurrence of recurrent spontaneous abortion. • Confirmed that the protective effect of Jiawei Shoutai pills may be related to the regulation of SGK1/ENaC pathway. [ABSTRACT FROM AUTHOR]

Background: The putative benefit of rhBMP-2 is in the setting of limb reconstruction using structural allografts, whether it be allograft-prosthetic composites, osteoarticular allografts, or intercalary segmental grafts. There are also potential advantages in augmenting osseointegration of uncemented endoprosthetics and in reducing infection. Recombinant human BMP-2 might mitigate nonunion in structural allograft augmented osteosarcoma limb salvage surgery; however, its use is limited because of concerns about the prooncogenic effects of the agent.Questions/purposes: (1) To assess if BMP-2 signaling influences osteosarcoma cell line growth. (2) To characterize degree of osteosarcoma cell line osteoblastic differentiation in response to BMP-2. (3) To assess if BMP-2 signaling has a consistent effect on local or systemic tumor burden in various orthotopic murine models of osteosarcoma.Methods: In this study, 143b, SaOS-2 and DLM8-M1 osteosarcoma cell lines were transfected with BMP-2 cDNA controlled by a constitutive promoter (experimental) or an empty vector (control) using a PiggyBac transposon system. Cellular proliferation was assessed using a quantitative MTT colorimetric assay. Osteoblastic differentiation was compared between control and experimental cell lines using quantitative real-time polymerase chain reaction of the osteoblastic markers connective tissue growth factor, Runx-2, Osterix, alkaline phosphatase and osteocalcin. Experimental and control cell lines were injected into the proximal tibia of either NOD-SCID (143b and SaOS-2 xenograft model), or C3H (DLM8-M1 syngeneic model) mice. Local tumor burden was quantitatively assessed using tumor volume caliper measurements and bioluminescence, and qualitatively assessed using post-mortem ex vivo microCT. Lung metastasis was qualitatively assessed by the presence of bioluminescence, and incidence was confirmed using histology. rhBMP-2 soaked absorbable collagen sponges (experimental) and sterile-H2O soaked absorbable collagen sponges (control) were implanted adjacent to 143b proximal tibial cell line injections to compare the effects of exogenous BMP-2 application with endogenous upregulation.Results: Constitutive expression of BMP-2 increased the in vitro proliferation of 143b cells (absorbance values 1.2 ± 0.1 versus 0.89 ± 0.1, mean difference 0.36 [95% CI 0.12 to 0.6]; p = 0.01), but had no effect on SaOS-2 and DLM8-M1 cell proliferation. In response to constitutive BMP-2 expression, 143b cells had no differences in osteoblastic differentiation, while DLM8-M1 cells downregulated the early marker connective tissue growth factor (mean ΔCt 0.2 ± 0.1 versus 0.6 ± 0.1; p = 0.002) and upregulated the early-mid range marker Runx-2 (mean ΔCt -0.8 ± 0.1 versus -1.1 ± 0.1; p = 0.002), and SaOS-2 cells upregulated the mid-range marker Osterix (mean ΔCt -2.1 ± 0.6 versus -3.9 ± 0.6; p = 0.002). Constitutive expression of BMP-2 resulted in greater 143b and DLM8-M1 local tumor volume (143b: 307.2 ± 106.8 mm versus 1316 ± 387.4 mm, mean difference 1009 mm [95% CI 674.5 to 1343]; p < 0.001, DLM8-M1 week four: 0 mm versus 326.1 ± 72.8 mm, mean difference 326.1 mm [95% CI 121.2 to 531]; p = 0.009), but modestly reduced local tumor growth in SaOS-2 (9.5 x 10 ± 8.3x10 photons/s versus 9.3 x 10 ± 1.5 x 10 photons/s, mean difference 8.6 x 10 photons/s [95% CI 5.1 x 10 to 1.2 x 10]; p < 0.001). Application of exogenous rhBMP-2 also increased 143b local tumor volume (495 ± 91.9 mm versus 1335 ± 102.7 mm, mean difference 840.3 mm [95% CI 671.7 to 1009]; p < 0.001). Incidence of lung metastases was not different between experimental or control groups for all experimental conditions.Conclusions: As demonstrated by others, ectopic BMP-2 signaling has unpredictable effects on local tumor proliferation in murine models of osteosarcoma and does not consistently result in osteosarcoma cell line differentiation. Further investigations into other methods of safe bone and soft tissue healing augmentation and the use of differentiation therapies is warranted.Clinical Relevance: Our results indicate that BMP-2 has the potential to stimulate the growth of osteosarcoma cells that are poorly responsive to BMP-2 mediated osteoblastic differentiation. As this differentiation potential is unpredictable in the clinical setting, BMP-2 may promote the growth of microscopic residual tumor burden after resection. Our study provides further support for the recommendation to avoid the use of BMP-2 after limb-salvage surgery in patients with osteosarcoma. [ABSTRACT FROM AUTHOR]

