肝硬化门脉高压时的肝星细胞和一氧化氮(文献综述)

肝星细胞是肝细胞重要组成部分 ,其收缩及舒张对门脉血流有重要的调节作用。一氧化氮是肝硬化门静脉高压时活跃的介质 ,对肝星细胞的收缩及舒张产生作用 ,对它们相互关系的研究有助于门静脉高压的治疗。

TGF-β1诱导肝星状细胞活化调控肝癌的炎症和免疫反应

目的通过TGF-β1诱导活化的肝星状细胞与肝癌细胞SMMC7721共培养,观察肝癌的炎症和免疫变化。方法选取厦门大学附属福州第二医院体外培养人肝星状细胞LX-2,用10μg/L TGF-β1处理LX-224 h,取活化LX-2上清液共培养SMMC7721细胞12 h后,用Disitetide处理共培养细胞,流式细胞术观察CD4+CD25+T细胞干预前后的表达变化,ELISA观察白细胞介素-8(IL-8)、白细胞介素-10(IL-10)干预前后的表达情况。结果10μg/L的TGF-β1刺激LX-2后,α-SMA基因和蛋白水平表达均明显增加。活化的LX-2上清液与SMMC7721共培养后,CD_(4)^(+)CD_(25)^(+)的表达比例较SMMC7721中明显增加(P=0.003),IL-8和IL-10的表达水平也明显上调(P<0.001),用Disitetide抑制TGF-β1后CD_(4)^(+)CD_(25)^(+)的表达比例较共培养组中明显减少(P=0.025),IL-8和IL-10的表达水平也明显下调(P<0.001)。结论TGF-β1可以诱导肝星状细胞活化从而促进肝癌细胞的炎症和免疫反应。

抗纤软肝冲剂药物血清对肝星状细胞胶原代谢及其相关基因表达的影响

目的 :从细胞分子学水平探讨抗纤软肝冲剂抗肝纤维化的作用机制。方法 :抗纤软肝冲剂药物血清温育传一代HSC ,以生理盐水和秋水仙碱为对照。ELISA测定细胞上清Ⅰ型胶原 ,并测定细胞层总蛋白以校正胶原含量 ;半定量法RT -PCR法检测Ⅰ型胶原、间质胶原酶 (MMP1)的mRNA表达。结果 :抗纤软肝冲剂组细胞上清中的Ⅰ型胶原含量明显低于其它两组 (P <0 .0 1) ;能明显抑制细胞的Ⅰ型胶原mRNA表达 (P <0 .0 1) ,且能促进MMP1的mRNA表达 (P <0 .0 1)。结论 :抗纤软肝冲剂能明显抑制HSC的胶原基因表达和促进间质胶原酶基因表达 ,从而可达到抑制胶原合成 ,促进胶原降解 ,这可能是抗纤软肝冲剂抗肝纤维化的细胞分子学机制之一。

Female hepatology:Favorable role of estrogen in chronic liver disease with hepatitis B virus infection

Chronic hepatitis B virus (HBV) infection is the most common cause of hepatic fibrosis and hepatocellular carcinoma (HCC),mainly as a result of chronic necroinflammatory liver disease. A characteristic feature of chronic hepatitis B infection,alcoholic liver disease and nonalcoholic fatty liver disease (NAFLD) is hepatic steatosis. Hepatic steatosis leads to an increase in lipid peroxidation in hepatocytes,which,in turn,activates hepatic stellate cells (HSCs). HSCs are the primary target cells for inflammatory and oxidative stimuli,and these cells produce extracellular matrix components. Chronic hepatitis B appears to progress more rapidly in males than in females,and NAFLD,cirrhosis and HCC are predominately diseases that tend to occur in men and postmenopausal women. Premenopausal women have lower hepatic iron stores and a decreased production of proinflammatory cytokines. Hepatic steatosis has been observed in aromatase-deficient mice,and has been shown to decrease in animals after estradiol treatment. Estradiol is a potent endogenous antioxidant which suppresses hepatic fibrosis in animal models,and attenuates induction of redox sensitive transcription factors,hepatocyte apoptosis and HSC activation by inhibiting a generation of reactive oxygen species in primary cultures. Variant estrogen receptors are expressed to a greater extent in male patients with chronic liver disease than in females. These lines of evidence suggest that the greater progression of hepatic fibrosis and HCC in men and postmenopausal women may be due,at least in part,to lower production of estradiol and a reduced response to the action of estradiol. A better understanding of the basic mechanisms underlying the sex-associated differences in hepatic fibrogenesis and carciogenesis may open up new avenues for the prevention and treatment of chronic liver disease.

