EGCG对日本血吸虫感染小鼠TIMP-1及α-SMA表达和肝纤维化的影响

Effects of EGCG on the expression of TIMP-1 and α-SMA and inhibition of hepatic fibrosis in mice infected with Schistosoma japonicum

目的观察表没食子儿茶素没食子酸酯(EGCG)对日本血吸虫感染小鼠干扰素-γ(IFN-γ)、白介素-13(IL-13)、金属蛋白酶组织抑制因子-1(TIMP-1)及α-平滑肌肌动蛋白(α-SMA)表达的影响,探讨EGCG抑制小鼠日本血吸虫病肝纤维化的作用及机制。方法随机将小鼠分为感染组及对照组(A组)。感染组小鼠用腹部贴片法感染日本血吸虫尾蚴(30±5)条/只后,随机分为感染模型组(B组)、EGCG治疗高剂量[400mg/(kg·d)]组(C组)、治疗低剂量[40mg/(kg·d)]组(D组)、高剂量佐吡喹酮治疗组(E组)、低剂量佐吡喹酮治疗组(F组),溶剂+吡喹酮对照组(G组),灌胃溶剂对照组(H组),每组10只。在血吸虫尾蚴攻击感染后第28d,用EGCG对应灌胃治疗,1次/d,连续治疗8周。其中E、F、G组在感染后第42、43d加用吡喹酮[500mg/(kg·d)]灌胃。实验结束后处死小鼠,测算肝体指数。留取肝脏组织和血清,采用ELISA法测定血清中IFN-γ、IL-13及TIMP-1含量,采用苏木素-伊红(HE)和Masson胶原纤维染色观察小鼠肝组织变性、虫卵肉芽肿与胶原沉积等病理改变,采用免疫组化法观察肝组织中α-SMA的表达。结果与对照组相比,各实验组小鼠肝体指数均升高,肝脏有虫卵肉芽肿形成、胶原沉积及肝纤维化,血清中IL-13、TIMP-1含量升高(P<0.05),肝细胞内α-SMA表达升高(P<0.05)。与感染模型组相比,EGCG高、低剂量治疗组及其佐吡喹酮组IL-13和TIMP-1含量及肝体指数均显著降低(P<0.05),虫卵肉芽肿面积减小,胶原沉积减轻。结论 EGCG可抑制α-SMA表达及TIMP-1分泌,其抗纤维化机制与抑制肝星状细胞(HSC)活化相关,EGCG与吡喹酮伍用可增强其抗血吸虫病肝纤维化作用。

Objective To investigate the effects of epigallocatechin-3-gallate（EGCG）in combination with praziquantel to treat hepatic fibrosis in mice infected with Schistosoma japonicum. Methods S.japonicumcercariae（30）were applied to the abdominal skin to infect mice.Uninfected mice and mice infected with the cercariae of S.japonicum were randomly divided into 8groups：the control group（A）,a disease model group（B）,agroup treated with a high dose of EGCG [400mg/（kg·d）]（C）,agroup treated with a low dose of EGCG [40mg/（kg·d）]（D）,agroup treated with a high dose of EGCG and praziquantel（E）,agroup treated with a low dose of EGCG and praziquantel（F）,a control group treated with praziquantel（G）,and a group treated with a solvent（H）.Each group consisted of 10 mice.On day 28 after infection,EGCG was orally gavaged to the mice once a day for 8weeks without interruption.On day 42 after infection,groups E,F,and G were treated with praziquantel[500mg/（kg·d）]for 2d.At the end of treatment,the mice were euthanized and their liver index was measured.Serum and liver tissue samples were collected for an enzyme-linked immunosorbent assay of interferon-gamma（IFN-gamma）,interleukin-13（IL-13）and tissue inhibitor of metalloproteinase（TIMP-1）.Liver tissues were subjected to hematoxylin and eosin（HE）staining and Masson staining.Collagen fibers in the liver were observed for degeneration,and pathologic changes such as egg granulomas and collagen deposition were measured.Immunohistochemical observation of liverα-smooth muscle actin（α-SMA）expression was performed. Re-sults Compared to group A（uninfected control）,the infected groups had liver tissue samples with a significant increase in egg granuloma formation and collagen deposition,as well as hepatic fibrosis.Levels of serum IL-13 and TIMP-1increased significantly（P〈0.05）,and expression ofα--SMA in liver cells increased（P〈0.05）.Compared to group B,groups C and D（treated with EGCG）and groups E and F（treated with EGCG combined with praziquantel）had significantly lower levels of IL-13 and TIMP-1and lower liver indices（P〈0.05）.In those groups,egg granulomas decreased significantly and collagen deposition decreased. Conclusion Orally administered EGCG inhibits liver fibrosis caused by schistosomiasis-related factors.The anti-fibrotic mechanism of EGCG appears to be via inhibition of α--SMA expression and secretion of TIMP-1,which are associated with the inhibition of hepatic stellate cell（HSC）activation.In addition,combined therapy with EGCG and praziquantel countered liver fibrosis induced by schistosomiasis.