Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-β/Smad signaling pathway

BACKGROUND Liver fibrosis is a refractory disease whose persistence can eventually induce cirrhosis or even liver cancer.Early liver fibrosis is reversible by intervention.As a member of the transforming growth factor-beta(TGF-β)superfamily,bone morphogenetic protein 7(BMP7)has anti-liver fibrosis functions.However,little is known about BMP7 expression changes and its potential regulatory mechanism as well as the relationship between BMP7 and TGF-βduring liver fibrosis.In addition,the mechanism underlying the anti-liver fibrosis function of BMP7 needs to be further explored.AIM To investigate changes in the dynamic expression of BMP7 during liver fibrosis,interactions between BMP7 and TGF-β1,and possible mechanisms underlying the anti-liver fibrosis function of BMP7.METHODS Changes in BMP7 expression during liver fibrosis and the interaction between BMP7 and TGF-β1 in mice were observed.Exogenous BMP7 was used to treat mouse primary hepatic stellate cells(HSCs)to observe its effect on activation,migration,and proliferation of HSCs and explore the possible mechanism underlying the anti-liver fibrosis function of BMP7.Mice with liver fibrosis received exogenous BMP7 intervention to observe improvement of liver fibrosis by using Masson’s trichrome staining and detecting the expression of the HSC activation indicator alpha-smooth muscle actin(α-SMA)and the collagen formation associated protein type I collagen(Col I).Changes in the dynamic expression of BMP7 during liver fibrosis in the human body were further observed.RESULTS In the process of liver fibrosis induced by carbon tetrachloride(CCl4)in mice,BMP7 protein expression first increased,followed by a decrease;there was a similar trend in the human body.This process was accompanied by a sustained increase in TGF-β1 protein expression.In vitro experiment results showed that TGF-β1 inhibited BMP7 expression in a time-and dose-dependent manner.In contrast,high doses of exogenous BMP7 inhibited TGF-β1-induced activation,migration,and proliferation of HSCs;this inhibitory effect was associated with upregulation of pSmad1/5/8 and downregulation of phosphorylation of Smad3 and p38 by BMP7.In vivo experiment results showed that exogenous BMP7 improved liver fibrosis in mice.CONCLUSION During liver fibrosis,BMP7 protein expression first increases and then decreases.This changing trend is associated with inhibition of BMP7 expression by sustained upregulation of TGF-β1 in a time-and dose-dependent manner.Exogenous BMP7 could selectively regulate TGF-β/Smad pathway-associated factors to inhibit activation,migration,and proliferation of HSCs and exert antiliver fibrosis functions.Exogenous BMP7 has the potential to be used as an antiliver fibrosis drug.     

Role of Tenascin-X in regulating TGF-β/Smad signaling pathway in pathogenesis of slow transit constipation

BACKGROUND Chronic constipation is a gastrointestinal functional disease that seriously harms physical and mental health and impacts the quality of life of patients.Its incidence rate is 2%-27%.Slow transit constipation(STC)is a common type of chronic functional constipation,accounting for 10.3%-45.5%of such cases.Scholars have performed many studies on the pathogenesis of STC.These studies have indicated that the occurrence of STC may be related to multiple factors,such as dysfunction of the enteric nervous system,interstitial cells of Cajal(ICC)damage,and changes in neurotransmitters regulating intestinal peristalsis.AIM To investigate the role of Tenascin-X(TNX)in regulating the TGF-β/Smad signaling pathway in the pathogenesis of STC.METHODS This study included an experimental group and a control group.The experimental group included 28 patients with severe colonic STC,and the control group included 18 patients with normal colon tissues.Immunohistochemistry(IHC)was used to detect c-Kit,a specific marker of the ICC.Western blot,immunofluorescence,and IHC were used to detect the localization and expression of TNX and TGF-β/Smad.RESULTS IHC showed that the number of ICC with positive c-Kit expression was significantly reduced in the colon of STC patients(22.17±3.28 vs 28.69±3.53,P<0.05)and that the distribution was abnormal.Western blot results showed that c-Kit and Smad7 levels were significantly decreased in the colon of STC patients(c-kit:0.462±0.099 vs 0.783±0.178,P<0.01;Smad7:0.626±0.058 vs 0.799±0.03,P<0.01)and that TNX and Smad2/3 levels were higher in the STC group(TNX:0.868±0.028 vs 0.482±0.032,P<0.01).There was no significant difference in TGF-βbetween the two groups(0.476±0.028 vs 0.511±0.044,P=0.272).Pearson correlation analysis showed that the TNX protein exhibited a strong correlation with Smad2/3 and Smad7(P<0.05,|R|>0.8)and TGF-β(P<0.05,|R|=0.7).CONCLUSION The extracellular matrix protein TNX may activate the TGF-β/Smad signaling pathway by upregulating the Smad 2/3 signaling protein and thereby induce slight or complete epithelial stromal cell transformation,leading to an abnormal distribution and dysfunction of ICC in the diseased colon,which promotes the occurrence and development of STC.     

