Protective effect of Acacia confusa bark extract and its active compound gallic acid against carbon tetrachloride-induced chronic liver injury in rats

Acacia confusa Merr. (Leguminosae), a species native to Taiwan, is widely distributed on the hills and lowlands of Taiwan, and has been traditionally used as a medicine. The hepatoprotective effects of A. confusa bark extract (ACBE) and its active constituent gallic acid were evaluated against carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats. CCl₄-induced hepatic pathological damage and significantly increased the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and malondialdehyde (MDA) in plasma, and cytochrome P4502E1 (CYP2E1) protein expression in hepatic samples, and decreased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) in erythrocytes. Treatment with ACBE, gallic acid or silymarin could decrease significantly the AST, ALT, and MDA levels in plasma, and CYP2E1 expression in liver tissues, and increase the activities of SOD and GPX in erythrocyte when compared with CCl₄-treated group. Liver histopathology also showed that ACBE, gallic acid or silymarin could significantly reduce the incidence of liver lesions induced by CCl₄. These results suggested that the ACBE and gallic acid exhibit potent hepatoprotection against CCl₄-induced liver damages in rats, and the hepatoprotective effects of ACBE and gallic acid may be due to the modulation of antioxidant enzymes activities and inhibition of lipid peroxidation and CYP2E1 activation.     

Transforming growth factor-β type I receptor/ALK5 contributes to doxazosin-induced apoptosis in H9C2 cells

The mechanism of doxazosin-induced apoptosis through α₁-adrenoceptor-independent pathway has been reported in various types of cell models. However, the molecular events involved in this effect are still not fully discovered. In present study, we proposed that the transforming growth factor-β type I receptor (TβRI/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell viability, apoptotic nuclei, and caspase-3 activity, we found that doxazosin induced concentration- and time-dependent apoptosis in H9C2 cells. The cell apoptosis induced by 30 μM doxazosin was exacerbated by the addition of 10 ng/ml transforming growth factor-β1 (TGF-β1). Doxazosin or TGF-β1 alone respectively elevated p38 mitogen-activated protein kinases (MAPK) and Smad3 protein phosphorylation in H9C2 cells. However, the cotreatment of doxazosin and TGF-β1 attenuated the TGF-β1-induced Smad3 protein phosphorylation and increased doxazosin-induced p38 MAPK protein phosphorylation. Furthermore, inhibitors of TβRI/ALK5 (SB431542) and p38 MAPK (SB202190) or TβRI/ALK5 knockdown all dramatically reduced the doxazosin-induced apoptosis in H9C2 cells. In conclusion, our results demonstrated that TβRI/ALK5-p38 MAPK phosphorylation signaling pathway could contribute to doxazosin-induced cell apoptosis, which could be further enhanced by TGF-β1 in association with attenuating Smad3 phosphorylation in H9C2 cells.     

FGF21对HepG2细胞TGF-β信号通路的影响

目的研究FGF21对TGF-β/Smads信号通路中相关蛋白和mRNA表达的影响。方法采用Western blot法检测HepG2 cells的TGF-1,TGF-βRⅡ,Smad 2,3,4,7的蛋白表达水平和采用Q-PCR方法检测HepG2 cells的TGF-β1,TGF-βRⅡ,Smad 2,3,4,7的mRNA表达水平。结果在不同FGF21浓度下,TGF-β1,TGF-βRⅡ,Smad 2,3,4,7蛋白和mRNA表达水平不同(P<0.05)。结论 FGF21通过促进TGF-β信号通路中的信号分子或受体的表达,或者通过下调此信号通路的阻断分子而抑制肿瘤发生。     

Transforming Growth Factor-beta signal responding in hepatic stem-like cells

Objective To investigate the effects of TGF-β on the expressions and distribution of phosphorated Smad2/3 and Smad7 in hepatic stem-like cells.Methods Using immunogold transmission electron microscopy,we observed the expressions and distribution of phosphorated Smad2/3,and Smad7 before and after TGF-β1(5 ng·mL-1)treatment for 4,8,and 24 hours in hepatic stem-like cells(WB cells).In addition,we also detected the apoptosis status after TGF-β1 stimulation by transmission electron microscopy.Results TGF-β1 stimulation can result in expression increasing of phosphorated Smad2/3 in WB cells,and reach the peak at 8 h,especially in the nuclear.After treatment with TGF-β1 for 24 h,the nuclear expression of phosphorated Smad2/3 gradually decreased.Additionally,we found that TGF-β1 treatment also contributed to increasing in protein level and alteration in cellular distribution of Smad7(translocation from the nucleus to the cytoplasm)in WB cells.Furthermore,we observed apoptotic body in WB cells after TGF-β1 treatment for 8 h.Conclusions These results indicate that TGF-β stimulation can alter the expression and cellular distribution of phosphorated Smad2/3 and Smad7 which are its downstream molecular,suggesting hepatic stem-like cells own intact responding to TGF-β.     

Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway

Liver fibrosis is considered to be a result of chronic liver pathological changes, and hepatic stellate cells (HSCs) play an important role during this process. Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities. This study focused on the effects of evodiamine on carbon tetrachloride (CCl₄)-induced liver fibrosis in rats and HSCs in vitro via the TGF-β1/Smad signaling pathway. A liver fibrosis rat model was established by the intraperitoneal injection of CCl₄ (3 ml/kg, 30% in olive oil). Evodiamine (15 and 25 mg/kg) was administered orally for 8 weeks. HSCs were treated with different evodiamine concentrations. The results indicated that evodiamine could improve the histopathological abnormalities in liver tissues and decrease the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hydroxyproline, and total bilirubin (TBIL). Concentrations of IL-6, tumor necrosis factor-α (TNF-α), collagen-I (COL-I), and collagen-III (COL-III) were reduced by evodiamine. Western blotting and real-time PCR showed that protein expression of transforming growth factor-β (TGF-β1), p-Smad 2/3 (phosphorylation of Smad 2/3), and smooth muscle alpha-actin (α-SMA) as well as mRNA expression of TGF-β1 and α-SMA in liver tissues were downregulated by evodiamine. The cell proliferation, production of hydroxyproline, and the protein expression of TGF-β1, p-Smad 2/3, and α-SMA in HSCs were dose-dependently reduced by evodiamine. Collectively, evodiamine had an antifibrosis effect in CCl₄-induced liver fibrosis, and reduced HSCs proliferation and collagen metabolism in vitro. The major mechanism was downregulation of relative expression of TGF-β1, p-Smad 2/3, and α-SMA.     

CCM111 prevents hepatic fibrosis via cooperative inhibition of TGF-β, Wnt and STAT3 signaling pathways

CCM111 is an aqueous extract of Antrodia cinnamomea (AC) that has exhibited anti-liver fibrosis functions. However, the detailed mechanisms of AC action against liver fibrosis have not been elucidated yet. The present research showed that CCM111 significantly lowered the levels of the hepatic enzyme markers glutamate oxaloacetate transaminase (GOT) and glutamic pyruvic transaminase (GPT), prevented liver damage and collagen deposition, and downregulated TGF-β/Smad signaling in a dose-dependent manner compared with CCl₄ treatment alone. CCM111 markedly inhibited TGF-β, Wnt and STAT3 signaling pathway-regulated downstream genes in the liver by next-generation sequencing. The antifibrotic mechanisms of CCM111 were further demonstrated in HSC-T6 cells. Our data demonstrated for the first time that CCM111 can protect against CCl₄-induced liver fibrosis by the cooperative inhibition of TGF-β-, Wnt- and STAT3-dependent proinflammatory and profibrotic mediators, suggesting that CCM111 might be a candidate for preventing and treating chronic fibrotic liver diseases.     

Hepatoprotective effects of the nitric oxide donor isosorbide-5-mononitrate alone and in combination with the natural hepatoprotectant, silymarin, on carbon tetrachloride-induced hepatic injury in rats

