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目的 蚓激酶同工酶(LKIs)作为肠溶胶囊的有效成分,用于治疗血栓性疾病已有30多年历史.近年来,LKIs在其他危重疾病中的研究时有报道.本文关注LKIs在乙型肝炎方面的作用.方法 乙型肝炎表面抗原(HBsAg)、核心抗原(HBcAg)和e抗原(HBeAg)分别与不同浓度LKIs孵育,观察这些蛋白质的降解和估计肽链的切割位点.HepG2.2.15细胞与LKIs孵育,采用酶联免疫吸附测定(ELISA)和蛋白质印迹(Western blotting)检测细胞分泌的HBsAg和HbeAg.LKIs灌胃Balb/c小鼠30天,采用ELISA和Western blotting检测其血清HBsAg和HBeAg,免疫组化染色检测肝组织中的HBcAg.采用苏木精-伊红染色分析乙肝病毒转基因小鼠肝组织的损害,并通过ELISA定量分析血清谷草转氨酶(GOT)和谷丙转氨酶(GPT).腹腔注射后,取大鼠血清和肝组织,测定其中的LKIs含量,从而观察LKIs的吸收.采用LKIs给龙岩麻鸭灌胃30天,通过PCR检测其血清HBV DNA.结果 蚓激酶肠溶胶囊的有效成分是含有6种LKIs的复方蛋白酶药物,可以降解HBV编码的蛋白质.LKIs降解HBsAg的位点为K141/P142及R160/F161;HBcAg为R142/E143;HBeAg为R122/E123.LKIs可显著抑制HepG2.2.15细胞分泌HBsAg和naeAg.LKIs灌胃,HBV转基因小鼠血清HBsAg和HBeAg水平及肝组织的HBcAg水平均降低,提示病毒的组装和释放可能受到了抑制.在LKIs处理的转基因小鼠中,血清GPT和GOT水平降低,肝组织溶解数量减少,表明LKIs对小鼠肝细胞具有保护作用.LKIs灌胃龙岩麻鸭,血清中DHBV DNA水平明显下降,停药后出现反弹.结论 LKIs通过降解HBs、HBc和HBe蛋白,可能干扰HBV的装配和释放,减少病毒在肝细胞之间的传递,从而对肝细胞起到保护作用.“,”Objective Lumbrokinase isozymes (LKIs),which were isolated from earthworm called Dilong in traditional Chinese medicine (TCM),have been used as the active ingredient of the enteric-coated capsule to treat clotting disease approximately 30 years.Recently,the study of LKIs on other critical diseases received much attention.Methods To demonstrate the efficacy of LKIs on hepatitis B proteins,we incubated surface antigen (HBsAg),core antigen (HBcAg) and e antigen (HBeAg) with LKIs at different concentrations for different time intervals,and then estimated their cleavage sites.HepG2.2.15 cells were incubated with LKIs and their HBsAg,HbeAg were determined by ELISA and Western blotting.HBV-transgenic mice (Balb/c) were gavaged with LKIs for 30 days.HBsAg and HBeAg in serum were detected by ELISA and Western blotting,and HBcAg in hepatic tissues were immunohistochemically stained.Hematoxylin-eosin (HE) staining was used to exhibit liver endolysis of LKI-treated HBV-transgenic mice.Serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were semi-quantitatively detected with ELISA.After intraperitoneal injection of LKIs into Sprague Dawley rats,LKIs in serum and liver tissue were assayed.Longyan sheldrakes (LYS) were gavaged with LKIs for 30 days,and their serum HBV DNA were assayed by PCR.Results We observed that the capsule ingredient is a compound drug containing 6 LKIs.By incubating with the HBV proteins,LKIs were probably estimated to degrade HBsAg at K141/P142 and R160/F161,HBcAg at R142/E143,and HBeAg at R122/E123.LKIs significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells.Levels of HBsAg and HBeAg in serum and those of HBcAg in hepatic tissues decreased in HBV-transgenic mice gavaged with LKIs,suggesting a suppression of viral assembly.Levels of GOT and GPT and the number of endolysis in liver exhibited by HE staining were decreased in the LKI-treated HBV-transgenic mice,demonstrating LKIs\' protecting mice hepatic cells.The activity of LKIs could be detected in serum and hepatic tissues of Sprague Dawley rats after being intraperitoneally injected with LKIs.After gavaged with LKIs,the ducks showed a decrease in their serum HBV DNA levels.Conclusion The current work indicates that LKIs degrade HBs,HBc and HBe proteins and may interfere with the virion assembly and release,leading to decrease in the virus transmission between hepatocytes,and to hepatic protection.