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM:To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells (HSC) in vitro. METHODS: The study in vitro was carried out in the culture of HSC lines. Various concentrations of Yigan Decoction were added and incubated. Cell proliferation was detected with MTT colorimetric assay. Cell apoptosis was detected by electron microscopy, flow cytometry and TUNEL. RESULTS: The proliferation of HSC was inhibited by Yigan Decoction, which depending on dose and time significantly. The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L(-1)) were 21.62% and 40.54% respectively, significantly lower than that of normal control group(P【0.01). The HSC proliferation rates of groups at the end concentrations 36, 18 and 9(g.L(-1)) were 54.05%, 45.95% and 51.35% respectively, lower than that of control group (P【0.05). When the end concentration was 4.5 g.L(-1), the proliferation rate was 83.78%, which appeared no significant differences compared with control group. At the same concentrations of 18 g.L(-1), the inhibitory effects of Yigan Decoction at 24 h, 48 h and 72 h time point were observed, the effects were time-dependent, and reached a peak at 72 h. Meanwhile, it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose-dependent and time-dependent. The apoptosis index(AI) was detected by TUNEL. After Yigan Decoction had been incubated for 48 h at the end concentration of 18 g.L(-1), the AI (14.5+/-3.1)% was significantly higher than that of control group (4.3+/-1.3)% (P【0.01). When visualized under transmission electron microscopy, some apoptotic stellate cells were found, i.e. dilated endoplasmic reticulum, irregular nuclei, chromatin condensation and heterochromatin ranked along inside of nuclear membrane. By flow cytometry detection, after HSC was treated with Yigan Decoction at different concentrations of 36, 18 and 9(g.L(-1)) for 48 h, AI (%) were 13.3+/-3.2, 10.7+/-2.7 and 10.1+/-2.5 respectively, which were significantly higher than that of control group(4.1+/-1.9) (P【0.01). At the same concentration of 18 g. L(-1) for 24h, 48 h and 72 h, AI (%) were 9.3+/-1.8,10.7+/-2.7 and 14.6+/-4.3 respectively, which were significantly higher than that of control group (P【0.01). CONCLUSION: Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently, which may be related to its mechanism of antifibrosis.

Expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic stellate cells during rat hepatic fibrosis and its intervention by IL-10

AIM: To investigate the expression of matrix metallopr-oteinase-2 and tissue inhibitor of metalloproteinase-1 in hepatic fibrosis and the antifibrogenic role of exogenous interleukin-10 (IL-10). METHODS: Hepatic fibrosis was induced by CCI4 administration and 60 male Sprague-Dawley rats were randomly divided into normal control group (group N, 8 rats), CCI4-induced group (group C, 28 rats) and IL-10-treated group (group I, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through portal vein catheter and the suspension was centrifuged by 11% Nycodenz density gradient to isolate hepatic stellate cells (HSCs). RT-PCR was used to analyze mRNA of MMP-2 and TIMP-1 from freshly isolated cells. Densitometric data were standardized with β-actin signals. Immunocytochemistry was performed to detect MMP-2 and TIMP-1 expression in HSC cultured for 72 h. RESULTS: Compared to group N in the 7th wk, MMP-2 and TIMP-1 mRNA increased in group C (P= 0.001/0.001) and group I (P= 0.001/0.009). The level of MMP-2 and TIMP-1 mRNA in group I was significantly lower than that in group C (P= 0.001/0.001). In the 11th wk, MMP-2 mRNA in group I was still lower than that in group C (P = 0.005), but both dropped compared with that in the 7th week (P = 0.001/0.004). TIMP-1 mRNA in group I was still lower than that in group C (P= 0.001), and increased in group C (P= 0.001) while decreased in group I (P = 0.042) compared with that in the 7th wk. Same results were found by immunocytochemistry. CONCLUSION: Expression of MMP-2 and TIMP-1 is increased in hepatic fibrosis. IL-10 exhibits an antifibrogenic effect by suppressing MMP-2 and TIMP-1 expression.

