柴芪益肝方对肝纤维化小鼠的保护作用机制研究

Protective mechanism of Chaiqiyigan formula on hepatic fibrosis in mice

目的探讨柴芪益肝方抗肝纤维化的作用机制。方法选取50只无特定病原体级C57BL/6雄性小鼠通过随机数字表法分成空白对照组、模型组、柴芪益肝方低、高剂量组、水飞蓟素阳性对照组,各10只。除空白对照组外,各组小鼠腹腔注射15%的四氯化碳橄榄油诱导肝纤维化模型。从造模第1天起,空白对照组和模型组以等体积的0.5%羧甲基纤维素钠溶液灌胃,其余3组每天分别灌胃低、高剂量柴芪益肝方和水飞蓟素。小鼠处死后,观察肝脏形态,苏木精-伊红、Masson、天狼星红染色观察肝组织和胶原纤维变化,生化法分析血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,酶联免疫吸附试验法检测肝组织中丙二醛、超氧化物歧化酶(SOD)、谷胱甘肽过氧化酶(GSH-Px)和羟脯氨酸的含量,蛋白质印迹法检测肝组织中α-平滑肌激动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)、转化生长因子β(TGF-β)、基质金属蛋白酶9(MMP-9)及MMP组织抑制因子1(TIMP-1)蛋白表达水平。结果水飞蓟素阳性对照组和柴芪益肝方低、高剂量组小鼠肝细胞变性、坏死及纤维化组织增生程度均较模型组明显减轻。柴芪益肝方低、高剂量组血清ALT、AST水平,肝组织中丙二醛、羟脯氨酸、α-SMA、CollagenⅠ、TGF-β及TIMP-1蛋白表达较模型组呈现剂量依赖性降低,而柴芪益肝方高剂量组SOD、GSH-Px、MMP-9的蛋白表达水平显著高于模型组,差异均有统计学意义[(125±14)U/mg比(41±11)U/mg、(267±19)U/g比(82±22)U/g、(0.43±0.04)比(0.12±0.01)](P<0.01或P<0.05)。结论柴芪益肝方对肝纤维化小鼠有一定的抗肝纤维化及肝脏保护作用,其机制可能与其调节脂质代谢、抗氧化应激反应、抑制TGF-β蛋白表达,促进细胞外基质降解等相关。

Objective To investigate the mechanism of Chaiqiyigan formula(CQYG)on hepatic fibrosis.Methods Totally 50 specific pathogen free C57BL/6 male mice were selected and randomly divided into blank control group,model group,low dose of CQYG treatment(CQYG-L)group,high dose of CQYG treatment(CQYG-H)group,and silymarin positive control group,with 10 mice in each group.Except the blank control group,mice in each group were intraperitoneally injected with 15%carbon tetrachloride olive oil to induce hepatic fibrosis model.From the first day of modeling,the blank control group and model group were given the same volume of 0.5%sodium carboxymethyl cellulose solution by gavage,and other three groups were given CQYG(low and high dose)and silymarin by gavage,respectively.After the mice sacrificed,the hepatic morphology were observed,and hematoxylin-eosin,Masson and Sirius red staining were performed to observe the progression of hepatic tissue and collagen fiber changes.The serum levels of alanine aminotransferase(ALT)and aspartate transaminase(AST)were detected by biochemical technology,malondialdehyde,superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and hydroxyproline in hepatic tissue were detected by enzyme-linked immunosorbent assay,and protein expression levels ofα-smooth muscle activator protein(α-SMA),CollagenⅠ,transforming growth factor-β(TGF-β),matrix metalloproteinase-9(MMP-9)and tissue inhibitor of MMP-1(TIMP-1)were determined by western blot.Results Compared with the model group,silymarin positive control group,CQYG-L group and CQYG-H group had significantly less hepatic cell degeneration,necrosis and fibrosis tissue proliferation degrees.Compared with the model group,the serum levels of ALT and AST and the protein expressions of malondialdehyde,hydroxyproline,α-SMA,CollagenⅠ,TGF-βand TIMP-1 in hepatic tissue of CQYG-L group and CQYG-H group decreased in a dose-dependent manner,but the protein expression levels of SOD,GSH-Px and MMP-9 in CQYG-H group were significantly higher than those in model group,and the differences were statistically significant[(125±14)U/mg vs(41±11)U/mg,(267±19)U/g vs(82±22)U/g,(0.43±0.04)vs(0.12±0.01)](P<0.01 or P<0.05).Conclusion CQYG has anti-hepatic fibrosis and protective effect on the mice with hepatic fibrosis,which may be due to its ability to regulate lipid metabolism and anti-oxidative stress response,also may inhibit the protein expression of TGF-βand promote extracellular matrix degradation.