基于线粒体自噬研究正肝方治疗肝癌的作用机制  

Study on the mechanism of Zhenggan Decoction in the treatment of liver cancer based on mitophagy

作　　者：王红 曾昊天 冉云 WANG Hong;ZENG Hao-tian;RAN Yun(Dongzhimen Hospital,Beijing University of Chinese Medicine(Beijing,100700),China.;不详)

机构地区：[1]北京中医药大学东直门医院,北京100700 [2]北京中医药大学深圳医院(龙岗)

出　　处：《中西医结合肝病杂志》2024年第12期1101-1106,共6页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：深圳市科技计划项目(No.JCYJ20220530172804010);深圳市“医疗卫生三名工程”项目资助(No.SZZYSM202311018)。

摘　　要：目的:探讨正肝方调节线粒体自噬抑制H22肝癌小鼠移植瘤的作用机制。方法:构建小鼠H22肝癌移植瘤模型,随机分成空白对照组、模型组、正肝方低、中、高剂量组及索拉非尼组,给药干预14 d。末次给药12 h后处死小鼠,剥离肿瘤、胸腺及脾脏后称重记录并计算抑瘤率、胸腺及脾脏指数。苏木精-伊红(HE)染色观察肿瘤组织结构;原位末端转移酶标记(TUNEL)染色观察肿瘤细胞凋亡水平;酶联免疫吸附(ELISA)法检测血清中甲胎蛋白(AFP)表达水平;透射电镜观察肿瘤组织线粒体超微结构及线粒体自噬变化,免疫组化检测自噬相关蛋白微管相关蛋白轻链3(LC3)、P62表达水平。结果:与模型组相比,高剂量正肝方显著增加肝癌小鼠体重(P<0.05),并能有效减轻肿瘤重量(P<0.01)。HE染色结果提示,正肝方组小鼠肿瘤细胞减少、细胞膜破裂、细胞核碎裂,可见大小不等的坏死区域;肝癌细胞凋亡水平升高(P<0.01),并明显降低血清中AFP水平(P<0.01),且高剂量组与索拉非尼组药效相当;透射电镜结果显示,正肝方组肿瘤组织线粒体肿胀、嵴断裂、嵴结构紊乱,并可见空泡及自噬小体;免疫组化结果显示,自噬相关蛋白LC3蛋白表达水平升高、P62蛋白表达水平降低,呈现剂量依赖性趋势。结论:正肝方能有效抑制H22肝癌小鼠移植瘤生长。并且能够升高自噬蛋白LC3表达水平,降低P62表达水平,增加自噬小体,表明其作用机制可能与激活线粒体自噬有关。Objective:To investigate the mechanism by which Zhenggan formula regulates mitophagy to inhibit the growth of H22 hepatocellular carcinoma(HCC)xenografts in mice.Methods:A mouse model of H22 HCC xenografts was established and randomly divided into six groups:blank,model,low-dose Zhenggan formula,medium-dose Zhenggan formula,high-dose Zhenggan formula,and sorafenib.The treatment was administered for 14 days.The mice were euthanized 12 hours after the last administration.Tumors,thymus,and spleens were excised,weighed,and recorded.Tumor inhibition rate,thymus index,and spleen index were subsequently calculated.HE staining was used to observe tumor tissue structure;TUNEL staining was performed to assess apoptosis in tumor cells.ELISA was used to measure serum AFP levels.Transmission electron microscopy was employed to observe changes in mitochondrial ultrastructure and levels of mitophagy in tumor tissues,and immunohistochemistry was conducted to detect the expression of autophagy-related proteins LC3 and P62.Results:Compared to the model group,the high-dose Zhenggan formula significantly increased the body weight of HCC mice(P<0.05)and effectively reduced the tumor weight(P<0.01).HE staining indicated a reduction in tumor cells,membrane rupture,nuclear fragmentation,and necrotic areas of varying sizes in the Zhenggan formula groups.The apoptosis level of liver cancer cells in all drug-treated groups increased significantly(P<0.01),accompanied by a notable decrease in serum AFP levels(P<0.01).The therapeutic efficacy of the high-dose group was comparable to that of the sorafenib group.The apoptosis level of liver cancer cells in all drug-treated groups increased significantly(P<0.01),and a notable decrease in serum AFP levels(P<0.01).The therapeutic efficacy of the high-dose group was comparable to that of the sorafenib group.Transmission electron microscopy showed mitochondrial swelling,cristae fracture,disordered cristae structure,vacuoles,and autophagosomes in the ZGF-treated tumor tissues.Immunohistochemistry res

关 键 词：正肝方 线粒体自噬 肝癌 凋亡 甲胎蛋白 

分 类 号：R259[医药卫生—中西医结合]