Notch3 regulates the activation of hepatic stellate cells

AIM： To investigate whether Notch signaling is involved in liver fibrosis by regulating the activation of hepatic stellate cells （HSCs）.METHODS： Immunohistochemistry was used to detect the expression of Notch3 in fibrotic liver tissues of patients with chronic active hepatitis. The expression of Notch3 in HSC-T6 cells treated or not with transforming growth factor （TGF）-β1 was analyzed by iramunofluorescence staining, The expression of Notch3 and myofibroblastic marker （z-smooth muscle actin （（z-SMA） and collagen 1 in HSC-T6 cells transfected with pcDNA3. I-N3ICD or control vector were detected by Western blotting and immunofluorescence staining. Moreover, effects of Notch3 knockdown in HSC-T6 by Notch3 siRNA were investigated by Western blotting and immunofluorescence staining.RESULTS： The expression of Notch3 was significantly up-regulated in fibrotic liver tissues of patients withchronic active hepatitis, but not detected in normal liver tissues. Active Notch signaling was found in HSC-T6 cells. TGF-β1 treatment led to up-regulation of Notch3 expression in HSC-T6 cells, and over-expression of Notch3 increased the expression of α-SMA and collagen I in HSC-T6 without TGF-β1 treatment. Interestingly, transient knockdown of Notch3 decreased the expression of myofibroblastic marker and antagonized TGF-β1 induced expression of α-SMA and collagen I in HSC-T6.CONCLUSION： Notch3 may regulate the activation of HSCs, and the selective interruption of Notch3 may provide an anti -fibrotic strategy in hepatic fibrosis.

Role of ethanol in the regulation of hepatic stellate cell function

Evidence has accumulated to suggest an important role of ethanol and/or its metabolites in the pathogenesis of alcohol-related liver disease. In this review, the fibrogenic effects of ethanol and its metabolites on hepatic stellate cells (HSCs) are discussed. In brief, ethanol interferes with retinoid metabolism and its signaling, induces the release of fibrogenic cytokines such as transforming growth factor β-1 (TGFβ-1) from HSCs, up-regulates the gene expression of collagen I and enhances type I collagen protein production by HSCs. Ethanol further perpetuates an activated HSC phenotype through extracellular matrix remodeling. The underlying pathophysiologic mechanisms by which ethanol exerts these pro-fibrogenic effects on HSCs are reviewed.

