BM-MSCs overexpressing the Numb enhance the therapeutic effect on cholestatic liver fibrosis by inhibiting the ductular reaction.

BACKGROUND: Cholestatic liver fibrosis (CLF) is caused by inflammatory destruction of the intrahepatic bile duct and abnormal proliferation of the small bile duct after cholestasis. Activation of the Notch signaling pathway is required for hepatic stem cells to differentiate into cholangiocytes during the pathogenesis of CLF. Our previous research found that the expression of the Numb protein, a negative regulator of Notch signaling, was significantly reduced in the livers of patients with primary biliary cholangitis and CLF rats. However, the relationship between the Numb gene and CLF is largely unclear. In this study, we investigated the role of the Numb gene in the treatment of bile duct ligation (BDL)-induced CLF. METHODS: In vivo, bone marrow-derived mesenchymal stem cells (BM-MSCs) with Numb gene overexpression or knockdown obtained using lentivirus transfection were transplanted into the livers of rats with BDL-induced CLF. The effects of the Numb gene on stem cell differentiation and CLF were evaluated by performing histology, tests of liver function, and measurements of liver hydroxyproline, cytokine gene and protein levels. In vitro, the Numb gene was overexpressed or knocked down in the WB-F344 cell line by lentivirus transfection, Then, cells were subjected immunofluorescence staining and the detection of mRNA levels of related factors, which provided further evidence supporting the results from in vivo experiments. RESULTS: BM-MSCs overexpressing the Numb gene differentiated into hepatocytes, thereby inhibiting CLF progression. Conversely, BM-MSCs with Numb knockdown differentiated into biliary epithelial cells (BECs), thereby promoting the ductular reaction (DR) and the progression of CLF. In addition, we confirmed that knockdown of Numb in sodium butyrate-treated WB-F344 cells aggravated WB-F344 cell differentiation into BECs, while overexpression of Numb inhibited this process. CONCLUSIONS: The transplantation of BM-MSCs overexpressing Numb may be a useful new treatment strategy for CLF.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-ß/Smad Signaling.

OBJECTIVE: To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro. METHODS: Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor ß (TGF-ß)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-ß1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed. RESULTS: High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFß R1, TGFß R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01). CONCLUSIONS: Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-ß/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

SNS-032 attenuates liver fibrosis by anti-active hepatic stellate cells via inhibition of cyclin dependent kinase 9.

Liver fibrosis is a common pathological process of all chronic liver diseases. Hepatic stellate cells (HSCs) play a central role in the development of liver fibrosis. Cyclin-dependent kinase 9 (CDK9) is a cell cycle kinase that regulates mRNA transcription and elongation. A CDK9 inhibitor SNS-032 has been reported to have good effects in anti-tumor. However, the role of SNS-032 in the development of liver fibrosis is unclear. In this study, SNS-032 was found to alleviate hepatic fibrosis by inhibiting the activation and inducing the apoptosis of active HSCs in carbon tetrachloride-induced model mice. In vitro, SNS-032 inhibited the activation and proliferation of active HSCs and induced the apoptosis of active HSCs by downregulating the expression of CDK9 and its downstream signal transductors, such phosphorylated RNA polymerase II and Bcl-2. CDK9 short hairpin RNA was transfected into active HSCs to further elucidate the mechanism of the above effects. Similar results were observed in active HSCs after CDK9 knockdown. In active HSCs with CDK9 knockdown, the expression levels of CDK9, phosphorylated RNA polymerase II, XIAP, Bcl-2, Mcl-1, and ɑ-SMA significantly decreased, whereas those of cleaved-PARP1 and Bax decreased prominently. These results indicated that SNS-032 is a potential drug and CDK9 might be a new prospective target for the treatment of liver fibrosis.

Research Progress in Chinese Medicine Preparations for Promoting Blood Circulation and Removing Blood Stasis for Cirrhotic Patients with Portal Vein Thrombosis Following Splenectomy.