Caffeine mitigates experimental nonalcoholic steatohepatitis and the progression of thioacetamide-induced liver fibrosis by blocking the MAPK and TGF-β/Smad3 signaling pathways

Introduction and objectivesCaffeine 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.MethodsNASH 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.ResultsRats 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-β), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) expression and by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and Smad3 phosphorylation.ConclusionsCaffeine attenuates NASH and the progression of liver fibrosis due to its antifibrotic effects and modulating the MAPK and TGF-β pathways. Therefore, caffeine could be a suitable candidate for treating liver diseases associated with fibrosis.     

Inhibition of Fas/FasL mRNA expression and TNF-a release in concanavalin A-induced liver injury in mice by bicyclol

AIM:Bicyclol,4,4'-dimethoxy-5,6,5',6'-dimethylene-dioxy-2-hydroxymethyl-2'-carbonyl biphenyl,is a new anti-hepatitisdrug.The aim of the present study was to investigate theprotective effect of bicyclol on concanavalin A(Con A)-inducedimmunological liver injury in mice and its mechanism.METHODS:Liver injury was induced by injection of Con Avia tail vein of mice and assessed biochemically andhistologically.Serum transaminase and tumor necrosis factoralpha(TNF-α)were determined.Liver lesions were observedby light microscope.Expressions of TNF-α,interferon gamma(IFN-γ),Fas and Fas ligand(FasL)mRNA in the livers weremeasured by RT-PCR.RESULTS:Serum transaminase level and liver lesions inCon A-induced mice were markedly reduced by oraladministration of 100,200 mg/kg of bicyclol.TNF-α level inserum was also reduced by bicyclol.Con A injection inducedup-regulation of TNF-α,IFN-γ,Fas and FasL mRNA expressionin liver tissues.Bicyclol significantly down-regulated theexpression of IFN-γ,Fas and FasL mRNA,but only slightlyaffected TNF-α mRNA expression in liver tissues.CONCLUSION:Bicyclol protects against Con A-induced liverinjury mainly through inhibition of Fas/FasL mRNA expressionin liver tissues and TNF-α release in mice.     

The Wnt/β-catenin signaling pathway in liver biology and disease

The Wnt signaling pathway is an evolutionarily conserved, highly complex signaling pathway that is critical for development, differentiation and cellular homeostasis. The protein β-catenin is the central player in one major arm of the Wnt pathway called the canonical Wnt pathway. As in other organs, the Wnt/β-catenin pathway is critical for liver development. However, recent research suggests that the pathway is also important in liver regeneration, liver metabolism and maintenance of normal function in the adult liver. Aberrant activation of β-catenin has also been implicated in the pathogenesis of hepatobiliary neoplasia, ranging from benign lesions to liver cancer. The explosion of research into the many roles of the Wnt/β-catenin pathway promises to change our fundamental understanding of normal liver biology and the aberrations that lead to disease and cancer.     

