吴茱萸碱对小鼠肝纤维化的影响

Effect of evodiamine on liver fibrosis in mice

目的探究吴茱萸碱对小鼠肝纤维化的影响及作用机制。方法32只SPF级C57BL/6J小鼠按照随机数字表法分为对照组、模型组、吴茱萸碱低剂量组、吴茱萸碱高剂量组,每组8只。采用10%四氯化碳(CCL4)在模型组、吴茱萸碱低剂量组、吴茱萸碱高剂量组中复制小鼠肝纤维化模型。每周1、3、5按0.5 mL/kg的剂量经腹腔注射10%CCL4,重复12周。造模过程中,吴茱萸碱低剂量组给予吴茱萸碱溶液20 mg/(kg·d)、吴茱萸碱高剂量组给予吴茱萸碱溶液25 mg/(kg·d)进行灌胃干预,模型组和对照组只注射10%CCL4混合溶剂。造模结束后,小鼠摘眼球采集血液标本,检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平;取肝组织,制备肝组织匀浆,检测丙二醛(MDA)水平;免疫组化法检测肝组织中Ⅲ型胶原蛋白(Col-Ⅲ)、α-平滑肌肌动蛋白(α-SMA)表达情况;免疫印迹法检测肝组织中增殖细胞核抗原(PCNA)蛋白、细胞基质金属蛋白酶-9(MMP-9)表达情况。结果与模型组比较,吴茱萸碱低剂量组、吴茱萸碱高剂量组ALT、AST、MDA水平均降低,差异有统计学意义(P<0.05);免疫组化法检测结果显示,与模型组比较,吴茱萸碱低剂量组、吴茱萸碱高剂量组Col-Ⅲ及α-SMA表达下调;免疫印迹法检测结果显示,与模型组比较,吴茱萸碱低剂量组、吴茱萸碱高剂量组PCNA蛋白表达下调,MMP-9表达上调,差异均有统计学意义(P<0.05)。结论吴茱萸碱对于CCL4诱导小鼠肝纤维化有一定的保护作用,其作用机制可能与降低MDA水平,抑制机体氧化应激反应,抑制肝星状细胞活化并下调PCNA蛋白、Col-Ⅲ、α-SMA表达,上调MMP-9表达有关。

Objective To investigate the effect and mechanism of evodiamine on liver fibrosis in mice.Methods A total of 32 cases of SPF C57BL/6J mice were divided into control group,model group,evodiamine low-dose group and evodiamine high-dose group according to the random number table method,with 8 mice in each group,with 10%carbon tetrachloride(CCL4)copy of liver fibrosis mice model in model group,evodiamine low-dose group and evodiamine high-dose group.Every Monday,Wednesday and Friday,10%CCL4 was injected intraperitoneally at a dose of 0.5 mL/kg body weight for 12 weeks.During the molding process,the evodiamine low-dose group was given 20 mg/(kg·d)and the evodiamine high-dose group was given 25 mg/(kg·d)fro gavage intervention,the model group and the control group were given only solvent.After the modeling,the eyeballs of the mice were collected and the contents of serum glutamate aminotransferase(ALT)and aspartate aminotransferase(AST)were detected.Liver tissue was collected and liver tissue homogenate was prepared for detection of malondialdehyde(MDA)content.TypeⅢcollagen(Col-Ⅲ)andα-smooth muscle actin(α-SMA)were detected by immunohistochemistry.Proliferating cell nuclear antigen(PCNA)protein and cell matrix metalloproteinase-9(MMP-9)were detected by Western blotting.Results Compared with the model group,the levels of ALT,AST and MDA in the evodiamine low-dose group and the evodiamine high-dose group decreased significantly,the differences were statistically significant(P<0.05).The results of immunohistochemistry showed that the expression of Col-Ⅲandα-SMA in the evodiamine low-dose group and the evodiamine high-dose group decreased significantly compared with those in the model group.Western blotting results showed that compared with the model group,the expression of PCNA protein down-regulated and the expression of MMP-9 up-regulated in the evodiamine low-dose group and the evodiamine high-dose group,the differences were statistically significant(P<0.05).Conclusion Evodiamine has a certain protective effect on CCL4-induced liver fibrosis in mice,and its mechanism may be related to reducing MDA content,inhibiting oxidative stress,inhibiting the activation of hepatic stellate cells,down-regulating the expression of PCNA protein,Col-Ⅲandα-SMA,and up-regulating the expression of MMP-9.