This article presented an overview of the therapeutic effects of Chinese medicine (CM) preparations for promoting blood circulation and removing blood stasis for patients with portal vein thrombosis (PVT) after splenectomy. Based on published clinical researches of CM preparations for PVT after splenectomy in patients with cirrhotic portal hypertension (CPH), this paper evaluated the incidence of PVT, and explored potential active components and mechanisms of CM preparations. Safflower Yellow Injection, Danshen Injection () Danhong Injection (), and Compound Danshen Dropping Pill () achieved good curative effect alone or combined with anticoagulant therapy. In addition, Compound Biejia Ruangan Tablet () and Anluo Huaxian Pill () can also significantly improve the hemodynamic disorders of portal vein system in patients with cirrhosis. Considering the role of CM preparations in ameliorating the incidence of PVT after splenectomy in patients with CPH, we suggested that future research should provide more attention to CM alone or CM combined with anticoagulant for cirrhosis with PVT.

Pharmacological Effects of Salvianolic Acid B Against Oxidative Damage.

Salvianolic acid B (Sal B) is one of the main active ingredients of Salvia miltiorrhiza, with strong antioxidant effects. Recent findings have shown that Sal B has anti-inflammatory, anti-apoptotic, anti-fibrotic effects and can promote stem cell proliferation and differentiation, and has a beneficial effect on cardiovascular and cerebrovascular diseases, aging, and liver fibrosis. Reactive oxygen species (ROS) include oxygen free radicals and oxygen-containing non-free radicals. ROS can regulate cell proliferation, survival, death and differentiation to regulate inflammation, and immunity, while Sal B can scavenge oxygen free radicals by providing hydrogen atoms and reduce the production of oxygen free radicals and oxygen-containing non-radicals by regulating the expression of antioxidant enzymes. The many pharmacological effects of Sal B may be closely related to its elimination and inhibition of ROS generation, and Nuclear factor E2-related factor 2/Kelch-like ECH-related protein 1 may be the core link in its regulation of the expression of antioxidant enzyme to exert its antioxidant effect. What is confusing and interesting is that Sal B exhibits the opposite mechanisms in tumors. To clarify the specific target of Sal B and the correlation between its regulation of oxidative stress and energy metabolism homeostasis will help to further understand its role in different pathological conditions, and provide a scientific basis for its further clinical application and new drug development. Although Sal B has broad prospects in clinical application due to its extensive pharmacological effects, the low bioavailability is a serious obstacle to further improving its efficacy in vivo and promoting clinical application. Therefore, how to improve the availability of Sal B in vivo requires the joint efforts of many interdisciplinary subjects.

Hepatic stem cell Numb gene is a potential target of Huang Qi Decoction against cholestatic liver fibrosis.

Numb is a negative regulator of Notch signal pathway. Previous study has demonstrated that Notch signal pathway activation is required for hepatic progenitor cell (HPC) differentiating into cholangiocytes in cholestatic liver fibrosis (CLF), and Huang Qi Decoction (HQD) could prevent CLF through inhibition of the Notch signal pathway. However, the role of Numb in HQD against CLF is yet unclear. Thus, CLF rats transplanted into rat bone marrow-derived mesenchymal stem cells with knocked down Numb gene (BMSCNumb-KD) were treated with HQD. Simultaneously, Numb gene knockdown was also performed in WB-F344 cell line and then treated with refined HQD in vitro. In vivo study revealed that liver fibrosis was inhibited by HQD plus BMSCNumb-KD treatment, while Hyp content in liver tissue, the gene and protein expression of α-SMA, gene expression of Col I, TNF-α, and TGF-ß1 were increased compared to that in HQD group. Furthermore, Notch signal pathway was inhibited by HQD plus BMSCNumb-KD, while the protein expression of Numb was decreased and RBP-Jκ and Hes1 was increased compared to that in HQD group. In vitro, HQD reduced the differentiation of WB-F344 cells into cholangiocyte phenotype, while this effect was attenuated after Numb-knockdown. This study highlights that the absence of hepatic stem cell Numb gene decreases effect of HQD against CLF, which give rise the conclusion that Numb might be a potential target for HQD against CLF.