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM： TO study the effects of interleukin-10 （IL-10） on the expression of o-smooth muscle actin （α-SMA）, nuclear factor-κB（NF-κB） and Fas/Fas ligand （FasL） in hepatic stellate cells of experimental rats with hepatic fibrosis. METHODS： Sixty clean SD rats were randomly divided into control group （group N）, liver fibrotic group （group C） and IL-10 treatment group （group I）. Control group received intraperitoneal injection of saline （2ml·kg^-1）, twice a week. Fibrotic group was injected intraperitoneally with 50% carbon tetrachloride （CCh） （2 ml·kg^-1）, twice a week. IL-10 treatment group was given IL-10 at a dose of 4 pg·kg^-1 20 minutes before CCl4 administration from the third week. Hepatic stellate cells （HSCs） were isolated from these rats at the seventh and eleventh weeks during the course of liver fibrosis, respectively. The expression of α-SMA and NF-κB in HSCs was measured by S-P immunohistochemistry. The expression of Fas and FasL mRNA was measured by RT-PCR. Furthermore, liver tissues were harvested from three groups at the same time. RESULTS： The CCh- induced experimental rat hepatic fibrosis model was established successfully. The purity of extracted hepatic stellate cells was about 95% and the yield of hepatic stellate cells was 1.2-2.3×10^6/g liver tissue averagely. The positive expression of α-SMA and NF-κB was 36.5% and 28.5% respectively in group N. The positive levels of α-SMA and NF-κB were increased significantly in group C compared to group N （P〈0.01）. The positive signals decreased significantly （P〈0.05） in group I. In the 11^th week, the HSCs of group I became round with visible pyknotic nuclei. The expression of NF-κB in group C was significantly increased in a timedependentmanner （P〈0.01）, but there was no difference in the α-SMA expression （P〉0.05）. The mRNA of Fas and FasL in group C was significantly increased in a timedependent manner compared to that in control group. After treated with IL-10, the expression level of Fas and FasL was higher in group I than in group C. CONCLUSION： The positive expression of α-SMA and NF-κB in hepatic stellate cells is decreased by ectogenic IL-10 in liver fibrosis induced by CCh. The expression of Fas and FasL is increased in the course of liver fibrosis, and is further increased by IL-10. IL-10 could inhibit the activation of HSCs and cause apoptosis of activated HSCs.

Phosphatidylinositol 3-kinase/Akt pathway regulates hepatic stellate cell apoptosis

AIM: To investigate the role of phosphatidylinositol 3-kinase (PI 3-K)/Akt signaling pathway in the balance of HSC activation and apoptosis in rat hepatic stellate cells (HSC). METHODS: An activated HSC cell line was used in this study. LY 294002, the PI 3-K/Akt signal pathway block-er was used to investigate the molecular events on apoptosis in HSC and to interpret the role of this path-way in HSC apoptosis. Immunocytochemistry, Western blot and reverse transcription polymerase chain reac-tion (RT-PCR) analysis were applied to detect the ex-pression of PI 3-K, and simultaneously phosphorylated-Akt (p-Akt) and total-Akt were determined by Western blot. The HSC apoptosis was examined by annexin-V/ propidium iodide double-labelled flow cytometry and transmission electron microscopy. RESULTS: The apoptosis rates in LY 294002 (30.82% ± 2.90%) and LY 294002 + PDGF-BB (28.16% ± 2.58%) groups were signif icantly increased compared with those of control (9.02% ± 1.81%) and PDGF-BB (4.35% ± 1.18%). PDGF-BB augmented PI 3-K and p-Akt expres-sion. LY 294002 signif icantly reduced the contents of PI 3-K and p-Akt. mRNA transcription evaluated by RT-PCR showed similar tendencies as protein expression. CONCLUSION: Inhibition of PI 3-K/Akt signaling path-way induces apoptosis in HSC.

Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells

Despite the initial belief that non-alcoholic fatty liver disease is a benign disorder, it is now recognized that fibrosis progression occurs in a significant number of patients. Furthermore, hepatic steatosis has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of other liver diseases. Here, we established an in vitro model to study the effect of hepatic lipid accumulation on hepatic stellate cells （HSCs）, the central mediators of liver fibrogenesis. Primary human hepatocytes were incubated with the saturated fatty acid palmitate to induce intracellular lipid accumulation. Subsequently, human HSCs were incubated with conditioned media （CM） from steatotic or control hepatocytes. Lipid accumulation in hepatocytes induced the release of factors that accelerated the activation and proliferation of HSC, and enhanced their resistance to apoptosis, largely mediated via activation of the PI-3-kinase pathway. Furthermore, CM from steatotic hepatocytes induced the expression of the profibrogenic genes TGF-β, tissue inhibitor of metallo-proteinase-1 （TIMP-1）, TIMP-2 and matrix-metallo-proteinase-2, as well as nuclear-factor κB-dependent MCP-1 expression in HSC. In summary, our in vitro data indicate a potential mechanism for the pathophysiological link between hepatic steatosis and fibrogenesis in vivo. Herewith, this study provides an attractive in vitro model to study the molecular mechanisms of steatosis-induced fibrogenesis, and to identify and test novel targets for antifibrotic therapies in fatty liver disease.