The aim of this study was to investigate the effect of the nitric oxide donor isosorbide-5-mononitrate (5-ISMN) alone or in combination with the natural hepatoprotectant with anti-oxidant activity silymarin on the carbon tetrachloride (CCl₄)-induced hepatic injury in rats. 5-ISMN (1.8, 3.6 or 7.2 mg/kg), silymarin (25 mg/kg) or 5-ISMN (1.8, 3.6 or 7.2 mg/kg) combined with silymarin was given once daily orally simultaneously with CCl₄ and for 15 days thereafter. Liver damage was assessed by determining serum enzyme activities and hepatic histopathology. 5-ISMN given at the above doses conferred significant protection against the hepatotoxic actions of CCl₄ in rats, reducing serum alanine aminotransferase (ALT) levels by 31.2, 39.3 and 61.6%, respectively, when compared with controls. Serum aspartate aminotransferase (AST) levels decreased by 19.8, 22.7 and 59.4%, respectively, while alkaline phosphatase (ALP) decreased by 26.1 and 32.6% by the drug at 3.6 and 7.2 mg/kg, respectively. When silymarin was added to 5-ISMN (1.8, 3.6 or 7.2 mg/kg), ALT decreased by 32.8, 59.6, 70.2% and AST by 28.7, 50.3, 60%, when compared with CCl₄ control group levels. Silymarin in combination with 3.6 or 7.2 mg/kg 5-ISMN resulted in 37.5 and 39.2% reductions in ALP when compared with CCl₄ control group. Meanwhile, silymarin alone reduced ALT, AST and ALP levels by 65.9, 52 and 62.3%, respectively. Blood levels of reduced glutathione were markedly decreased in CCl₄-treated rats. Reduced glutathione levels were increased by the administration of 5-ISMN and restored to near normal values by silymarin treatment. Histopathological alterations by CCl₄ were markedly reduced after treatment with 5-ISMN alone or in combination with silymarin. Histopathologic examination of the livers of CCl₄-treated rats administered 5-ISMN at 7.2 mg/kg showed marked restoration of the normal architecture of the liver tissue and minimal fibrosis. Silymarin co-administered with 5-ISMN resulted in further improvement of the histologic picture. These results indicates that treatment with 5-ISMN protects against hepatocellular necrosis induced by CCl₄. The study suggests a potential therapeutic use for 5-ISMN in combination with silymarin in liver injury.     

止喘胶囊对哮喘大鼠气道重建的TGF-β1及Smads蛋白表达的影响

目的:探讨止喘胶囊对哮喘大鼠气道重建TGF-β1及其胞内信号转导分子Smads蛋白的影响。方法:将40只雄性SD大鼠随机分为5组:正常对照组、哮喘模型组、止喘胶囊组、地塞米松组和止喘胶囊+地塞米松组,每组8只。通过ip卵清蛋白及右腿im氢氧化铝凝胶致敏,并雾化吸入卯清蛋白,建立哮喘模型,在哮喘激发4 wk后检测肺组织TGF-β1蛋白及mRNA以及Smad-2、Smad-7 mRNA表达。计量资料用ANOVA程序进行分析,并用LSD进行两两比较。结果:哮喘模型组气道上皮TGF-β1以及TGF-β1 mRNA表达显著高于正常对照组(P＜0．05,P＜0．01)。哮喘模型组Smad-2 mRNA较正常对照组表达明显升高(P＜0．05),止喘胶囊组、止喘胶囊+地塞米松组和地塞米松组TGF-β1,TGF-β1 mRNA,Smad-2 mR- NA表达显著低于哮喘模型组(P＜0．01或P＜0．05),止喘胶囊组可显著上调Smad-7 mRNA表达水平,与哮喘模型组相比有显著的差异(P＜0．05)。结论:止喘胶囊可下调哮喘大鼠生长因子TGF-β1蛋白及mR- NA、Smad-2 mRNA表达,上调Smad-7 mRNA的表达,阻断TGF-β1的信号转导,从而减轻哮喘大鼠气道重建,为止喘胶囊的临床使用提供实验依据。     

Curcumin ameliorates intrahepatic angiogenesis and capillarization of the sinusoids in carbon tetrachloride-induced rat liver fibrosis

Neoangiogenesis and the development of an abnormal angio-architecture in the liver are strongly linked with progressive fibrogenesis. This study aimed to evaluate the ability of curcumin to protect liver fibrosis-associated angiogenesis and capillarization of the sinusoids in experimental rats. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without curcumin for 6 weeks. The results suggest that curcumin treatment markedly attenuated CCl₄-induced liver fibrosis, as assessed by histology and hydroxyproline content, and inhibited hepatic stellate cell activation. Curcumin ameliorated hepatic angiogenesis, as assessed by measuring microvessel density using Von Willebrand factor staining and by examining the expression of the endothelial cell markers CD31 and vascular endothelial growth factor receptor (VEGFR)-2 in the livers. Pathologic remodeling of liver sinusoidal capillarization, as assessed by electron-microscopic analysis of Disse's space and by evaluation of the levels of basement membrane protein expression, was also attenuated by curcumin administration. The intrahepatic gene or protein expression of hypoxia-inducible factor-1α, VEGFR-1, placental growth factor, and cyclooxygenase-2 decreased with treatment with curcumin in fibrotic rats. In conclusion, curcumin ameliorates hepatic angiogenesis and sinusoidal capillarization in CCl₄-induced rat liver fibrosis through suppressing multiple proangiogenic factors.     