Yiguanjian decoction enhances fetal liver stem/progenitor cell-mediated repair of liver cirrhosis through regulation of macrophage activation state.

AIM: To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation. METHODS: A rat model of liver cirrhosis was established via subcutaneous injection of carbon tetrachloride (CCl4) for 8 wk. From the beginning of the ninth week, the rats received 2-acetylaminofluorene (2-AAF) by oral gavage and a DLK-1+ fetal liver stem/progenitor cell (FLSPC) transplant or an FLSPC transplant in combination with YGJ treatment for 4 wk. In vitro, lipopolysaccharide (LPS)-activated macrophages were co-cultured with WB-F344 cells, and the differentiation of WB-F344 cells was observed in the presence and absence of YGJ treatment. RESULTS: FLSPC transplantation improved liver function and histopathology, and inhibited the activation of the non-canonical Wnt signaling pathway, while activating the canonical Wnt signaling pathway. YGJ enhanced the therapeutic effects of FLSPCs and also promoted the liver regeneration differentiation of FLSPCs into hepatocytes. In vitro, LPS-activated macrophages promoted the differentiation of WB-F344 cells into myofibroblasts, and the canonical Wnt signaling was inhibited while the non-canonical Wnt signaling was activated in WB-F344 cells. YGJ suppressed the activation of macrophages and then inhibited non-canonical Wnt signaling and promoted canonical Wnt signaling. CONCLUSION: YGJ enhances FLSPC-mediated repair of liver cirrhosis through regulation of macrophage activation state, and YGJ in combination with stem cell transplantation may be a suitable treatment for end-stage liver cirrhosis.

Research progress on signaling pathways in cirrhotic portal hypertension.

Portal hypertension (PHT) is an important consequence of liver cirrhosis, which can lead to complications that adversely affect a patient's quality of life and survival, such as upper gastrointestinal bleeding, ascites, and portosystemic encephalopathy. In recent years, advances in molecular biology have led to major discoveries in the pathological processes of PHT, including the signaling pathways that may be involved: PI3K-AKT-mTOR, RhoA/Rho-kinase, JAK2/STAT3, and farnesoid X receptor. However, the pathogenesis of PHT is complex and there are numerous pathways involved. Therefore, the targeting of signaling pathways for medical management is lagging. This article summarizes the progress that has been made in understanding the signaling pathways in PHT, and provides ideas for treatment of the disorder.

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.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

AIM: To investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism. METHODS: Paired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined. RESULTS: GCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs (P < 0.01). Astragaloside IV treatment strongly inhibited the proliferation-, migration- and invasion-promoting capacities of GCAFs (P < 0.05 for 10 µmol/L, P < 0.01 for 20 µmol/L and 40 µmol/L). Compared with GNFs, GCAFs expressed a lower level of microRNA-214 (P < 0.01) and a higher level of microRNA-301a (P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression (P < 0.01) and down-regulated microRNA-301a expression (P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production (P < 0.01) and secretion (P < 0.05), and elevated TIMP2 production (P < 0.01) and secretion (P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV. CONCLUSION: Astragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is promisingly a potent therapeutic agent regulating tumor microenvironment.

Yiguanjian decoction and its ingredients inhibit angiogenesis in carbon tetrachloride-induced cirrhosis mice.