Background and Objective: During cyclosporine‐induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan‐binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin‐8 (Gal‐8) in the response of gingival connective tissue cells to cyclosporine. Methods: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal‐8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real‐time polymerase chain reaction, ELISA and immunofluorescence, pull‐down with Gal‐8‐Sepharose for Gal‐8‐to‐cell surface glycoprotein interactions, short hairpin RNA for Gal‐8 silencing and Student's t test and ANOVA for statistical analysis. Results: Galectin‐8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF‐β1‐stimulated human gingival fibroblasts. Gal‐8 interacted with α5β1‐integrin and type II TGF‐β receptor. Gal‐8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF‐α) increased Gal‐8 protein levels. Finally, silencing of galectin‐8 in NIH3T3 cells abolished cyclosporine‐induced fibronectin protein levels. Conclusion: Taken together, these results reveal for the first time Gal‐8 as a fibrogenic stimulus exerted through β1‐integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal‐8 in human gingival tissues and its role in drug‐induced gingival overgrowth. [ABSTRACT FROM AUTHOR]

The purpose of this study was to evaluate the effects of diosgenin on the D-galactose-induced cerebral cortical widely dispersed apoptosis. Male 12-week-old Wistar rats were divided into four groups: Control (1 mg/kg/day of saline, i.p.), DD0 (150 mg/kg/day of D-galactose, i.p.), DD10, and DD50 (D-galactose + 1 0 or 50 mg/kg/day of diosgenin orally). After eight weeks, histopathological analysis, positive TUNEL and Western blotting assays were performed on the excised cerebral cortex from all four groups. The TUNEL-positive apoptotic cells, the components of Fas pathway (Fas, FADD, active caspase-8 and active caspase-3), and mitochondria pathway (t-Bid, Bax, cytochrome c , active caspase-9 and active caspase-3) were increased in the DD0 group compared with the control group, whereas they were decreased in the DD50 group. The components of survival pathway (p-Bad, Bcl-2, Bcl-xL, IGF-1, p-PI3K and p-AKT) were increased in the DD50 group compared to the control group, whereas the levels of Bcl-xL, p-PI3K, and p-AKT were also compensatorily increased in the DD0 group compared to the control group. Taken together, diosgenin suppressed D-galactose-induced neuronal Fas-dependent and mitochondria-dependent apoptotic pathways and enhanced the Bcl-2 family associated pro-survival and IGF-1-PI3K-AKT survival pathways, which might provide neuroprotective effects of diosgenin for prevention of the D-galactose-induced aging brain. [ABSTRACT FROM AUTHOR]

