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Objective To investigate the relationship between nuclear factor(NF)-κB signaling pathway and gender differences in alcoholic liver fibrosis. Methods C57BL/6 N mice at 7-8 weeks of age were randomly divided into: male normal group, male model group, female normal group and female model group of 20 mice each. The normal group was fed with control liquid diet for 8 weeks, and the model group was fed with alcoholic liquid diet for 8 weeks combined with 31.5% ethanol gavage (5g/kg twice a week) to establish an alcoholic liver fibrosis model. The mice were executed at the end of 8 weekends, and the alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity, estradiol (E
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AIM To investigate the protective effects of Dahuang Zhechong (DHZC) Pills (Rhei Radix et Rhizoma,Eupolyphaga seu Steleophaga,Hirudo,etc.) against alcoholic liver fibrosis (ALF) injury in mice and to explore the underlying mechanisms.METHODS C57BL/6 male mice were used to build up ALF injury model,intervened with DHZC Pills.The serum of mice was examined for changes in alanine transaminase (ALT),aspartate aminotransferase (AST),interleukin-6 (IL-6),interleukin-10 (IL-10),interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α).Simultaneously,the deposit of collagen 1 (COL-1) and apoptotic cell death in liver tissues were analyzed by immunofluorescent and TUNEL assay,respectively.The expressions of cleaved caspase-3 (CC3) in livers were measured by Western blot.RESULTS Compared with the model group,the levels of serum ALT,AST,IL-6,IFN-γand TNF-α of mice in DHZC group were decreased significantly.And the level of serum IL-10 of mice in DHZC group was increased significantly.Mice in DHZC group had higher rates of COL-1 deposition and apoptotic cell death in liver tissues than those in the model group.Mice treated with DHZC Pills showed lower expression of CC3.CONCLUSION DHZC Pills confers protection against ALF injury in mice by inhibiting the generation of COL-1 and down-regulating apoptosis of liver cells death as a result of adjusting the levels of inflammatory factors.
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OBJECTIVE:To investigate the preventive and therapeutic effect of Shuganning injection on alcoholic liver fibrosis (ALF) in model rats,and provide experimental basis for its clinical application for alcoholic liver disease. METHODS:50 rats were enrolled and intraperitoneally given mixed liquid of 60% alcohol-corn oil-pyrazole to reduce ALF model. Another 10 rats were enrolled and intraperitoneally given normal saline,as normal control group. After 16 weeks,survived model rats(n=40)were ran-domly divided into model group,positive control group(Anluo huaxian pill 0.75 g/kg,ig),Shuganning injection high-dose,medi-um-dose,low-dose groups(4.8,2.4,1.2 mL/kg,ip),8 in each group. Normal control group and model group were intraperitone-ally injected equal volume of normal saline (5 mL/kg),administration groups were given relevant medicines,once a day,for 8 weeks;and modeling was contiuously conducted at the same time. After administration,body mass of rats was weighed,and the levels of liver function indexes [aspartate aminotransferase(AST),alanine aminotransferase(ALT)] and liver fibrosis indexes [hyal-uronic acid(HA),laminin(LN),type Ⅲ procollagen(PⅢNP),type Ⅳ collagen(Ⅳ-C)] in serum of rats were detected. Liver index of rats was determined and pathological changes of liver tissue were observed. RESULTS:Compared with normal control group,body mass of rats in model group was significantly decreased(P<0.05);liver index,and liver function index,liver fibro-sis index levels in serum were significantly increased (P<0.05 or P<0.01). Liver tissue showed steatosis,hepatocyte vacuoliza-tion,a large number of fibrous tissue deposition around portal areas and other pathological changes. Compared with model group,above-mentioned changes were improved significantly in administration groups (P<0.05 or P<0.01). CONCLUSIONS:Shugan-ning injection can obviously improve liver tissue damage of model rats with ALF,showing certain preventive and therapeutic effect on alcoholic liver disease.
