Hydronidone ameliorates liver fibrosis by inhibiting activation of hepatic stellate cells via Smad7-mediated degradation of TGFßRI.

BACKGROUND AND PURPOSE: Liver fibrosis is a wound-healing reaction that eventually leads to cirrhosis. Hydronidone is a new pyridine derivative with the potential to treat liver fibrosis. In this study, we explored the antifibrotic effects of hydronidone and its potential mode of action. METHODS: The anti-hepatic fibrosis effects of hydronidone were studied in carbon tetrachloride (CCl4 )- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)- induced animal liver fibrosis. The antifibrotic mechanisms of hydronidone were investigated in hepatic stellate cells (HSCs). The antifibrotic effect of hydronidone was further tested after Smad7 knockdown in HSCs in mouse models of fibrosis. RESULTS: In animal models, hydronidone attenuated liver damage and collagen accumulation, and reduced the expression of fibrosis-related genes. Hydronidone decreased the expression of fibrotic genes in HSCs. Impressively, hydronidone significantly upregulated Smad7 expression and promoted the degradation of transforming growth factor ß receptor I (TGFßRI) in HSCs and thus inhibited the TGFß-Smad signalling pathway. Specific knockdown of Smad7 in HSCs in vivo blocked the antifibrotic effect of hydronidone. CONCLUSION: Hydronidone ameliorates liver fibrosis by inhibiting HSCs activation via Smad7-mediated TGFßRI degradation. Hydronidone is a potential drug candidate for the treatment of liver fibrosis.

Targeting of Smad7 in Mesenchymal Cells Does Not Exacerbate Fibrosis During Experimental Chronic Pancreatitis.

OBJECTIVES: Transforming growth factor-ß (TGF-ß)-mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-ß signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-ß signaling in this cell type in mice. METHODS: A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis. RESULTS: Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice was only slightly increased, without statistical significance. Except for fibronectin, the expression of different extracellular matrix proteins and the numbers of fibroblasts and inflammatory cells were similar between Smad7-mutant and control mice. CONCLUSIONS: There was no clear evidence that the lack of Smad7 in pancreatic stellate cells plays a major role in experimental pancreatitis, at least in the mouse model investigated here.

Dexamethasone Effect on the Expression of Transforming Growth Factor-ß1/Smads Signaling Pathway in Benign Biliary Stricture Fibroblasts in Rodent.

OBJECTIVE: To investigate the effects of dexamethasone on transforming growth factor (TGF)-ß1/Smads signaling pathway in benign biliary stricture (BBS) fibroblasts. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Guizhou Medical University, Guiyang, Guizhou, China, from January to August 2016. METHODOLOGY: Fibroblasts derived from rabbit BBS model were cultured and identified, then treated by different concentration of dexamethasone (0.02, 0.1 and 0.5 mg/ml). Dexamethasone-treated cells and non-treated control groups were incubated respectively for 48 hours. Cell proliferation was assessed using cell counting kit-8. Relative mRNA expression of TGF-ß1, Smad4 and Smad7 were assessed by quantitative RT-PCR. Protein expression of TGF-ß1 and Smad4 were investigated by Western blotting. RESULTS: Treatment with dexamethasone significantly inhibited the proliferation of BBS fibroblasts, significantly attenuated both the mRNA and protein expression of TGF-ß1 and Smad4, and significantly up-regulated the mRNA expression of Smad7 in BBS fibroblasts (all p<0.05, 0.1-0.5 mg/ml), and exhibited in a dose-dependent manner. CONCLUSION: TGF-ß1/Smads signaling pathway may play an important role in BBS progression; dexamethasone significantly altered the expression of TGF-ß1/Smads signaling pathway and significantly inhibited cell proliferation in rabbit BBS fibroblasts. Therefore, dexamethasone may be a therapeutic option for the prevention of BBS.

Huang Qi Decoction Prevents BDL-Induced Liver Fibrosis Through Inhibition of Notch Signaling Activation.