Qishen granules (QSG) are a famous formula with cardioprotective properties to heart failure (HF). The aim of this study was to investigate the underlying mechanism of QSG on apoptosis and fibrosis in the treatment of HF. HF model was induced by left anterior descending artery ligation on Sprague-Dawley rats. Transcriptome analysis was used to investigate the regulatory pathways of QSG on HF. Interestingly, downregulated genes of QSG were significantly enriched in Hippo pathway which plays a crucial role in regulating cell apoptosis and proliferation. We found that QSG inhibited the expressions of proapoptotic key proteins P-53 and fibrosis-related proteins TGF-β1, SMAD3, and CTGF. Further, we conducted research on the key proteins in the Hippo pathway upstream of CTGF and P-53. The results showed that MST1, P-MST1, P-LATS1, and RASSF1A that exert proapoptotic function were downregulated after QSG intervention. Similarly, P-YAP and P-TAZ, mediating self-degradation and apoptosis, were both observably decreased after QSG administration. Taken together, QSG are shown to be likely to exert cardioprotective effects by inhibiting the progression of apoptosis and fibrosis through Hippo pathway. [ABSTRACT FROM AUTHOR]

Vitamin D receptor expression and associated function have been reported in various muscle models, including C2C12, L6 cell lines and primary human skeletal muscle cells. It is believed that 1,25-hydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, has a direct regulatory role in skeletal muscle function, where it participates in myogenesis, cell proliferation, differentiation, regulation of protein synthesis and mitochondrial metabolism through activation of various cellular signalling cascades, including the mitogen-activated protein kinase pathway(s). It has also been suggested that 1,25(OH)2D3 and its associated receptor have genomic targets, resulting in regulation of gene expression, as well as non-genomic functions that can alter cellular behaviour through binding and modification of targets not directly associated with transcriptional regulation. The molecular mechanisms of vitamin D signalling, however, have not been fully clarified. Vitamin D inadequacy or deficiency is associated with muscle fibre atrophy, increased risk of chronic musculoskeletal pain, sarcopenia and associated falls, and may also decrease RMR. The main purpose of the present review is to describe the molecular role of vitamin D in skeletal muscle tissue function and metabolism, specifically in relation to proliferation, differentiation and protein synthesis processes. In addition, the present review also includes discussion of possible genomic and non-genomic pathways of vitamin D action. [ABSTRACT FROM AUTHOR]

Objective: To assess the preclinical efficacy and mechanism of action of an anti‐CX3CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). Methods: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti‐CX3CL1 mAb on fibroblasts. In addition, bleomycin‐induced and growth factor–induced models of SSc were used to investigate the effect of anti‐CX3CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. Results: Anti‐CX3CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor β1 (TGFβ1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3CL1 levels (P < 0.05) and augmented lesional CX3CL1 expression. Simultaneous administration of anti‐CX3CL1 mAb or CX3CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFβ1 in the skin, which was inhibited by anti‐CX3CL1 mAb. Further, the dermal infiltration of CX3CR1+ cells, macrophages (inflammatory and alternatively activated [M2‐like] subsets), and CD3+ cells significantly decreased following anti‐CX3CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti‐mCX3CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFβ and connective tissue growth factor (P < 0.01). Conclusion: Anti‐CX3CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation‐driven fibrotic skin disorders such as SSc. [ABSTRACT FROM AUTHOR]

