蚓激酶同工酶降解乙型肝炎抗原并保护肝功能  

Compound Lumbrokinase Isozymes Decrease Hepatitis B Antigens and Protect Hepatic Function

作　　者：周园[2] 牟丽娴 范士超 王秀梅 曹潇 王学清[3] 张奉学 赵静[2] 魏艳[2] 赫荣乔 ZHOU Yuan;MOU Li-Xian;FAN Shi-Chao;WANG Xiu-Mei;CAO Xiao;WANG Xue-Qing;ZHANG Feng-Xue;ZHAO Jing;WEI Yan;HE Rong-Qiao(Basic College of Medicine,Southwest Medical University,Luzhou 646000,China;Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China;Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems,School of Pharmaceutical Science,Peking University,Beijing 100191,China;School of Basic Medical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 510099,China)

机构地区：[1]西南医科大学基础医学院,泸州646000 [2]中国科学院生物物理研究所,北京100101 [3]北京大学药学院,分子药剂学与新释药系统北京市重点实验室,北京100191 [4]广州中医药大学基础医学院,广州510099

出　　处：《生物化学与生物物理进展》2022年第1期202-218,共17页Progress In Biochemistry and Biophysics

基　　金：国家自然科学(31470036)基金资助项目。

摘　　要：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(HBs Ag)、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。Hep G2.2.15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;HBc Ag为R142/E143;HBeAg为R122/E123。LKIs可显著抑制Hep G2.2.15细胞分泌HBsAg和HBeAg。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(HBs Ag),core antigen(HBc Ag)and e antigen(HBe Ag)with LKIs at different concentrations for different time intervals,and then estimated their cleavage sites.Hep G2.2.15 cells were incubated with LKIs and their HBs Ag,Hbe Ag were determined by ELISA and Western blotting.HBV-transgenic mice(Balb/c)were gavaged with LKIs for30 days.HBs Ag and HBe Ag in serum were detected by ELISA and Western blotting,and HBc Ag 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 HBs Ag at K141/P142 and R160/F161,HBc Ag at R142/E143,and HBe Ag at R122/E123.LKIs significantly inhibited HBs Ag and HBe Ag secretion from Hep G2.2.15 cells.Levels of HBs Ag and HBe Ag in serum and those of HBc Ag 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

关 键 词：HBEAG HBCAG HBSAG 蚓激酶同工酶 乙肝病毒 同工酶 

分 类 号：Q55[生物学—生物化学] R511[医药卫生—内科学]