BACKGROUND: Cirrhosis is associated with angiogenesis and disruption of hepatic vascular architecture. Yiguanjian (YGJ) decoction, a prescription from traditional Chinese medicine, is widely used for treating liver diseases. We studied whether YGJ or its ingredients (iYGJ) had an anti-angiogenic effect and explored possible mechanisms underlying this process. METHODS: Cirrhosis was induced with carbon tetrachloride (CCl4) (ip) in C57BL/6 mice for 6 weeks. From week 4 to week 6, cirrhotic mice were randomly divided into four groups: sorafenib-treated, YGJ-treated and iYGJ-treated mice and placebo. Serum biochemistries, hydroxyproline (Hyp) content and histopathological changes of hepatic tissues were measured as were α-smooth muscle actin (α-SMA), collagen I, CD31, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) 2 and hypoxia-inducible factor (HIF)-1α. RESULTS: Both YGJ and iYGJ improved serum biochemistries. Changes of histopathology showed that YGJ and iYGJ reduced hepatic tissue necroinflammatory and collagen fiber deposition in cirrhosis mice. Compared to the CCl4 treated animals, Hyp, α-SMA, collagen I, CD31, VEGF, VEGFR, and HIF-1α expression decreased in YGJ and iYGJ groups. CONCLUSIONS: YGJ and iYGJ inhibited liver angiogenesis in cirrhotic mice treated with CCl4 by inhibiting the HIF-1α/VEGF signaling pathway, suggesting that anti-angiogenic effects of YGJ and iYGJ are associated with improving the hepatic hypoxic microenvironment.

MicroRNAs May Serve as Emerging Molecular Biomarkers for Diagnosis and Prognostic Assessment or as Targets for Therapy in Gastric Cancer.

Gastric cancer (GC) is one of the most common cancers, with high incidences in East Asia countries. Most GC patients have been reported with low early diagnosis rate and show extremely poor prognosis. Therefore, it is necessary to develop novel and more sensitive biomarkers to improve early diagnosis and therapy in order to provide longer survival and better quality of life for gastric cancer patients. MicroRNAs (miRNAs) play crucial roles in GC development and progression. miRNAs have emerged as a novel molecular biomarker for cancer diagnosis, prognosis and therapy with surprising stability in tissues, serum or other body fluids. This review summarizes major advances in our current knowledge about potential miRNA biomarkers for GC that have been reported in the past two years.

Association of miR-193b down-regulation and miR-196a up-regulation with clinicopathological features and prognosis in gastric cancer.

Dysregulated expression of microRNAs (miRNAs) has been shown to be closely associated with tumor development, progression, and carcinogenesis. However, their clinical implications for gastric cancer remain elusive. To investigate the hypothesis that genome-wide alternations of miRNAs differentiate gastric cancer tissues from those matched adjacent non-tumor tissues (ANTTs), miRNA arrays were employed to examine miRNA expression profiles for the 5-pair discovery stage, and the quantitative real-time polymerase chain reaction (qRT- PCR) was applied to validate candidate miRNAs for 48-pair validation stage. Furthermore, the relationship between altered miRNA and clinicopathological features and prognosis of gastric cancer was explored. Among a total of 1,146 miRNAs analyzed, 16 miRNAs were found to be significantly different expressed in tissues from gastric cancer compared to ANTTs (p<0.05). qRT-PCR further confirmed the variation in expression of miR-193b and miR-196a in the validation stage. Down-expression of miR-193b was significantly correlated with Lauren type, differentiation, UICC stage, invasion, and metastasis of gastric cancer (p<0.05), while over-expression of miR-196a was significantly associated with poor differentiation (p=0.022). Moreover, binary logistic regression analysis demonstrated that the UICC stage was a significant risk factor for down-expression of miR-193b (adjusted OR=8.69; 95%CI=1.06-56.91; p=0.043). Additionally, Kaplan-Meier survival curves indicated that patients with a high fold-change of down-regulated miR-193b had a significantly shorter survival time (n=19; median survival=29 months) compared to patients with a low fold-change of down-regulated miR-193b (n=29; median survival=54 months) (p=0.001). Overall survival time of patients with a low fold-change of up-regulated miR- 196a (n=27; median survival=52 months) was significantly longer than that of patients with a high fold-change of up-regulated miR-196a (n=21; median survival=46 months) (p=0.003). Hence, miR-193b and miR-196a may be applied as novel and promising prognostic markers in gastric cancer.

Xiayuxue Decoction ([symbols; see text]) attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4-induced fibrotic liver of mice.