Effect of IL-10 on the expression of HSC growth factors in hepatic fibrosis rat

AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor （PDGF） of hepatic stellate cells （HSCs） of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by CCI4 administration intra-peritoneally. Sixty clean male Sprague-Dawley （SD） rats were randomly divided into three groups： normal control group （GN, 8 rats）, hepatic fibrosis model group （GC, 28 rats） and IL-10 treated group （GI, 24 rats）. At the beginning of the 7^th and 11^th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS： Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7^th and 11^th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN （P = 0.001/0.042, 0.001/0.001, 0.001/0.001） and GI （P= 0.001/0.007, 0.002/0.001, 0.001/0.001）. For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7^th wk （P= 0.005） and 11^th wk （P= 0.049）. For HGF, mRNA level in GI decreased compared with GN at the 7^th wk （P = 0.001） and 11^th wk （P= 0.021）. Between these two time points, TGF-β1 expression at the 7^th wk was higher than that of the 11^th wk （P = 0.049）, but for EGF, the former was lower than the latter （P = 0.022）. As for PDGF mRNA, there was no significant difference between thesegroups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION： The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

Tetrandrine stimulates the apoptosis of hepatic stellate cells and ameliorates development of fibrosis in a thioacetamide rat model

AIM： To investigate the therapeutic effect of tetrandrine on liver fibrosis induced by thioacetamide in rats in vivo and in vitro. METHODS： In vitro study： we investigated the effect of tetrandrine on the apoptosis of rat hepatic stellate cells transformed by simian virus 40 （T-HSC/CI-6）, which retains the features of activated cells. In vivo study： hepatic fibrosis was induced in rats by thioacetamide. Tetrandrine was given orally to rats at doses of 5, 10 or 20 mg/kg for 4 wk compared with intraperitoneal injection of interferon-r. RESULTS： In vitro study： 5, 10 or 25 μg/mL of tetrandrine-induced activation of caspase-3 in t-HSC/CI-6 cells occurred dose-dependently. In vivo study： tetrandrine treatment as well as interferon-r significantly ameliorated the development of fibrosis as determined by lowered serum levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT）, total bilirubin （T-Bil） and the levels of liver hydroxyproline （Hyp）, hyaluronic acid （HA）, laminin （LN） and also improved histological findings. The effects of tetrandrine at the concentration of 20 mg/kg were better than the other concentration groups. CONCLUSION： Tetrandrine promotes the apoptosis of activated HSCs in vitro. Tetrandrine administration can prevent liver fibrosis and liver damage induced by thioacetamide in rats in vivo, indicating that it might exert a direct effect on rat HSCs.

Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-pi and a-smooth muscle actin in rats with hepatic fibrosis

AIM:To investigate the effect of interferon-a (IFN-α) on preventing or reversing hepatic fibrosis in rat experimental model induced by CCI4. METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-a in saline daily at the doses of 1× 105 U for 6 wk, group D (IFN-a treatment, n = 24) treated with intra-muscular injection of IFN-a in saline daily at the doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemical and electron microscopic studies for the expressions of transforming growth factor-pi (TGF- μ41) and α-smooth muscle actin (α-SMA). RESULTS: The expressions of TGF-pl, the number of activated hepatic stellate cells and a-SMA in hepatic tissue of group C were significantly less than those of group B (P<0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P<0.01). CONCLUSION: IFN-a can inhibit the production of TGF-pl, decrease HSC activation and stimulate its apoptosis.