Corilagin is a polyphenol that can be extracted from many medicinal plants and shows multiple pharmacological effects. We aimed to investigate the role of corilagin on miR-21-regulated hepatic fibrosis, especially miR-21-regulated TGF- β 1/Smad signaling pathway, in hepatic stellate LX2 cell line and Sprague–Dawley rats. The mRNA or protein levels of miR-21, Smad7, connective tissue growth factor (CTGF), α -smooth muscle actin (α -SMA), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), collagen type I alpha 1 (COL1A1), Smad2, Smad3, Smad2/3, p-Smad2, p-Smad3, p-Smad2/3, and transforming growth factor- β 1 (TGF- β 1) in LX2 cells and liver tissues were determined. Furthermore, gain-of and loss-of function of miR-21 in miR-21-regulated TGF- β 1/Smad signaling pathway were analyzed in LX2 cells. Liver tissues and serum were collected for pathological analysis, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Corilagin treatment reduced mRNA or protein levels of miR-21, CTGF, α -SMA, TIMP-1, TGF- β 1, COL1A1, p-Smad2, p-Smad3, and p-Smad2/3 both in vitro and in vivo. While corilagin increased mRNA and protein levels of Smad7 and MMP-9. After gain-of and loss-of function of miR-21, the downstream effectors of miR-21-regulated TGF- β 1/Smad signaling pathway in LX2 cells changed accordingly, and the changes were inhibited by corilagin. Simultaneously, administration of corilagin not only ameliorated pathological manifestation of liver fibrosis but also reduced levels of α -SMA and COL1A1 in liver tissues and TGF- β 1, ALT levels in serum. Corilagin is able to potentially prevent liver fibrosis by blocking the miR-21-regulated TGF- β 1/Smad signaling pathway in LX2 cells and CCl4-induced liver fibrosis rats, which may provide a novel therapeutic strategy for liver fibrosis. [ABSTRACT FROM AUTHOR]

Objectives: Fibrosis diseases are one of the leading causes of suffering and death. However, no systematic investigation has been carried out on fibrosis‐related genes. Materials and Methods: By querying PubMed using keywords "fibrosis" and "gene" or "protein," we identified fibrosis‐related genes in the last decade. Bioinformatics analysis was performed by MAS 3.0 software. Key miRNA was selected to assess its relationship with oral submucous fibrosis (OSF) and fibroblast functions. Results: A total of 1,310 genes related to fibrosis were identified. TGF‐β1, CTGF, MMP9, HSP47, and S1P were found to be associated with mainly fibrotic organs. In total, 244 cellular components terms, 595 molecular function terms, 1,816 cellular component terms, and 136 KEGG pathway annotations were predicted. miR‐199‐5p was selected as the key miRNA, which has higher level in OSF. Upregulated miR‐199‐5p was significantly related to OSF duration and OSF histological grade (p = 0.028 and 0.012, respectively). Overexpressive miR‐199‐5p reduced proliferation and induced apoptosis in buccal fibroblasts. Additionally, expression levels of collagen I (COL I) and III (COL III) were promoted by overexpressive miR‐199‐5p in buccal fibroblasts. Conclusion: These results indicate that fibrosis‐related genes are related to a series of complex mechanisms. The characteristics of miR‐199‐5p may supply important clues for developing therapeutic strategy for OSF. [ABSTRACT FROM AUTHOR]

Objective: Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule–associated protein with epidermal growth factor– and factor VIII–like domains (MFG‐E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG‐E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG‐E8 in skin fibrosis in SSc. Methods: We assessed expression of MFG‐E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG‐E8 (rMFG‐E8) on latent transforming growth factor β (TGFβ)–induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG‐E8 on fibrosis mouse models. Results: We demonstrated that MFG‐E8 expression around dermal blood vessels and the serum MFG‐E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG‐E8 significantly inhibited latent TGFβ–induced expression of type I collagen, α‐smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3–8), which suggested that MFG‐E8 inhibited activation of latent TGFβ as well as TGFβ signaling via binding to αv integrin. In a mouse model of bleomycin‐induced fibrosis (n = 5–8) and in a TSK mouse model (a genetic model of SSc) (n = 5–10), deficient expression of MFG‐E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG‐E8 significantly inhibited bleomycin‐induced dermal fibrosis. Conclusion: These results suggest that vasculopathy‐induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG‐E8, may be associated with the decreased expression of MFG‐E8 in SSc and that the deficient inhibitory regulation of latent TGFβ–induced skin fibrosis by MFG‐E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients. [ABSTRACT FROM AUTHOR]

Thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves' disease, is a process in which orbital connective tissues and extraocular muscles undergo inflammation and remodeling. The condition seems to result from autoimmune responses to antigens shared by the thyroid and orbit. The thyrotropin receptor (TSHR), expressed at low levels in orbital tissues, is a leading candidate antigen. Recent evidence suggests that another protein, the insulin-like growth factor-I receptor (IGF-IR), is overexpressed in TAO, and antibodies against IGF-IR have been detected in patients with the disease. Furthermore, TSHR and IGF-IR form a physical and functional complex, and signaling initiated at TSHR requires IGF-IR activity. Identification of therapy for this rare disease has proven challenging and currently relies on nonspecific and inadequate agents, thus representing an important unmet need. A recently completed therapeutic trial suggests that inhibiting IGF-IR activity with a monoclonal antibody may be an effective and safe treatment for active TAO. [ABSTRACT FROM AUTHOR]

Background/purpose: Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts.Methods: The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting.Results: CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2.Conclusion: Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. [ABSTRACT FROM AUTHOR]

Objectives To investigate the mechanism of 2-methoxyestradiol (2-ME) in inhibiting hypoxia-induced collagen synthesis of fibroblasts in SSc. Methods The expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and connective tissue growth factor (CTGF) in skin specimens derived from SSc patients and healthy volunteers were examined by immunohistochemistry. HIF-1α was knocked down by lentiviral transduction, and SSc dermal fibroblasts cultured under normoxic (21% O2) or hypoxic (1% O2) condition were treated with PI3K inhibitor LY294002, rapamycin or 2-ME (25 μM). The protein levels of HIF-1α, CTGF, collagen I, p-Akt and p-mTOR were examined by western blotting or immunofluorescence. Apoptosis and cell cycle of fibroblasts were assessed by flow cytometry and by measuring caspase 3 activity, and cell proliferation was evaluated by Cell Counting Kit-8. Results The expressions of HIF-1α and CTGF were increased in skins of SSc patients compared with healthy controls. Hypoxia up-regulated the protein levels of HIF-1α, CTGF and collagen I in SSc fibroblasts. In contrast, 2-ME inhibited PI3K/Akt/mTOR pathway and down-regulated protein levels of HIF-1α, CTGF and collagen I. Knockdown of HIF-1α reduced expressions of CTGF and collagen I, which were further down-regulated by 2-ME intervention. Moreover, 2-ME promoted the apoptosis and inhibited the proliferation of SSc fibroblasts by arresting the cell cycle at the G2/M phase. Conclusion 2-ME reduced the production of CTGF and collagen I in SSc fibroblasts induced by hypoxia through PI3K/Akt/mTOR/HIF-1α signalling and inhibited the proliferation of fibroblasts. These findings suggested that 2-ME could be employed as a promising antifibrotic therapy for SSc. [ABSTRACT FROM AUTHOR]

We hypothesised that a feedback mechanism of the transforming growth factor (TGF)‐β1 signalling pathway, triggered by high‐level TGF‐β1, activates platelet‐rich plasma (PRP) release to reduce connective tissue growth factor (CTGF) production and expression of CTGF mRNA in hypertrophic scar dermal fibroblasts. Primary dermal fibroblasts were isolated from cultures of hypertrophic scars. Cells were cultured after addition of serum‐free Dulbecco's modified Eagle's medium supplemented with 5% (wt/vol) PRP or platelet‐poor plasma (PPP). At 1, 4, 6, 8, 11, and 13 days after addition of PRP or PPP, the TGF‐β1 and CTGF levels in supernatants were determined using solid‐phase enzyme‐linked immunosorbent assays. Quantitative reverse transcription polymerase chain reactions were performed to quantify TGF‐β1 and CTGF mRNA expression levels. TGF‐β1 mRNA expression in the PRP groups was lower than in the PPP groups from 4 to 13 days of culture, and there was statistically significant difference (P < .01). CTGF level and mRNA expression in the PRP groups was lower than in the PPP groups, and there were statistically significant differences (P < .01). Although further experiments will focus on clarifying the second messenger of the TGF‐β1 negative feedback mechanism, the in vitro data presented show that PRP can potentially reduce CTGF and CTGF gene transcription by triggering the TGF‐β1 signalling negative feedback mechanism. [ABSTRACT FROM AUTHOR]