OBJECTIVE: To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice. METHODS: High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively. RESULTS: Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 µg/mL, 50 µg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05). CONCLUSIONS: XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.

Modification of a smoking motivation questionnaire for Chinese medical students.

INTRODUCTION: Smoking prevalence among the medical students is high in China. Therefore, understanding the smoking motivations of medical students is crucial for smoking control, but currently there are no scales questionnaires customized for probing the smoking motivations of medical students. This aim of study was to test and modify a questionnaire for investigating smoking motivations among medical students. METHODS: A cross-sectional survey was conducted among 1,125 medical students at Xuzhou Medical College in China in 2012.The model fit and validity was assessed by confirmatory factor analysis (CFA) and the reliability was tested by single-item reliability, composite reliability, and item-total correlation. RESULTS: The prevalence of smoking was 9.84 % among study population. In the modified scales, the global fit indices identified a CFI value of 0.96, TLI was 0.96, and the RMSEA was 0.063. CFA supported the two dimensional structure of the instrument. The average variance extracted ranged from 0.45 to 0.62. All single-item reliability scores were greater than 0.20, and the composite reliability ranged from 0.74 to 0.91. CONCLUSION: Modified scales could be the preliminary instrument used in evaluating the smoking motivations of medical students. However, it should be further assessed using other forms and methods of validity and reliability, additional motivations of smoking, and the survey of other medical colleges in China.

Xuefuzhuyu decoction inhibition of angiogenesis attenuates liver fibrosis induced by CCl4 in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuefuzhuyu decoction (XFZY) is a well-known traditional Chinese herbal formulation composed of 11 herbs. It is an effective treatment for cardiovascular and chronic liver diseases. The aim of the study is to investigate the role of XFZY on angiogensis in hepatic fibrogenesis, and identify the possible mechanism. MATERIAL AND METHODS: Liver fibrosis was induced by intraperitoneal injection of Carbon tetrachloride (CCl4) in C57BL/6 mice for 6 weeks. From week 4 to week 6, the CCl4-injected mice were randomly divided into three groups, followed by oral administration of Sorafenib, XFZY and water for 3 weeks. Biochemical parameters, hydroxyproline (Hyp) content and histological changes of the liver were determined. The expressions of alpha smooth muscle actin (α-SMA), collagen I, CD31 and vascular endothelial grow factor (VEGF) were assessed by immunohistochemistry and western blot. The protein expressions of VEGFR-2, hypoxia inducing factor (HIF)-1α, asymmetric dimethylarginine (ADMA) and dimethylarginine hydrolase (DDAH) 1 were determined by western blot. The mRNA levels of α-SMA, VEGF and HIF-1α were measured by RT-PCR. RESULTS: Both Sorafenib and XFZY improved biochemical parameters of the liver fibrosis mice. A significant reduction in Hyp content was found in the XFZY-treated mice as well as the Sorafenib-treated mice. Changes in histopathology showed that Sorafenib and XFZY decreased inflammatory and fibrotic stages of the liver in fibrosis mice. Compared to CCl4 model group, Sorafenib and XFZY decreased α-SMA, collagen I, CD31, VEGF, VEGFR-2, HIF-1α and ADMA, and increased the expression of DDAH1. CONCLUSION: XFZY inhibits liver fibrosis not only through inhibiting collagen deposition but also through an antiangiogenic effect on the fibrotic liver. Moreover, the antiangiogenic mechanism of XFZY involves alleviating hypoxia and protecting liver sinusoidal endothelial cell function.

[Effects of Chinese herbal medicine Xiaopi Pill in preventing rats from dimethylnitrosamine-induced liver fibrosis].