Role of iron in hepatic fibrosis: One piece in the puzzle

Iron is an essential element involved in various biological pathways. When present in excess within the cell, iron can be toxic due to its ability to catalyse the formation of damaging radicals, which promote cellular injury and cell death. Within the liver, iron related oxidative stress can lead to fibrosis and ultimately to cirrhosis. Here we review the role of excessive iron in the pathologies associated with various chronic diseases of the liver. We also describe the molecular mechanism by which iron contributes to the development of hepatic fibrosis.

Wnt5a participates in hepatic stellate cell activation observed by gene expression profile and functional assays

AIM:To identify differentially expressed genes in quiescent and activated hepatic stellate cells(HSCs)and explore their functions.METHODS:HSCs were isolated from the normal Sprague Dawley rats by in suit perfusion of collagenase and pronase and density Nycodenz gradient centrifugation.Total RNA and mRNA of quiescent HSCs,and cultureactivated HSCs were extracted,quantified and reversely transcripted into cDNA.The global gene expression profile was analyzed by microarray with Affymetrix rat genechip.Differentially expressed genes were annotated with Gene Ontology(GO)and analyzed with Kyoto encyclopedia of genes and genomes(KEGG)pathway using the Database for Annotation,Visualization and Integrated Discovery.Microarray data were validated by quantitative real-time polymerase chain reaction(qRTPCR).The function of Wnt5a on human HSCs line LX-2 was assessed with lentivirus-mediated Wnt5a RNAi.The expression of Wnt5a in fibrotic liver of a carbon tetrachloride(CCl4)-induced fibrosis rat model was also analyzed with Western blotting.RESULTS:Of the 28 700 genes represented on this chip,2566 genes displayed at least a 2-fold increase or decrease in expression at a P<0.01 level with a false discovery rate.Of these,1396 genes were upregulated,while 1170 genes were downregulated in culture-activated HSCs.These differentially expressed transcripts were grouped into 545 GO based on biological process GO terms.The most enriched GO terms included response to wounding,wound healing,regulation of cell growth,vasculature development and actin cytoskeleton organization.KEGG pathway analysis revealed that Wnt5a signaling pathway participated in the activation of HSCs.Wnt5a was significantly increased in cultureactivated HSCs as compared with quiescent HSCs.qRTPCR validated the microarray data.Lentivirus-mediated suppression of Wnt5a expression in activated LX-2 resulted in significantly impaired proliferation,downregulated expressions of typeⅠcollagen and transforming growth factor-β1.Wnt5a was upregulated in the fibrotic liver of a CCl4-induced fibrosis rat model.CONCLUSION:Wnt5a is involved in the activation of HSCs,and it may serve as a novel therapeutic target in the treatment of liver fibrosis.

Hepatic stellate cells and innate immunity in alcoholic liver disease

Constant alcohol consumption is a major cause of chronic liver disease, and there has been a growing concern regarding the increased mortality rates worldwide. Alcoholic liver diseases (ALDs) range from mild to more severe conditions, such as steatosis, steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The liver is enriched with innate immune cells (e.g. natural killer cells and Kupffer cells) and hepatic stellate cells (HSCs), and interestingly, emerging evidence suggests that innate immunity contributes to the development of ALDs (e.g. steatohepatitis and liver fibrosis). Indeed, HSCs play a crucial role in alcoholic steatosis via production of endocannabinoid and retinol metabolites. This review describes the roles of the innate immunity and HSCs in the pathogenesis of ALDs, and suggests therapeutic targets and strategies to assist in the reduction of ALD.