Effect of three stage sequential therapy on TGF-β_1/Smad signaling pathway in asthma model rats in the process of steroid withdrawal

正Objective To observe the effect of three stage sequential therapy on protein expressions of TGF-β_1,Smad2,Smad3,Smad6,Smad7 on TGF-β_1/Smad signaling pathway in asthma model rats in the process of steroid withdrawal.Methods Totally 150 SD rats were randomly divided into the normal control group(A),the airway remodeling group(B),the Dexamethasone interve-     

Metformin ameliorates liver fibrosis induced by congestive hepatopathy via the mTOR/HIF-1α signaling pathway

Introduction and ObjectivesCongestive hepatopathy (CH) is a hepatic vascular disease that results in chronic liver congestion, which can lead to liver fibrosis. New uses of metformin have been discovered over the years. However, the function of metformin in congestive liver fibrosis is not yet fully understood. This study aimed to investigate the effect of metformin on liver fibrosis in a mouse model of CH.Materials and MethodsPartial ligation of the inferior vena cava (pIVCL) was used to establish a mouse model of liver congestion. Metformin (0.1%) was added to the daily drinking water of the animals, and the effect of metformin on liver tissue was studied after 6 weeks. Hepatic stellate cells (HSCs) were also stimulated with CoCl₂ to investigate the inhibitory impact of metformin on the mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) pathway.ResultsMetformin attenuated liver congestion; decreased the expression of collagen, fibronectin, α-smooth muscle actin (α-SMA), and HIF-1α; and ameliorated liver fibrosis in pIVCL mice. The proliferation and migration of HSCs were inhibited by metformin in vitro, which prevented α-SMA expression and restrained HSC activation. The expression levels of phosphorylated-mTOR, HIF-1α, and vascular endothelial growth factor were also decreased.ConclusionsMetformin inhibits CH-induced liver fibrosis. Functionally, this beneficial effect may be the result of inhibition of HSC activation and of the mTOR/HIF-1α signaling pathway.     

Inhibition of Fas／FasL mRNA expression and TNF-a release in concanavalin A-induced liver injury in mice by bicyclol

AIM: Bicyclol, 4,4‘-dimethoxy-5,6,5‘,6‘-dimethylene-dioxy-2-hydroxymethy1-2‘-carbonyl biphenyl, is a new anti-hepatitis drug. The aim of the present study was to investigate the protective effect of bicyclol on concanavalin A (Con A)-induced immunological liver injury in mice and its mechanism. METHODS: Liver injury was induced by injection of Con A via tail vein of mice and assessed biochemically and histologically. Serum transaminase and tumor necrosis factor alpha (TNF-a were determined. Liver lesions were observed by light microscope. Expressions of TNF-a, interferon gamma (IFN-y), Fas and Fas ligand (FasL) mRNA in the livers were measured by RT-PCR. RESULTS: Serum transaminase level and liver lesions in Con A-induced mice were markedly reduced by oral administration of 100, 200 mg/kg of bicyclol. TNF-a level inserum was also reduced by bicyclol. Con A injection in ducedup-regulation of TNF-a, IFN-7, Fas and FasL mRNA expression in liver tissues. Bicyclol significantly down-regulated the expression of IFN-y, Fas and FasL mRNA, but only slightly affected TNF-a mRNA expression in liver tissues. CONCLUSION: Bicyclol protects against Con A-induced liver injury mainly through inhibition of Fas/FasL mRNA expression in liver tissues and TNF-a release in mice.     

Paclitaxel ameliorates fibrosis in hepatic stellate cells via inhibition of TGF-β/Smad activity

AIM: To investigated if paclitaxel can attenuate hepatic fi brosis in rat hepatic stellate cells (RHSCs). METHODS: RHSCs were cultured in vitro and randomly assigned to four groups: normal control group (treated only with Dulbecco's Modified Eagle's Medium), Taxol group (200 nmol/L paclitaxel was added to the cell culture), transforming growth factor (TGF)-β group (5 ng/mL recombinant human TGF-β1 was added to the cell culture), and TGF-β + Taxol group. TGF-β signaling cascade and status of various extracel...     

