虫草菌丝通过抑制TLR4/NF-κB信号通路与血管生成素样蛋白4对肝纤维化小鼠模型的保护作用  

Cultured mycelia of Cordyceps sinensis exerts a protective effect on a mouse model of liver fibrosis by inhibiting the Toll-like receptor 4/nuclear transcription factor-κB signaling pathway and angiopoietin-like protein 4

作　　者：张霖璋 张定棋 徐莹 杨海琳 戚胜兰 张聪聪 陈佳美 刘平 刘伟 ZHANG Linzhang;ZHANG Dingqi;XU Ying;YANG Hailin;QI Shenglan;ZHANG Congcong;CHEN Jiamei;LIU Ping;LIU Wei(Institute of Liver Diseases,Key Laboratory of Liver and Kidney Diseases of Ministry of Education,Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Teaching and Experimental Center,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院,肝病研究所,肝肾疾病病证教育部重点实验室,上海市中医临床重点实验室,上海201203 [2]上海中医药大学交叉科学研究院,上海201203 [3]上海中医药大学教学实验中心,上海201203

出　　处：《临床肝胆病杂志》2022年第7期1540-1547,共8页Journal of Clinical Hepatology

基　　金：上海市教育委员会和上海市教育发展基金会“晨光计划”项目(20CG50);中国科协青年托举工程项目(2020QNRC001);国家自然科学基金项目(81530101,82004162);上海市科技创新行动计划(20S21902600);上海市青年科技英才扬帆计划资助项目(20YF14495000);中国博士后基金面上项目(2020M68136)。

摘　　要：目的探究虫草菌丝对CCl_(4)诱导的肝纤维化小鼠模型的干预作用,及对Toll样受体4(TLR4)/核转录因子-κB(NF-κB)通路与血管生成素样蛋白4(ANGPTL4)的影响。方法将60只SPF级雄性C57/BL6J小鼠随机分为正常组、模型组、扶正化瘀方组及虫草菌丝低、中、高剂量组,每组各10只。模型组和各用药组注射15%CCl_(4)-橄榄油,造模6周。在第4周首日开始给药,连续3周。分别采用血生化法检测小鼠血清ALT、AST、TBil水平或活性,HE和SR染色观察肝组织病理学,碱水解法测定肝组织羟脯氨酸(HYP)含量情况,PCR检测α-SMA、Col-Ⅰ的mRNA表达情况,免疫组化测定肝组织α-SMA、Col-Ⅰ与ANGPTL4蛋白表达,Western Blot检测α-SMA、ANGPTL4、TLR4、NF-κB/P-NF-κB、CD163蛋白的表达。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果与正常组比较,模型组小鼠血清ALT、AST、TBil水平均显著增加(P值均<0.01);与模型组比较,虫草菌丝高剂量组显著改善小鼠肝纤维化程度,血清ALT、AST、TBil水平均显著减少(P值均<0.05)。HE染色发现虫草菌丝高剂量组能显著减少肝组织炎性细胞浸润。SR染色发现,虫草菌丝高剂量组明显减轻肝脏胶原沉积。与正常组比较,模型组肝组织HYP含量、SR阳性染色面积比均显著增加(P值均<0.01);与模型组比较,虫草菌丝高剂量组肝组织HYP含量、SR阳性染色面积比均显著减小(P值均<0.05)。与正常组比较,免疫组化、PCR和Western Blot结果显示模型组肝组织中α-SMA和Col-ⅠmRNA及蛋白表达显著增加(P值均<0.01);与模型组比较,虫草菌丝高剂量组肝组织中α-SMA、Col-Ⅰ胶原纤维表达均显著减少(P值均<0.05)。与正常组比较,模型组肝组织ANGPTL4、TLR4、P-NF-κB/NF-κB、CD163蛋白表达均显著增加(P值均<0.01);与模型组比较,虫草菌丝高剂量组肝组织ANGPTL4、TLR4、P-NF-κB/NF-κB、CD163蛋白含量均显著降低(P值均<0.05)。�Objective To investigate the interventional effect of cultured mycelia of Cordyceps sinensis on a mouse model of CCl_(4)-induced liver fibrosis and its effect on the Toll-like receptor 4(TLR4)/nuclear transcription factor-κB(NF-κB)pathway and angiopoietin-like protein 4(ANGPTL4).Methods A total of 60 specific pathogen-free male C57/BL6J mice were randomly divided into normal group,model group,Fuzheng Huayu group,and low-,middle-,and high-dose Cordyceps sinensis groups,with 10 mice in each group.The mice in the model group and each medication group were intraperitoneally injected with 15%CCl_(4)-olive oil for 6 weeks of modeling,and drug intervention was started on the first day of week 4 and lasted for 3 consecutive weeks.Blood biochemistry was used to measure the serum level or activity of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil);HE and SR staining was used to observe liver histopathology;alkaline hydrolysis was used to measure the content of hydroxyproline(HYP)in liver tissue;PCR was used to measure the mRNA expression of alpha-smooth muscle actin(α-SMA)and collagen typeⅠ(Col-Ⅰ),immunohistochemistry was used to measure the protein expression ofα-SMA,Col-Ⅰ,and ANGPTL4 in liver tissue,and Western blot(WB)was used to measure the protein expression ofα-SMA,ANGPTL4,TLR4,NF-κB/phosphorylated NF-κB(P-NF-κB),and CD163.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the LSD-t test was used for further comparison between two groups.Results Compared with the normal group,the model group had significant increases in the serum levels of ALT,AST,and TBil(all P<0.01),and compared with the model group,the high-dose Cordyceps sinensis group had a significant improvement in the degree of liver fibrosis and significant reductions in the serum levels of ALT,AST,and TBil(all P<0.05).HE staining revealed that the high-dose Cordyceps sinensis group had a significant reduction in inflammatory cell infiltration in liver ti

关 键 词：肝硬化 血管生成素类 TOLL样受体4 NF-ΚB 

分 类 号：R285.5[医药卫生—中药学]