Notch signaling has been demonstrated to be involved in ductular reactions and fibrosis. Previous studies have shown that Huang Qi Decoction (HQD) can prevent the progression of cholestatic liver fibrosis (CLF). However, whether HQD affects the Notch signaling pathway is unclear. In this study, CLF was established by common bile duct ligation (BDL) in rats. At the end of the first week, the rats were randomly divided into a model group (i.e., BDL), an HQD group, and a sorafenib positive control group (SORA) and were treated for 3 weeks. Bile duct proliferation and liver fibrosis were determined by tissue staining. Activation of the Notch signaling pathway was evaluated by analyzing expressions of Notch-1, -2, -3, and -4, Jagged (JAG) 1, and Delta like (DLL)-1, -3, and -4. The results showed that HQD significantly reduced the deposition of collagen and the Hyp content of liver tissue and inhibited the activation of HSCs compared with the BDL group. In addition, HQD significantly decreased the protein and mRNA expressions of TGF-[Formula: see text]1 and [Formula: see text]-SMA. In contrast, HQD significantly enhanced expression of the Smad 7 protein. HQD also reduced biliary epithelial cell proliferation, and reduced the mRNA levels of CK7, CK8, CK18, SRY-related high mobility group-box gene (SOX) 9, epithelial cell adhesion molecule (EpCAM) and the positive areas of CK19 and OV6. In addition, the mRNA and protein expressions of Notch-3, -4, JAG1, and DLL-1, -3 were significantly reduced in the HQD compared to the BDL group. These results demonstrated that HQD may prevent biliary liver fibrosis through inhibition of the Notch signaling pathway, and it may be a potential treatment for cholestatic liver disease.

Synergistic effect of vitamin D and low concentration of transforming growth factor beta 1, a potential role in dermal wound healing.

Dermal wound healing, in which transforming growth factor beta 1 (TGFß1) plays an important role, is a complex process. Previous studies suggest that vitamin D has a potential regulatory role in TGFß1 induced activation in bone formation, and there is cross-talk between their signaling pathways, but research on their effects in other types of wound healing is limited. The authors therefore wanted to explore the role of vitamin D and its interaction with low concentration of TGFß1 in dermal fibroblast-mediated wound healing through an in vitro study. Human dermal fibroblasts were treated with vitamin D, TGFß1, both, or vehicle, and then the wound healing functions of dermal fibroblasts were measured. To further explore possible mechanisms explaining the synergistic effect of vitamin D and TGFß1, targeted gene silencing of the vitamin D receptor was performed. Compared to either factor alone, treatment of fibroblasts with both vitamin D and low concentration of TGFß1 increased gene expression of TGFß1, connective tissue growth factor, and fibronectin 1, and enhanced fibroblast migration, myofibroblast formation, and collagen production. Vitamin D receptor gene silencing blocked this synergistic effect of vitamin D and TGFß1 on both collagen production and myofibroblast differentiation. Thus a synergistic effect of vitamin D and low TGFß1 concentration was found in dermal fibroblast-mediated wound healing in vitro. This study suggests that supplementation of vitamin D may be an important step to improve wound healing and regeneration in patients with a vitamin D deficiency.

Effect of BMP7 on podocyte transdifferentiation and Smad7 expression induced by hyperglycemia.

OBJECTIVE: To investigate the effect of BMP7 on the transdifferentiation and Smad7 expression of podocytes induced by high glucose in vitro and to explore its possible protective mechanisms. METHODS: Mouse podocytes were cultured and divided into normal glucose group (NG), high glucose group (HG), mannitol group, NG+BMP7 group, and HG+BMP7 group. Real-time PCR and Western blot were applied respectively to detect the mRNA and protein expression levels of synaptopodin, desmin, and Smad7. RESULTS: The cells significantly up-regulated the mRNA and protein expression of desmin and reduced the expression of both synaptopodin and Smad7 after 48 hours (vs. NG, p < 0.01). BMP7 dramatically suppressed the mRNA and protein expression of desmin and protected the expression of synaptopodin and Smad7 after incubation with high glucose for 48 hours (vs. HG, p < 0.01). CONCLUSIONS: BMP7 can inhibit the epithelial-to-mesenchymal cell transformation (EMT) of podocytes induced by high glucose; Smad7 may mediate the blunting effects of BMP7 on high glucose in podocytes.