Thymosinβ4 alleviates cholestatic liver fibrosis in mice through downregulating PDGF/PDGFR and TGFβ/Smad pathways

Liver fibrosis is an important health problem without adequate and effective therapeutics. In this study, effects of thymosinβ4 (Tβ4) on hepatic fibrogenesis and the underlying molecular mechanisms were explored in bile duct ligation (BDL)-induced mice cholestatic liver fibrosis model. Results showed exogenous Tβ4 significantly reduced the mortality and liver/body weight ratio in BDL mice. Histological examinations and biochemical analyses demonstrated that BDL induced evident portal fibrosis and a significant increase in hepatic collagen contents. However, these changes were significantly attenuated by exogenous Tβ4. Quantitative real-time PCR assays showed that Tβ4 suppressed BDL-induced increases in many fibrotic genes expression including α-smooth muscle actin (α-SMA), collagen I, III and fibronectin, TGFβ1, TGFβR II, Smad2, Smad3, and PDGFRβ. Results from immunohistochemistry and Western blots also showed that Tβ4 reduced TGFβ1 and PDGFRβ protein levels in the liver tissues of BDL mice. In vitro studies using LX-2 cells demonstrated that Tβ4 could decrease PDGFRβ and TGFβR II levels in hepatic stellate cells. Taken together, findings in our present studies suggested that exogenous Tβ4 alleviated BDL-induced cholestatic liver fibrosis through downregulating PDGF/PDGFR and TGFβ/Smad pathways.     

Role of TGF-β1/Smads pathway in carotid artery remodeling in renovascular hypertensive rats and prevention by Enalapril and Amlodipine

Objective To investigate the role of transforming growth factor-β1 (TGF-β1), Smad2/3 and Smad7 expressions in carotid artery remodeling in renovascular hypertensive rats, and also the therapeutic effect of Enalapril and Amlodipine. Methods The renovascular hypertensive rat (RHR) models with “two-kidney and one-clip” were established, including model group (n = 6), sham-operated group (n = 6), Enalapril group (10 mg/kg per day, n = 6), Amlodipine group (5 mg/kg per day, n = 6) and combination group (Amlodipine 2.5 mg/kg per day + Enalapril 5mg/kg per day, n = 6). The medication were continuous administrated for six weeks. Carotid artery morphological and structural changes in the media were observed by HE staining, Masson staining and immuno histochemical staining. Media thickness (MT), MT and lumen diameter ratio (MT/LD), and the expression levels of media α-smooth muscle actin (α-actin), proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in carotid arteries were measured. Results The media of carotid arteries in RHR model group was significantly thickened, the volume of smooth muscle cell was increased, and the array was in disorder; MT, MT/LD, the proliferation index of smooth muscle cell and collagen fiber area percentage of carotid arteries in the model group were significantly higher than those in the sham-operated group (P < 0.01). Compared to sham-operated group, the model group had significantly higher expressions of TGF-β1 and p-Smad2/3 (P < 0.05) and lower Smad7 expression. Both Enalapril and Amlodipine improved smooth muscle hypertrophy and collagen deposition, reduced RHR carotid MT, MT/LD, proliferation index of smooth muscle cell, collagen fiber area percentage and the expressions of TGF-β1 and p-Smad2/3 (P < 0.05), increased Smad7 expression (P < 0.05). Moreover, the combination treatment of Enalapril and Amlodipine had significantly better effects than single Amlodipine group (P < 0.05), but not single Enalapril group. Conclusions TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodeling in RHR; the role of Amlodipine and Enalapril in inversing carotid artery remodeling may be related to the change of TGF-β1/Smads pathway, the combination treatment of Amlodipine and Enalapril had better effects than single administration of Amlodipine.     

Qishen Yiqi dripping pill inhibited myocardial fibrosis after myocardial infarction in rats and its effects on TGF-β_1/Smads pathway

正Objective To observe the effect of Qishen Yiqi Dripping Pill(QYDP)on myocardial fibrosis(MF)after myocardial infarction(MI)in rats,and to study its mechanism based on TGF-β_1/Smads pathway.Methods The left anterior descending coronary artery was ligated to establish MI model in male Wistar rats.The successful     

Role of Wnt signaling pathway hypofunction mediated by dephosphorylation of β-catenin in impaired wound healing of type 1 diabetic rats