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Objective@#To investigate the effect of interleukin-22 (IL-22) on the activation and proliferation of hepatic stellate cells (HSCs) induced by acetaldehyde, as well as the role of the antioxidant axis Nrf2-keap1-ARE.@*Methods@#Hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and after 24 and 48 hours of acetaldehyde stimulation at various concentrations (25, 50, 100, 200, and 400 μmol/L), MTT assay was used to measure cell proliferation rate to screen out the optimal conditions for model establishment. HSC-T6 cells were treated first with the optimal concentration of acetaldehyde (200 μmol/L) for 24 hours and then with different concentrations of IL-22 (10, 20, and 50 ng/ml) for 24 hours. MTT assay was used to measure cell proliferation, Western blot and cell immunohistochemistry were used to measure the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and α-smooth muscle actin (α-SMA), and spectrophotometry was used to measure the changes in the content of malondialdehyde (MDA) and reduced glutathione (GSH) in culture supernatant. SPSS 17.0 was used for statistical analysis and data were expressed as mean±SD. P < 0.05 was considered statistically significant. A one-way analysis of variance was used for comparison of means between any two groups.@*Results@#HSCs had significantly enhanced proliferation and activation after being treated with acetaldehyde, especially at 200 μmol/L for 48 hours. After the intervention with gradient concentrations of IL-22, the proliferation and activation of HSCs were inhibited in a dose-dependent manner, and the proliferation and migration rates in the 10, 20, and 50 ng/ml IL-22 groups were 14%, 25%, and 35%, respectively (all P < 0.05). The results of Western blot and immunohistochemistry showed that there was no significant difference in the expression of Nrf2 total protein in HSCs between groups, while there was extremely low expression of Nrf2 nucleoprotein in the blank control group. There was increased expression of Nrf2 nucleoprotein after acetaldehyde stimulation (compared with the blank control group, P < 0.05), and after the intervention with gradient concentrations of IL-22, the expression of Nrf2 nucleoprotein was further increased (all P < 0.05). The results of spectrophotometry showed that compared with the blank control group, the model group had increased levels of MDA and GSH in culture supernatant after acetaldehyde stimulation; after the intervention with gradient concentrations of IL-22, there was a significant reduction in the MDA level and a significant increase in the GSH level in a dose-dependent manner (all P < 0.05).@*Conclusion@#The activation and proliferation of HSCs induced by acetaldehyde helps with the successful establishment of an in vitro model of alcoholic liver fibrosis. IL-22 effectively inhibits the activation and proliferation of HSCs induced by acetaldehyde, and its mechanism may be related to promoting Nrf2 nuclear translocation in HSCs and expression of the downstream target gene GSH and increasing the activity of the antioxidant axis Nrf2-keap1-ARE.
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Aim To investigate the influence of down-regulating adenosine A1 receptor and adenosine A2 A receptor gene expression on proliferation and activation of acetaldehyde-induced hepatic stellate cell-T6 cells through siRNA. Methods Alcoholic liver fibrosis in vitro model was constructed by inducing HSC-T6 cells with acetaldehyde. siRNA targeting A1R and A2AR were designed and synthesized according to its mRNA. The siRNA was transfected into rat HSC-T6 cells by li-posome LipofectamineTM 2000. HSC cell proliferation was measured by MTT. The mRNA levels of A1R, A2AR, α-SMA, Collagen I in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. The protein levels of A1R, A2AR, α-SMA, Collagen I were measured by Western blot. Results A1 R and A2 AR siRNA effectively inhibited the cell proliferation, and they also significantly decreased the levels of A1R, A2AR,α-SMA, Collagen I, suggesting that A1 R and A2 AR might be potential target genes in the alcoholic liver fibrosis. Conclusions Silencing A1 R or A2 AR by RNAi can significantly inhibit the HSC proliferation, A1R and A2AR may be potential therapeutic target genes for alcoholic liver fibrosis.
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Objective To investigate the correlation between the distribution of the charactcristics of TCM syndrome of alcoholic liver fibrosis and alcoholic consumption. Methods Our group studied clinical cases in multi-center prospective method. In virtue of Factor Analysis, we explored the characteristics of TCM syndrome of alcoholic liver fibrosis, and the forming characteristics of alcoholic liver fibrosis in inland. Rerults Among the subjects, the characteristics of TCM syndrome of alcoholic liver fibrosis mainly manifested excess syndrome with the common manifestations of deficiency and excess. The visceral position was mainly in liver and spleen, related to gallbladder and stomach. The result suggested that stagnation of Qi stagnation, blood stasis, damp heat, with deficiency of genuine Qi were the interpretation of the causes of alcoholic liver fibrosis. The total alcoholic consumption was one of the important factors which effected on the distribution of thecharacteristics of TCM syndrome of alcoholic liver fibrosis. Total bilirubin had mostly striking effect on the distribution of the characteristics of TCM syndrome. Conclusions The total alcoholic consumption was one of the important factors which effected on the distribution of the characteristics of TCM syndrome of alcoholic liver fibrosis.