Shensong Yangxin Capsule prevents diabetic myocardial fibrosis by inhibiting TGF-ß1/Smad signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Shensong Yangxin Capsule (SSYX), a traditional Chinese herbal medicine, has long been used clinically to treat arrhythmias in China. However, the effect of SSYX on interstitial fibrosis in diabetic cardiomyopathy is unknown. The objective of this study was to investigate the effects of SSYX on myocardial fibrosis in diabetic rats. MATERIALS AND METHODS: The antifibrotic effect of SSYX was investigated in streptozocin (STZ)-induced diabetic rats with high fat-diet (HFD). Fasting blood glucose, heart weight/body weight (HW/BW) ratio, total cholesterol (TC), triglycerides (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were measured. Echocardiography and histology examination were carried out to evaluate heart function. Expressions of Smad7, TGF-ß1, collagen I (col-1), collagen III (col-3), MMP-2, MMP-9 and α-SMA mRNA in heart tissues were measured by real time polymerase chain reaction (PCR). TGF-ß1, Smad2/3, p-Smad2/3 and Smad7 protein levels were measured by western blot analysis. Proliferation of cardiac fibroblast was detected via immunofluorescence. RESULTS: SSYX markedly decreased HW/BW ratio and improved the impaired cardiac function of type-2 diabetes mellitus (T2DM) rats. Transmission electron microscopy (TEM), haematoxylin and eosin (HE) and Masson staining results showed that SSYX attenuated cardiac fibrosis and collagen deposition in T2DM rats. Moreover, mRNA levels of TGF-ß1, col-1, col-3, MMP-2, MMP-9 and α-SMA were downregulated, whereas Smad7 expression was upregulated after treatment with SSYX in rats with cardiac fibrosis. Furthermore, SSYX decreased protein levels of TGF-ß1 and p-Smad2/3, and increased Smad7 expression. CONCLUSION: TGF-ß1/Smad signaling is involved in the cardiac fibrosis in diabetic cardiomyopathy and SSYX inhibits fibrosis and improves cardiac function via suppressing this pathway. Therefore, SSYX might be considered as an alternative therapeutic remedy for diabetic cardiomyopathy.

Compound Astragalus and Salvia miltiorrhiza extract suppresses rabbits' hypertrophic scar by modulating the TGF-ß/Smad signal.

BACKGROUND: Hypertrophic scar is a fibro-proliferative disease. Our previous studies demonstrate that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits proliferation and invasion in keloid fibroblasts. OBJECTIVE: To investigate the effects of CASE on hypertrophic scar. METHODS: Rabbits were divided into the control, model and three dosage groups of CASE (0.94, 1.88, 3.76%). An animal model of hypertrophic scar was established and treated with CASE ointment or ointment base. The histopathological detection by hematoxylin & eosin and Masson's trichrome staining and protein expression of scars by Western blot were performed. RESULTS: The hydroxyproline content was decreased under CASE treatment. Transforming growth factor beta 1 (TGF-ß1) protein expression increased in the model group while it decreased under CASE treatment. The elevated expression of Smad4 protein was decreased under CASE treatment. Additionally, CASE promoted Smad7 protein expression. CONCLUSION: CASE could inhibit formation of hypertrophic scar by modulating TGF-ß/Smad signal and may be useful for the treatment of hyperplastic scars.

Oxymatrine inhibits collagen synthesis in keloid fibroblasts via inhibition of transforming growth factor-ß1/Smad signaling pathway.

BACKGROUND: Keloids are benign dermal tumors characterized by fibroblastic proliferation and excessive accumulation of collagen. Oxymatrine (OMT) is an alkaloid extracted from the Chinese herb Sophora japonica with capacities of anti-fibrosis. OBJECTIVE: To evaluate the effects of OMT on collagen production and to explore its mechanisms. METHODS: OMT was applied to human keloid fibroblasts in vitro. Collagen, transforming growth factor (TGF)-ß1, TGF-ß receptor, and Smads were analyzed by Western Blot, reverse transcription polymerase chain reaction, and immunofluorescence. RESULTS: We found that both collagen synthesis and Smad3 production were significantly suppressed in a dose-dependent administration of OMT. However, expression of TGF-ß1, TGF-ß receptor1, TGF-ß receptor2, Smad4, and Smad7 was unchanged. We also found that OMT reversed phosphorylation and nuclear translocation of Smad3 induced by TGF-ß1. CONCLUSIONS: OMT inhibited collagen synthesis, which might be associated with TGF-ß/Smad signaling pathway. These findings suggest that OMT may be a promising candidate to prevent keloid and other fibrotic diseases.

