| ObjectiveTo observe the effect of qi-tonifying stasis-removing phlegm-resolving on liver tissue structure,extracellular matrix and nuclear NF-kappa B,TGFbeta 1 in rats with hepatic fibrosis,and to explore the possible mechanism of improving hepatic fibrosis by qi-tonifying stasis-removing phlegm-resolving.MethodsSelecting 10 rats randomly from 105 male SD rats as the normal control group,and the remaining 95 rats were treated by intraperitoneal injection of 40% carbon tetrachloride(CCL4)corn oil solution combined with eating the high fat low protein diet to establish the rat model of hepatic fibrosis.Masson staining was used to test the results of the rat model.The rat model of liver fibrosis were randomly divided into 6 groups: the model group with 10 rats,the colchicine group,the qi-tonifying group,the stasis-removing group,the phlegm-resolving group,qi-tonifying stasis-removing and phlegm-resolving group,9 rats in each group.The rats were given intragastric administration respectively for 12 weeks,while the model group and the normal group were given equal amount of saline.After the intervention,the morphological changes of liver tissues were observed by HE,Masson and Sirius red staining.The levels of ALT,AST and ALB in serum were measured by automatic biochemical analyzer.The content of Col I,P III NP and FN in serum of rats in each group were detected by Enzyme-linked immunosorbent assay.The expression level of TGF-beta 1,T beta R I,Smad3,Samd7,alpha-SMA,HGF,KLF15,NF-kappa B,KLF15 and NF-alpha in liver tissue were detected by real time quantitative PCR.Results1.pathological morphological observation of liver tissueThe results of HE staining showed that hepatocytes were swollen and necrotic,hepatic sinusoids disappeared,and accompanied by extensive infiltration of inflammatory cells in the model group.The infiltration of inflammatory cells in the treatment groups were decreased compared with the model group,maintaining normal hepatic tissue structure.Masson staining and Sirius red staining showed that compared with normal rats,a large number of collagen fibers in the liver tissue of rats of the model group proliferated and deposited,resulting in the formation of false lobules.Compared with the model group,the degree of liver fibrosis in each drug intervention group was reduced.2.Effect of the liver function index on liver fibrosis in ratsCompared with the normal group,the levels of ALT and AST in the model group in rat serum were increased significantly(all P < 0.05),and the ALB level was decreased significantly(all P < 0.05).Compared with the model group,the levels of ALT and AST in each treatment group in rat serum were significantly decreased(all P < 0.05),and the level of ALB was increased significantly(all P < 0.05).Among them,the mixture group had the most significant improvement compared with the other four groups(all P < 0.05).3.Effects of hepatic fibrosis on related indexes of liver fibrosis in ratCompared with the normal group,the contents of Col I,P III,NP and FN in the model group were increased significantly(all P < 0.05).Compared with the model group,the contents of Col I and P III NP in each group were significantly decreased(all P < 0.05).The content of FN in the qi-tonifying group,stasis-removing group and the mixture group were decreased significantly(all P < 0.05).The content of FN in the phlegm-resolving group and colchicine group were not statistically different.Among them,the expression level of Col I,P III NP and FN in the mixture group was the most obvious,which was significantly different from the other four groups(all P < 0.05).4.The relative expressions of TNF-α、NF-κB、IL-1β、KLF15 and TGF-β1、TβRⅠ、Smad3、Samd7、α-SMA、HGF mRNA in liver tissuesCompared with normal group,the expression of NF-kappa B,IL-1 beta,TNF-alpha,TGF-beta 1,T beta R I,Smad3,and alpha-SMA genes in the liver tissues of the model group were significantly up-regulated(all P <0.05).Compared with the model group,the expression of NF-kappa B,IL-1 beta,TNF-a,TGF-beta 1,T beta R I,Smad3,and alpha-SMA genes were down regulated in varying degrees.The expression of KLF15,Samd7 and HGF genes were up-regulated.Among them,the improvement of the indicators in the mixture group was the most obvious,and there was a significant difference compared with the other four groups(all P < 0.05).ConclusionQi-tonifying stasis-removing phlegm-resolving method and colchicine can reduce the degree of fibrosis in liver tissue.Its mechanism may be related to inhibit the expression of NF-kappa B and other inflammatory related factors in liver tissue and the TGF-beta 1/Smad signaling pathway.Although the single agent has good curative effect and varying degrees,it can play an auxiliary role in the combination of the three methods,which is superior and more beneficial to clinical treatment. |