Chronic CCl4 intoxication causes liver and bone damage similar to the human pathology of hepatic osteodystrophy: a mouse model to analyse the liver–bone axis

Patients with chronic liver diseases frequently exhibit decreased bone mineral densities (BMD), which is defined as hepatic osteodystrophy (HOD). HOD is a multifactorial disease whose regulatory mechanisms are barely understood. Thus, an early diagnosis and therapy is hardly possible. Therefore, the aim of our study consisted in characterizing a mouse model reflecting the human pathomechanism. Serum samples were collected from patients with chronic liver diseases and 12-week old C57Bl6/N mice after 6-week treatment with carbon tetrachloride (CCl₄). Repetitive injections of CCl₄ induced liver damage in mice, resembling liver fibrosis in patients, as assessed by serum analysis and histological staining. Although CCl₄ did not affect primary osteoblast cultures, μCT analysis revealed significantly decreased BMD, bone volume, trabecular number and thickness in CCl₄-treated mice. In both HOD patients and CCl₄-treated mice, an altered vitamin D metabolism with decreased CYP27A1, CYP2R1, vitamin D-binding protein GC and increased 7-dehydrocholesterol reductase hepatic gene expression, results in decreased 25-OH vitamin D serum levels. Moreover, both groups exhibit excessively high active transforming growth factor-beta (TGF-β) serum levels, inhibiting osteoblast function in vitro. Summarizing, our mouse model presents possible mediators of HOD, e.g. altered vitamin D metabolism and increased active TGF-β. Liver damage and significant changes in bone structure and mineralization are already visible by μCT analysis after 6 weeks of CCl₄ treatment. This fast response and easy transferability makes it an ideal model to investigate specific gene functions in HOD.     

Betaine treatment decreased oxidative stress,inflammation, and stellate cell activation in rats with alcoholic liver fibrosis

The aim of this study was to investigate the effect of betaine (BET) on alcoholic liver fibrosis in rats. Fibrosis was experimentally generated with ethanol plus carbon tetrachloride (ETH + CCl₄) treatment. Rats were treated with ETH (5% v/v in drinking water) for 14 weeks. CCl₄ was administered intraperitoneally (i.p.) 0.2 mL/kg twice a week to rats in the last 6 weeks with/without commercial food containing BET (2% w/w). Serum hepatic damage markers, tumor necrosis factor-α, hepatic triglyceride (TG) and hydroxyproline (HYP) levels, and oxidative stress parameters were measured together with histopathologic observations. In addition, α-smooth muscle-actin (α-SMA), transforming growth factor-β1 (TGF-β1) and type I collagen (COL1A1) protein expressions were assayed immunohistochemically to evaluate stellate cell (HSC) activation. mRNA expressions of matrix metalloproteinase-2 (MMP-2) and its inhibitors (TIMP-1 and TIMP-2) were also determined. BET treatment diminished TG and HYP levels; prooxidant status and fibrotic changes; α-SMA, COL1A1 and TGF-β protein expressions; MMP-2, TIMP-1 and TIMP-2 mRNA expressions in the liver of fibrotic rats. In conclusion, these results indicate that the antifibrotic effect of BET may be related to its suppressive effects on oxidant and inflammatory processes together with HSC activation in alcoholic liver fibrosis.     

