| Objective:In this study,the therapeutic effect of ASAs(Alstonia scholaris total alkaloids)against the extracellular matrix deposition,anti-inflammatory and hepatic stellate cell activation on the liver fibrosis was induced by thioacetamide(TAA).To observe the effect of ASAs in scutellaria chinensis on liver fibrosis,reveal the intrinsic relationship between ASAs liver fibrosis,hepatic stellate cells and related signaling factors,and to explore the related signaling pathways of ASAs in the treatment of liver fibrosis To provide new methods and ideas for the prevention and treatment of liver fibrosis by traditional Chinese medicine.Method:Experiment 1 100 male SD rats were randomly divided into 6 groups.The normal group: 20,intraperitoneal injection of 0.9% saline,the injection time is the same as the TAA injection time,free to eat standard feed and water,corresponding to ASAs group corresponding to distilled water;model group;10,intraperitoneal injection of TAA solution(According to the weight of rats,200mg/kg),corresponding to the lamp base total alkali group,the distilled water;carboxymethyl cellulose sodium(Carboxymethyl cellulose sodium,CMC-Na)group: 10,intraperitoneal injection of TAA solution,with the lamp leaf total The alkali group was administered with 0.5% CMC-Na solution;the low concentration group;20 rats were given daily spleen total alkal administration(dose 7.5 mg/kg)from the second day of modeling;medium concentration group: 20 Only,ASAs were administered daily(dose was 15 mg/kg);in the high concentration group: 20,ASAs were administered daily(dose of 30 mg/kg).The rats were weighed weekly,and the serum and liver samples of each group were collected for 4 weeks.The serum and liver samples of each group were collected for 9 weeks.The serum and liver samples of each group were collected for 14 weeks.The net weight of the liver was weighed;the serum biochemical parameters were detected by ELISA kit;the pathological changes of liver tissue were observed by HE and Masson staining;the rats of each group were detected by quantitative real-time PCR(PCR).Vimentin(vimentin),COL1A1(type I collagen α1 chain),COL1A2(type I collagen α2 chain),TIMP-1(metalloproteinase tissue inhibitor),TGF-β1(transforming growth factor-β),PDGF in liver tissue(Platelet-derived growth factor)andα-SMA(α-smooth muscle actin)were expressed at the mRNA level;protein expression of α-SMA in liver tissue of each group was detected by Western blot.Experiment 2 Rat hepatic stellate cells(HSC-T6)were cultured and subcultured into normal group,DMSO group,10 ug/ml group,1 ug/ml group,1 ng/ml group,0.1 ng/ml group and 0.01ng/ml group.The cells were uniformly seeded in a 96-well cell culture plate,and different concentrations of the lamp leaf total alkali solution were added to each group,and cell proliferation was detected by Days,Day 1,Day 2,Day 3,and Day 7 by MTS method.The optimal concentration of cell proliferation was used to affect the cells.The expression of TIMP-1 and α-SMA in each group was detected by qRT-PCR.The protein expression of α-SMA was detected by Western blot.Results:Experiment 1 The changes of serum ALT,AST,TBIL and IL-6 levels in rats at the first 14 weeks of experiment: the indexes of the model group and CMC-Na group were significantly higher than those of the normal group,the difference was statistically significant(P<0.01);Compared with the model group,the concentration of ASAs in each concentration group decreased significantly,and the difference was statistically significant(P<0.01).The results of HE and Masson staining in14-week liver of rats showed that the hepatic cords of the normal group were neatly arranged,the liver cells were not degenerated or necrotic,the cells were well-proportioned,the nuclei were full and round,and there was no inflammatory cell infiltration in the portal area.The fibrosis of the tube area is enlarged,the fiber spacing is accompanied by lobular structure disorder,part of the liver pseudolobule is formed,and it is in the S4 stage of liver fibrosis;the fibrosis of the liver tissue area of the high concentration lamp stage leaf group is enlarged,the fiber spacing is formed,the lobular structure is retained,and the liver fiber is S2 phase.The results of qRT-PCR detection of Vimentin,COL1A1,COL1A2,TIMP-1,TGF-β1,PDGF and α-SMA in the liver tissues of rats in each group showed that the model group and the CMC-Na control group were compared with the normal group.Significantly enhanced and the difference was statistically significant(P<0.01);the concentration of ASAs in the lamp stage was significantly lower than that in the model group,and the difference was statistically significant(P<0.01).The results of Western blot analysis showed that the indexes of the model group and the CMC-Na control group were significantly increased compared with the normal group(P<0.01).The concentration group of ASAs in the lamp stage Compared with the model group,the above indicators were significantly lower and the differences were statistically significant(P<0.05,P<0.01).Experiment 2 The results of cell experiments showed that from the second day,there was a significant difference in the cell proliferation level of the total alkali solution of the lamp stage leaf:compared with the normal group,the cell proliferation after the lamp stage treatment was significantly reduced.On the seventh day,the difference in cell proliferation between the control group and the lamp stage treatment group was the most significant.The difference between the normal group and the 10 ug/ml group was statistically significant(P<0.01);the normal group and the 0.01 ng/ml group.In comparison,the difference was statistically significant(P < 0.05).The results of qRT-PCR detection of TIMP-1 and α-SMA in rat hepatic stellate cells were significantly lower in the 10ug/ml group than in the normal group(P<0.01).Western blot analysis showed that the index of the 10ug/ml group was significantly lower than that of the normal group(P<0.01).Conclusion:1 ASAs have the effect of promoting TAA-induced rat liver fibrosis recovery and slowing the progression of TAA-induced liver fibrosis in rats.ASAs has the function of preventing liver fibrosis.2 The mechanism of ASAs treatment of liver fibrosis is related to the following ways:protection of HC(hepatic parenchymal cells);inhibition of HSC(hepatic stellate cells)activation;promotion of ECM(extracellular matrix)degradation;inhibition of TIMP-1 activity;inhibition TGF-β1 and PDGF activity.3 The total alkali solution of lampholder leaves can inhibit the proliferation of rat hepatic stellate cells;the total alkali solution of lamp leaf may inhibit the proliferation of HSC and reduce the apoptosis of HC by inhibiting the activity of TIMP-1 and α-SMA.The role of fibrosis. |
PDF Full Text Request