Arg-gly-asp-mannose-6-phosphate inhibits activation and proliferation of hepatic stellate cells in vitro

AIM： To investigate the effect of arg-gly-asp-mannose-6 phosphate （RGD-M6P） on the activation and proliferation of primary hepatic stellate cells in vitro. METHODS： Hepatic steUate cells （HSCs） were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum （FBS/DMEM） before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor 131, M6P, RGD, or RGD- M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle α-actin （α-SMA） was detected by immunocytochemistry, type Ⅲ procollagen （PCⅢ） in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry. RESULTS： RGD-M6P significantly inhibited the morphological transformation and the α-SMA and PC Ⅲ expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6R CONCLUSION： The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFβ1, suppresses the expression of PCIII and decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs, which may be a new approach to the prevention and treatment of liver fibrosis.

Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

AIM： To investigate the potential mechanism of Arg- Gly-Asp （RGD） peptide-labeled liposome loading oxy- matrine （OM） therapy in CCI4-induced hepatic fibrosis in rats. METHODS： We constructed a rat model of CCh- induced hepatic fibrosis and treated the rats with dif- ferent formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phospha- tase, hepatic histopathology （hematoxylin and eosin stain and Masson staining） and fibrosis-related gene expression of matrix metallopeptidase （MMP）-2, tis- sue inhibitor of metalloproteinase （TIMP）-I as well as type I procollagen via quantitative real-time poly- merase chain reaction. To detect cell viability and apop- tosis of hepatic stellate cells （HSCs）, we performed 3-（4,5）-dimethylthiahiazo（-z-yl）-3,5-diphenytetrazoli- umromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy. RESULTS： OM attenuated CCh-induced hepatic fibro- sis, as defined by reducing serum alkaline phosphatase （344.47± 27.52 U/L vs 550.69 ± 43.78 U/L, P 〈 0.05）, attenuating liver injury and improving collagen deposits （2.36% ± 0.09% vs 7.70% ±0.60%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and en- hanced the therapeutic effect of OM in terms of serum alkaline phosphatase （272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P 〈 0.05）, liver injury, collagen deposits （0.26%± 0.09% vs 2.36% ± 0.09%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. Moreover, in vitro assay demonstrated that RGD en- hanced the effect of OM on HSC viability and apoptosis. CONCLUSION： OM attenuated hepatic fibrosis by in- hibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting effi- ciency for HSCs and the therapeutic effect.

Insulin-like growth factor binding protein-7 induces activation and transdifferentiation of hepatic stellate cells in vitro

AIM:To investigate the role of insulin-like growth factor binding protein-7 (IGFBP-7) in the activation and transdifferentiation of hepatic stellate cells (HSC) in vitro.METHODS:Rat HSC-T6 cells were cultured in separate dishes and treated with various concentration of transforming growth factor (TGF)-β1,IGFBP-7 or antiIGFBP-7 antibody for 24 h.The supernatant or a cytoplasm suspension was obtained from cultured HSC,followed by transfer of cells to form cell-coated dishes.Immunocytochemistry and Western blotting were used to analyze the expression of IGFBP-7 induced by TGF-β1 and the level of fibronectin,collagen and α-smooth muscle actin (SMA).The pro-apoptotic effect of antiIGFBP-7 antibody was determined by flow cytometry.RESULTS:Immunocytochemistry and Western blotting revealed that the expression of IGFBP-7 in TGF-β1 treated HSC was significantly up-regulated compared to that in the control group.In addition,fibronectin,collagen and α-SMA also showed enhanced expression in accordance with the transdifferentiation process in a dose-dependent manner to some extent.Moreover,flow cytometry suggested that anti-IGFBP-7 antibody induced apoptosis of activated HSC,which is responsible for the development of liver fibrosis,and may represent a novel pathway and target for therapeutic intervention.CONCLUSION:IGFBP-7 showed increased expression in activated HSC and played an important role in the activation and transdifferentiation process of HSC.AntiIGFBP-7 antibody may ameliorate liver fibrogenesis.