OBJECTIVE: To explore the intervention effects of Xiaopi Pill (XPW), a compound traditional Chinese herbal medicine, on the development progress of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. METHODS: Liver fibrosis model was established by intraperitoneal injection of 0.5% DMN 2 mL/kg thrice a week for 4 weeks. Rats were divided into control group given saline and treatment group given XPW during the 3rd week of DMN injections. Rats were sacrificed at the end of the experiment, and then liver histological changes, liver function and mRNA expression of the liver fibrosis-associated markers were observed. RESULTS: (1) At the end of the 2nd and 4th weeks of DMN injection, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) increased significantly in rats (P<0.01 or P<0.05); content of total bilirubin (TBil) increased significantly compared with the normal group until the end of the 4th week (P<0.05); compared with the model group after 4 weeks of DMN injection, the serum levels of ALT, AST, ALP and TBil were decreased remarkably in the XPW-treated group (P<0.01 or P<0.05). (2) The hepatic inflammation and collagen deposition in hepatic tissues increased by different degrees in experimental rats. Parts of pathological changes in the rat liver were found at the end of the 4th week, including a complete round structure of false flocculus round, meantime, the hydroxyproline content of hepatic tissue was increased significantly at the end of the 2nd and 4th weeks (P<0.05). Compared with the 4-week model group, the hepatic inflammation, collagen deposition and hydroxyproline content in hepatic tissues were alleviated dramatically (P<0.05). (3) Compared with the normal and 2nd week groups, protein expression of alpha-smooth muscle actin (α-SMA) was gradually increased, and that of the 4th week group were aggrandized significantly (P<0.01). Compared with the normal group, the mRNA expression of α-SMA, transforming growth factor-ß1 (TGF-ß1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and heme oxygenase-1 (HO-1) was gradually increased. Further changes in above-mentioned abnormalities were found in the model rats at the end of the 4th week (P<0.01); while compared to the 4th week group, protein and mRNA levels of α-SMA and mRNA levels of TGF-ß1, TIMP-1, and HO-1 were decreased significantly in the XPW group (P<0.01 or P<0.05). CONCLUSION: Progressive DMN-induced liver fibrosis in rats can be suppressed by XPW; the mechanism may be associated with inhibition of the activated hepatic stellate cells.

[Dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian decoction in a rat model of CCl4-induced liver fibrosis].