Background: Icariin, a major active flavonoid glucoside, is widely used for the treatment of bone injury and rebuilding in the clinic because of its roles in suppressing osteoblastogenesis and promoting osteogenesis. The senescence-accelerated mouse SAMP6 was accepted as a useful murine model to reveal the mechanism of senile osteoporosis and the therapeutic mechanism of drug activity. However, little is known about the characteristics of SAMP6 osteoblasts and the associated regulatory roles of icariin. Methods: We isolated and cultured osteoblasts from SAMP6 or SAMR1 mice and compared their proliferation, migration, and differentiation by performing the CCK-8 assay, cell counting assay, EdU staining, cell cycle analysis, ALP staining and activity measurement, Alizarin red staining, and RT-qPCR analysis to measure the levels of osteoblast markers, including RUNX2, Colla1 and Oc. To assess the effects of icariin on BMP-2-induced osteoblast differentiation, after BMP-2 treatment, osteoblast markers were analyzed by RT-qPCR and semi-quantitative Western blotting. The effects of icariin on connective tissue growth factor (CTGF) were measured by RT-qPCR. shRNA targeting CTGF mRNA was employed to knockdown its expression level in osteoblasts. Results: The SAMP6 osteoblasts presented decreased the development and differentiation activity compared with SAMR1 osteoblasts, indicating that they are the potential mechanisms underlying age-associated disease. Moreover, SAMP6 osteoblasts presented upregulated CTGF compared with SAMR1 osteoblasts. Icariin enhanced BMP-2-induced osteoblast differentiation by downregulating CTGF expression, which tightly regulates osteoblast differentiation. By downregulating CTGF, icariin treatment upregulated phosphate-Smad1/5/8, indicating its activating effects on the BMP signaling pathway. Conclusion: These results suggest that decreased osteoblast development and function potentially contributes to age-associated disease. Icariin exerts enhancing effects on BMP-2-mediated osteoblast development via downregulating CTGF. [ABSTRACT FROM AUTHOR]

Background/purpose: Oral submucous fibrosis (OSF) is a premalignant condition caused by the chewing of areca nut (AN). Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of OSF. Connective tissue growth factor (CTGF or CCN2) and early growth response-1 (Egr-1) are important mediators in the fibrotic response to TGFβ in several fibrotic disorders including OSF. Arecoline, a major AN alkaloid, induced the synthesis of CCN2 and Egr-1 in human buccal mucosal fibroblast (BMFs). The aims of this study were to investigate whether arecoline-induced CCN2 and Egr-1 syntheses are mediated through TGFβ1 signaling and to inspect the detailed mechanisms involved.Methods: Western blot and TGFβ1 Emax® ImmunoAssay were used to measure the effect of arecoline on the TGFβ signaling pathways. 2',7'-dichlorodihydrofluorescein diacetate and MitoSOX™ Red were used to measure the effect of arecoline on the cellular and mitochondrial reactive oxygen species (ROS).Results: Arecoline induced latent TGFβ1 activation, Smad2 phosphorylation, and mitochondrial and total cellular ROS in BMFs. TGFβ-neutralizing antibody completely inhibited the arecoline-induced synthesis of CCN2 and Egr-1. Mito-TEMPO, a mitochondria-targeted antioxidant, completely suppressed arecoline-induced latent TGFβ1 activation and mitochondrial and total cellular ROS. Epigallocatechin-3-gallate (EGCG) dose-dependently inhibited arecoline-induced TGFβ1 activation and mitochondrial ROS in BMFs.Conclusion: Our results indicated that arecoline-induced mitochondrial ROS plays pivotal roles in the activation of latent TGFβ1 leading to the initiation of TGFβ1 signaling and subsequent increase in the synthesis of CCN2 and Egr-1. EGCG can be a useful agent in the chemoprevention and treatment of OSF. [ABSTRACT FROM AUTHOR]