<正>Objective To investigate the role of Wnt signaling pathway hypofunction mediated by dephosphorylation ofβ-catenin in the impaired wound healing of type 1 diabetic rats.Methods The back skin defect wounds were produced in rats with type 1 diabetes.These rats were divided into control,diabetes,lithium chloride treatment,and epidermal growth factor(EGF)treatment     

Notch3 Ameliorates Cardiac Fibrosis After Myocardial Infarction by Inhibiting the TGF-β1/Smad3 Pathway

Notch3 and TGF-β1 signaling play a key role in the pathogenesis and progression of chronic cardiovascular disease. However, whether Notch3 protects against myocardial infarction (MI) and the underlying mechanisms remains unknown. C57BL/6 mice were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) before coronary artery ligation. Four weeks after constructing MI model, cardiac function and fibrosis were compared between groups. The cardiac fibroblast cells (CFs) were isolated from newborn C57BL/6 mice (1–3 days old) and transfected with lentivirus carrying Notch3 cDNA. TGF-β1 (5 ng/ml), a well-known pro-fibrotic factor, was administered 72 h after Notch3 cDNA administration in CFs. The related proteins of fibrosis such as a-smooth muscle actin (a-SMA), Type I collagen, metalloprotease (MMP)-9 and the tissue inhibitor of metalloproteinases (TIMP)-2 were examined by western blot analysis. Notch3 cDNA treatment attenuated cardiac damage and inhibited fibrosis in mice with MI. Meanwhile, Notch3 siRNA administration aggravated cardiac function damage and markedly enhanced cardiac fibrosis in mice with MI. Overexpression of Notch3 inhibited TGF-β1-induced fibroblast–myofibroblast transition of mouse cardiac fibroblast cells, as evidenced by down-regulating a-SMA and Type I collagen expression. Notch3 cDNA treatment also increased MMP-9 expression and decreased TIMP-2 expression in the TGF-β1-stimulated cells. This study indicates that Notch3 is an important protective factor for cardiac fibrosis in a MI model, and the protective effect of Notch3 is attributable to its action on TGF-β1/Smad3 signaling.     

Melatonin attenuates hepatic ischemia-reperfusion injury in rats by inhibiting NF-κB signaling pathway

BackgroundThe sterile inflammatory response is one of the key mechanisms leading to hepatic ischemia-reperfusion injury. Melatonin has been shown to prevent organ injuries, but its roles in the inflammatory response after hepatic ischemia-reperfusion injury have not been fully explored, especially in late ischemia-reperfusion injury. The present study aimed to investigate the roles and possible mechanisms of melatonin in the inflammatory response after hepatic ischemia-reperfusion injury.MethodsSixty Sprague-Dawley rats were randomly divided into a sham group, ischemia-reperfusion injury group (I/R group), and melatonin-treated group (M + I/R group). The rats in the I/R group were subjected to 70% hepatic ischemia for 45 min, followed by 5 or 24 h of reperfusion. The rats in the M + I/R group were injected with melatonin (10 mg/kg, intravenous injection) 15 min prior to ischemia and immediately before reperfusion. Serum and samples of ischemic liver lobes were harvested for future analysis, and the 7-day survival rate was assessed after hepatic ischemia-reperfusion surgery.ResultsIn comparison with the I/R group, the M + I/R group showed markedly decreased expression levels of inflammatory cytokines (IL-6 and TNF-α) and numbers of apoptotic hepatocytes (P < 0.05). Immunoblotting showed that the expression levels of IL-6, p-NF-κBp65/t-NF-κBp65 and p-IκB-α/t-IκB-α in the M + I/R group were significantly lower than those in the I/R group, and immunofluorescence staining showed that the expression level of p-NF-κBp65 in the M + I/R group was lower than that in the I/R group (P < 0.05). The 7-day survival rates were 20% in the I/R group and 50% in the M + I/R group (P < 0.05).ConclusionsMelatonin downregulated the activity of the NF-κB signaling pathway in the early and late stages of hepatic ischemia-reperfusion injury, alleviated the inflammatory response, protected the liver from ischemia-reperfusion injury, and increased the survival rate.