To investigate the dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian (YGJ) Decoction on liver fibrosis induced by CCl4 in rat. Fifty-seven male Wistar rats were randomly divided into a liver fibrosis group (n = 39) and a normal group (n = 18). The liver fibrosis was treated with peritoneal injection of 50% CCl4 for nine weeks. At the end of weeks 3 and 6 of CCl4 treatment, six rats were sacrificed to assess the status of liver fibrosis. At the end of week 7, the remaining -fibrotic rats were randomly divided into an untreated model group (M, n=15) and a YGJ-treated group (n = 12). The YGJ group was administered daily, oral YGJ Decoction for three weeks, concomitant with continued CCl4 treatment. The M group and normal group received the same treatment oral regimen and volume of distilled water. At the end of week 8, four rats in group M were sacrificed to observed the fibrosis status. At the end of week 9, the fibrotic rats were sacrificed for sampling. Liver function, histological changes, contents of hydroxyproline (Hyp) and malondialdehyde (MDA), activity of super oxidase dismutase (SOD) and L-glutathione (GSH), protein expression of heat shock protein (HSP)70, heme oxygenase (HO)-1, transferrin, peroxiredoxin (Prxd) 6 and liver fatty acid binding protein (L-FABP) were detected. Compared with normal group-, the MDA content was increased significantly in M group at week 6 (M: 4.23+/-0.45 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 60.13, P less than 0.01) and week 9 (M: 6.29+/-1.23 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 66.99, P less than 0.01), but the SOD activity was decreased significantly at the same time points [week 6: (M: 196.94+/-39.20 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 11.12, P less than 0.01]); [week 9: (M: 152.2+/-51.65 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 23.11, P less than 0.01)], as were the GSH content [week 6: (M: 48.47+/-7.27 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 6.71, P less than 0.05]]; [week 9: (M: 37.89+/-9.01 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 24.06, P less than 0.01]]. Compared with group M at week 9, the YGH-treated model group had markedly decreased MDA (YGJ: 4.25+/-0.86 nmol/mg vs. M: 6.29+/-1.23 nmol/mg, F = 19.52, P less than 0.01], but significantly increased SOD (YGJ: 198.35+/-46.48 U/mg vs. 152.21+/-51.65 U/mg, F = 4.65, P less than 0.05] and GSH (YGJ: 53.73+/-7.54 nmol/mg vs. M: 37.89+/-9.01 nmol/mg, F = 19.23, P less than 0.01). Compared to normal rats at week 9, group M had significantly higher protein levels of HSP70 (normal: 1.21+/-0.06 vs. M: 0.58+/-0.07, F = 166.87, P less than 0.01) and HO-1 (normal: 1.11+/-0.06 vs. M: 0.58+/-0.06, F = 123.96, P less than 0.01), but significantly decreased levels of Prxd6 (normal: 0.04+/-0.05 vs. M: 1.49+/-0.05, F = 1215.85, P less than 0.01), transferrin (normal: 0.67+/-0.03 vs. M: 1.67+/-0.04, F = 301.35, P less than 0.01), and L-FABP (normal: 0.24+/-0.02 vs. M: 1.44+/-0.14, F = 219.05, P less than 0.01). Compared to group M at week 9, the YGJ treatment group showed significantly reduced HSP70 (YGJ: 0.82+/-0.04 vs. M: 1.21+/-0.06, F = 92.31, P less than 0.01) and HO-1 (YGJ: 0.90+/-0.04 vs. 1.11+/-0.06, F = 26.89, P less than 0.01), but significantly increased Prxd6 (YGJ: 0.88+/-0.11 vs. 0.04+/-0.05, F = 150.17, P less than 0.01), transferrin (YGJ: 1.36+/-0.13 vs. 0.24+/-0.02, F = 237.19, P less than 0.01), and L-FABP (YGJ: 1.04+/-0.12 vs. 0.67+/-0.03, F = 27.53, P less than 0.01). YGJ treatment of fibrotic liver rats reduces lipid peroxidation damage by preventing generation of oxidizing substances.

Ingredients of Huangqi decoction slow biliary fibrosis progression by inhibiting the activation of the transforming growth factor-beta signaling pathway.

BACKGROUND: Huangqi decoction was first described in Prescriptions of the Bureau of Taiping People's Welfare Pharmacy in Song Dynasty (AD 1078), and it is an effective recipe that is usually used to treat consumptive disease, anorexia, and chronic liver diseases. Transforming growth factor beta 1 (TGFß1) plays a key role in the progression of liver fibrosis, and Huangqi decoction and its ingredients (IHQD) markedly ameliorated hepatic fibrotic lesions induced by ligation of the common bile duct (BDL). However, the mechanism of IHQD on hepatic fibrotic lesions is not yet clear. The purpose of the present study is to elucidate the roles of TGFß1 activation, Smad-signaling pathway, and extracellular signal-regulated kinase (ERK) in the pathogenesis of biliary fibrosis progression and the antifibrotic mechanism of IHQD. METHODS: A liver fibrosis model was induced by ligation of the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the IHQD group. IHQD was administrated intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed for sampling of blood serum and liver tissue. The effect of IHQD on the TGFß1 signaling pathway was evaluated by western blotting and laser confocal microscopy. RESULTS: Decreased content of hepatic hydroxyproline and improved liver function and histopathology were observed in IHQD rats. Hepatocytes, cholangiocytes, and myofibroblasts in the cholestatic liver injury released TGFß1, and activated TGFß1 receptors can accelerate liver fibrosis. IHQD markedly inhibited the protein expression of TGFß1, TGFß1 receptors, Smad3, and p-ERK1/2 expression with no change of Smad7 expression. CONCLUSION: IHQD exert significant therapeutic effects on BDL-induced fibrosis in rats through inhibition of the activation of TGFß1-Smad3 and TGFß1-ERK1/2 signaling pathways.