We aimed to study the cardiac expression of bone morphogenetic protein 2, its receptor 1 b, and connective tissue growth factor, factors implicated in cardiac embryogenesis, following ischemia/hypoxia, heart failure, and in remodeling hearts from humans and mice. Biopsies from the left ventricle of patients with end-stage heart failure due to dilated cardiomyopathy or coronary artery disease were compared with donor hearts and biopsies from patients with normal heart function undergoing coronary artery bypass grafting. Mouse model of post-infarction remodeling was made by permanent ligation of the left coronary artery. Hearts were analyzed by real-time polymerase chain reaction and Western blotting after 24 hours and after 2 and 4 weeks. Patients with dilated cardiomyopathy and mice post-infarction had increased cardiac expression of connective tissue growth factor. Bone morphogenetic protein 2 was increased in human hearts failing due to coronary artery disease and in mice post-infarction. Gene expression of bone morphogenetic protein receptor 1 beta was reduced in hearts of patients with failure, but increased two weeks following permanent ligation of the left coronary artery in mice. In conclusion, connective tissue growth factor is upregulated in hearts of humans with dilated cardiomyopathy, bone morphogenetic protein 2 is upregulated in remodeling due to myocardial infarction while its receptor 1 b in human failing hearts is downregulated. A potential explanation might be an attempt to engage regenerative processes, which should be addressed by further, mechanistic studies. [ABSTRACT FROM PUBLISHER]

Osteosarcoma is the most common primary solid tumor of bone. It has a high metastatic potential and occurs predominantly in adolescents and young adults. Angiopoietin 2 (Angpt2) is a key regulator in tumor angiogenesis, facilitating tumor growth and metastasis. Connective tissue growth factor (CTGF, also known as CCN2), is a cysteine-rich protein that has been reported to promote metastasis of osteosarcoma. However, the effect of CTGF on Angpt2 regulation and angiogenesis in human osteosarcoma remains largely unknown. We found that overexpression of CTGF in osteosarcoma cells increased Angpt2 production and induced angiogenesis, in vitro and in vivo. Our findings demonstrate that CTGF-enhanced Angpt2 expression and angiogenesis is mediated by the phospholipase C (PLC)/protein kinase C (PKCδ) signaling pathway. Moreover, endogenous microRNA-543 (miR-543) expression was negatively regulated by CTGF via the PLC/PKCδ pathway. We also provide evidence showing clinical significance between CTGF, Angpt2, and miR-543 as well as tumor staging in human osteosarcoma tissue. CTGF may serve as a therapeutic target in the process of osteosarcoma metastasis and angiogenesis. [ABSTRACT FROM AUTHOR]

Introduction: Recent research has unravelled an increasing list of cardiac conditions and risk factors that may be responsible for the abnormal underlying atrial substrate that predisposes to atrial fibrillation (AF). Atrial fibrosis has been demonstrated as the pivotal structural abnormality underpinning conduction disturbances that promote AF in different disease models. Despite the advancement in our discoveries of the molecular mechanisms involved in the profibrotic milieu, targeted therapeutics against atrial fibrosis remain lacking. Areas covered: This review is focused on detailing the key molecular signalling pathways that contribute to atrial fibrosis including: angiotensin II, transforming growth factor (TGF- ß1), connective tissue growth factor (CTGF) and endothelin-1. We also discussed the potential therapeutic options that may be useful in modulating the abnormal atrial substrate. In addition, we examined the new paradigm of AF care in lifestyle and risk factor management that has been shown to arrest and reverse the atrial remodelling process leading to improved AF outcomes. Expert commentary: The future of AF care is likely to require an integrated approach consisting of aggressive risk factor management in addition to the established paradigm of rate and rhythm management and anticoagulation. Translational studies on molecular therapeutics to combat atrial fibrosis is urgently needed. [ABSTRACT FROM AUTHOR]