In vitro suppression of quercetin on hypertrophy and extracellular matrix accumulation in rat glomerular mesangial cells cultured by high glucose.

Quercetin's protective effects on the glomerulosclerosis of diabetic nephropathy (DN) in rat mesangial cells were investigated. The cell cycles, type IV collagen and laminin, TGF-ß(1) mRNA, Smad 2/3 and Smad 7, and activities of cell antioxidases were measured. Compared with the high glucose group, quercetin may decrease the cell percentages of G(0)/G(1) phase, Smad 2/3 expression, laminin and type IV collagen, and TGF-ß(1) mRNA level significantly. The antioxidant capacity, the cell percentages of S phase and Smad 7 expression was significantly increased by quercetin. These results suggest that quercetin is a protective agent against glomerulosclerosis in DN.

Interleukin-10 regulates transforming growth factor-beta signaling in cultured human bronchial epithelial cells.

BACKGROUND: The basic pathological features of bronchial asthma can be explained on the basis of chronic airway inflammation, involving inflammatory cells such as T cells (particularly type 2 helper T, Th2, cells) and mast cells, and airway remodeling. Many aspects of airway remodeling remain unclear at the molecular level. Recent attention has focused on the role of transforming growth factor (TGF)-beta, a fibrogenic cytokine, in airway remodeling. Currently available evidence suggests that airway remodeling is caused by an imbalance in regulatory mechanisms mediated by Smads, a family of signal-transducing molecules. OBJECTIVES: We studied the effects of the Th2 cytokines interleukin (IL)-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and the regulatory cytokine IL-10 on the expression of inhibitory Smad7 protein in bronchial epithelial cells. METHODS: Real-time reverse-transcriptase polymerase chain reaction was employed. RESULTS: Stimulation with IL-10 upregulated the expression of Smad7 compared with control. Neither IL-5 nor GM-CSF induced Smad7 expression. Smad7 expression was upregulated by IL-10 plus either IL-5 or GM-CSF. IL-10 inhibited the expression of TGF-beta-inducible early gene, which is known to downregulate Smad7 expression. CONCLUSIONS: Our results suggest that IL-10 acts as a regulatory cytokine in the inhibition of airway inflammation.

Effects of perindopril and valsartan on expression of transforming growth factor-beta-Smads in experimental hepatic fibrosis in rats.

BACKGROUND: Previous studies have shown that the renin-angiotensin system (RAS) plays an important role in the pathogenesis of hepatic fibrosis, and blockers of the RAS may be active as an antifibrogenic goal. However, the potential role of RAS inhibition on expression transforming growth factor (TGF)-beta-Smads in hepatic fibrosis remains unknown. The aim of this study was to investigate the effect and mechanism of an angiotensin-converting enzyme inhibitor (perindopril) and an angiotensin II receptor blocker (valsartan) on TGF-beta1 and TGF receptor II (TRII) mRNA, Smad3 and Smad7 in fibrotic hepatic livers in rats. METHODS: Sixty Wistar rats were randomly divided into four study groups (n = 15 for each group), including normal controls, hepatic fibrosis models, and two treated groups with either perindopril or valsartan, starting from the fourth week after being exposed to carbon tetrachloride (CCl(4)) for 4 weeks. The levels of TGF-beta and TRII mRNA in liver tissue were analyzed by RT-PCR. The expressions of TGF-beta1, Smad3 and Smad7 in liver tissues were evaluated by immunohistochemistry. The liver histopathology was examined by hematoxylin and eosin (HE) staining and by electron microscopy, respectively. The liver function and serum hyaluronic acid were also assayed by biochemistry and radioimmunoassay. RESULTS: Compared with the hepatic fibrosis models, the levels of TGF-beta1, TRII mRNA and the expression Smad3 significantly decreased in the two treated groups, and the expression of Smad7 was significantly increased in the liver of rats treated with perindopril or valsartan (P < 0.05 or P < 0.01). The histological changes and ultrastructure of fibrotic liver, liver function and hyaluronic acid also remarkably improved in the treated rats. CONCLUSIONS: The angiotensin-converting enzyme inhibitors perindopril and valsartan have a protective effect on liver injury and can ameliorate hepatic fibrosis in rats induced by CCl(4). The mechanisms may be associated with their effects of down-regulating TGF-beta1, TRII mRNA and smad3, and up-regulating Smad7.