| Objective:To explore the effects of Ganshen compound on liver fibrosis in rats of cholestasis liver fibrosis.At the same time,by observing the effects of Ganshen compound on the hepatic stellate cells?HSC?activation and the expression of phospho-extracellular regulated protein kinases ERK?p-ERK?protein and nuclear factor-kappa B?NF-?B?,to reveal the mechanism of anti-liver fibrosis of Ganshen compound.Methods:1.In vivo experiments:Rats were randomly divided into sham operation group,model group and Ganshen compound?GSFF?group.Rats of cholestasis liver fibrosis were subjected to bile duct ligation?BDL?.GSFF group were gave Ganshen compound(25mg.Kg-1)through intragastric administration,and all groups were sacrificed at 2 weeks and 4 weeks.The morphological changes of liver tissue were observed by HE staining.Masson staining was used to evaluate the cotent of fiber tissue of liver.The content of Tumor necrosis factor?TNF-??and Interleukin-1??IL-1??were detected by enzyme-linked immunosorbent assay?ELISA?.Immunoblotting was used to detect changes in the levels of collagen??Col.I?and p-ERK in liver tissue.2.In vitro experiments:The immortalized cell line HSC-T6?expressed as activated?was cultured in vitro.Observed the effect of Ganshen compound on the activation of HSC-T6:?1?The GSFF was diluted into 6 different concentration gradients,and the cell proliferation inhibition was detected by CCK-8 method to screen the optimal concentration of GSFF.?2?The cells were randomly divided into control group,the group of low,medium and high concentration of GSFF.ELISA method was used to examined the content of Col.?in the cell supernatant.?3?In order to further verify the effect of Ganshen compound on the activation of HSC-T6,HSC-T6 was stimulated by transforming growth factor-?1?TGF-?1?(2 ng·ml-1),and then the cells were treated with low,medium and high concentration of GSFF.CCK-8method was used to detected the cell proliferation.The expression of?-smooth muscle actin??-SMA?in HSC-T6 was detected by immunofluorescence.Western Blot was used to obseverd the the expression of p-ERK and NF-?B in HSC-T6.Furthermore,to observe the effect of GSFF on p-ERK,ERK inhibitor PD98059 and agonist platelet derived growth factor-BB?PDGF-BB?were used in the experiments.Results:1.GSFF significantly inhibited the formation of fibrous tissue in liver of BDL rats:?1?HE staining showed that degeneration and necrosis of hepatocyte,proliferation of bile ducts,and a large number of inflammatory cell infiltration in the portal area of BDL rats at the 2nd week.After 4 weeks,degeneration and necrosis of hepatocyte,and bile duct hyperplasia were more pronounced.A large amount of fibrous tissue could be seen in the portal area and necrotic areas.The fibrous tissues were connected to each other to form“pseudolobule”.GSFF could significantly inhibit liver tissue fibrosis at 2 weeks and 4 weeks,relieve liver structural disorder and inflammatory cell infiltration.?2?Masson staining showed that there were only a small amount of fibrous tissu around the central vein and portal tract in the sham rats.After 2weeks of BDL,fibrous tissue proliferated significantly.After 4 weeks,the proliferated fibrous tissues were connected to form pseudolobules.GSFF could significantly inhibit fibrous tissue regeneration at 2 and 4 weeks.?3?The ELISA results showed that the TNF-?and IL-1?contents in the liver tissue increased significantly after 2weeks and 4 weeks of BDL,which was significantly different from the sham group?P<0.01,P<0.001?.Compared with the model group,GSFF significantly inhibited TNF-?and IL-1?synthesis,which were significantly different from the model group?P<0.01,P<0.001?.?4?Western blot results showed that the synthesis of Col.?and p-ERK in the model group were increased significantly after 2 and 4weeks,and there were statistically significant differences compared with the sham group?P<0.001?.GSFF significantly inhibited the synthesis of Col.?and p-ERK at 2 and 4 weeks,which were statistically different compared with the model group?P<0.05 or P<0.01?.2.GSFF inhibited the activation of HSC-T6 and phosphorylation of ERK pathway:?1?After treatment with GSFF for 24h,0.03125,0.0625,0.125,0.25,0.5,1?mol·L-1 of GSFF had obvious inhibitory effect on the proliferation of HSC-T6.Compared with the control group,it was statistically significant?P<0.05?.On this basis,low(0.125?mol·L-1),medium(0.25?mol·L-1)and GSFF high(0.5?mol·L-1)concentration groups were screened out for following experiments.?2?The results of ELISA showed that 0.125,0.25,0.5?mol·L-1 of GSFF could inhibit the secretion of Col.?of HSC-T6,and 0.5?mol·L-11 was statistically different from the control group?P<0.05?.?3?HSC-T6 proliferation was significantly promoted as stimulated by TGF-?1,which was statistically significant compared with the control group?P<0.001?.The medium and high concentrations of GSFF could significantly inhibit the proliferation of cells stimulated by TGF-?1?P<0.05?.?4?The concentration of Col.?in the supernatant of cells and the expression of intracellular?-SMA after stimulation with TGF-?1 were significantly increased,and there was a statistically significant difference compared with the control group?P<0.01?.Compared with the TGF-?1 group,the three concentrations of GSFF can down-regulate synthesis of collagen and the production of?-SMA and there were statistically significance?P<0.05,P<0.01?.?5?GSFF has a down-regulation effect on activation of NF-?B/ERK signaling pathway in activated hepatic stellate cells:Compared with the control group,the expression of p-ERK and NF-?B in the low,middle and high concentration groups of GSFF was significantly decreased,and there were significant differences?P<0.05,P<0.01,P<0.001?.PDGF-BB promoted the expression of p-ERK protein in cells?P<0.05?.The content of p-ERK protein in HSC-T6 treated with PD98059 and PDGF-BB was significantly lower than that in PDGF-BB group?P<0.001?.Conclusions:1.GSFF could improve liver tissue morphology,reduce collagen fibrosis synthesis,inhibit liver inflammation,and achieve anti-fibrotic effect.The mechanism may be relataed with inhibiting the inflammatory response and inhibiting ERK-related pathways.2.GSFF could inhibit the activation of HSC-T6,the secretion of Col.I and the expression of?-SMA,suggesting that one of the intrinsic mechanisms of anti-fibrosis of GSFF may be inhibiting HSC activation.3.GSFF could inhibit the exprenssion of NF-?B down-regulate and the phosphorylation level of ERK in HSC-T6,which indicated that GSFF could alleviate inflammation and then inhibit phosphorylation of ERK and HSC activation. |
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