Effect of α-tocopherol on carbon tetrachloride intoxication in the rat liver

Carbon tetrachloride (1 ml/kg body weight as a 1:1 mixture of CCl₄ and mineral oil) was orally administered to rats. After 12 h, the activity of plasma ALT (alanine aminotransferase) was significantly higher than that of the control group, and plasma ALT and AST (aspartate aminotransferase) activities significantly increased 24 h after CCl₄ administration. These results indicated that the necrotic process had initiated at about 12 h and developed thereafter. After 6–24 h of CCl₄ administration, the hepatic level of vitamin C, the most sensitive indicator of oxidative stress, decreased significantly, indicating that oxidative stress was significantly enhanced 6 h after CCl₄ intoxication and thereafter. Oral administration of vitamin E (1 ml/kg body weight as a 1:1 mixture of α-tocopherol and mineral oil) 12 h before CCl₄ administration caused a significant elevation of liver vitamin E level and ameliorated liver necrosis 24 h after CCl₄ intoxication based on plasma AST and ALT. Vitamin E also significantly restored the hepatic vitamin C concentration 12 and 24 h after CCl₄ intoxication, demonstrating that vitamin E functioned as an antioxidant. The liver vitamin E concentration was not changed by vitamin E supplementation to rats that did not receive CCl₄. This result indicated that vitamin E accumulated in the damaged liver. The activation of JNK, ERK1/2 and p38 MAPK took place 1.5 h after CCl₄ administration. Co-administration of α-tocopherol with CCl₄ did not affect these early changes in MAPKs.     

Ⅱ°烧烫伤模型大鼠体内TGF-β、p38MAPK、IL-1β的表达分析

目的:探讨Ⅱ°烧烫伤模型大鼠体内转化生长因子β(TGF-β)、p38丝裂原活化蛋白激酶(MAPK)和白细胞介素1β(IL-1β)的表达情况。方法:取大鼠分为4组,每组14只,分别为正常对照组、NaOH烧伤组、水烫伤组和乙醇烧伤组,分别建立相应的Ⅱ°烧烫伤模型,24 h后观察各组大鼠病理变化,酶联免疫吸附剂测定法检测各组大鼠建模后第1、4、7天血清中TGF-β、p38 MAPK和IL-1β的水平。结果:与正常对照组比较,其余3组大鼠均可见表皮及真皮有不同程度的缺损或坏死,炎性细胞向肌层组织浸润,其中水烫伤组和乙醇烧伤组大鼠皮下水肿明显;NaOH烧伤组大鼠血清中TGF-β和p38 MAPK水平在第4、7天均明显升高(P<0.05或P<0.01),IL-1β表达无明显变化;水烫伤组大鼠血清中TGF-β水平在第1、4、7天均明显升高(P<0.05),p38 MAPK和IL-1β水平在第4、7天明显升高(P<0.05或P<0.01);乙醇烧伤组大鼠血清中TGF-β水平在第4天明显升高(P<0.05),p38 MAPK和IL-1β水平在第1、4、7天均明显升高(P<0.05或P<0.01)。结论:水烫伤和乙醇烧伤可致大鼠Ⅱ°烧烫伤模型中TGF-β、p38 MAPK和IL-1β水平均有不同程度的升高,并可能激发TGF-β、p38 MAPK和IL-1β参与的炎症信号通路。     

Involvement of the activation of Nrf2/HO-1, p38 MAPK signaling pathways and endoplasmic reticulum stress in furazolidone induced cytotoxicity and S phase arrest in human hepatocyte L02 cells: modulation of curcumin

Furazolidone (FZD) is extensively used as the antiprotozoal and antibacterial drug in clinic. The previous study has shown that curcumin pretreatment could improve FZD induced cytotoxicity by inhibiting oxidative stress and mitochondrial apoptotic pathway. The current study aimed to investigate the potential roles of endoplasmic reticulum (ER) stress, p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway in curcumin against FZD cytotoxicity by using human hepatocyte L02 cells. The results showed that curcumin could markedly attenuate FZD induced cytotoxicity. Compared with FZD alone group, curcumin pretreatment significantly reduced the expression of phospho (p)-p38, cyclin D1, p-checkpoint kinase 1 (ChK1) and breast cancer associated gene 1 (BRCA1) protein, followed to attenuate S phase arrest. Meanwhile, curcumin pretreatment prevented FZD induced ER stress, evidenced by the inhibition of glucose-regulated protein 78 and DNA damage inducible gene 153/C/EBP-homologous protein (GADD153/CHOP) protein expression. Moreover, compared with the control, FZD exposure activated the protein and mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which were further activated by curcumin treatment. These results reveal that curcumin could prevent FZD induced cytotoxicity and S phase arrest, which may involve the activation of Nrf2/HO-1 pathway and the inhibition of p38 MAPK pathway and ER stress.