温阳解毒化瘀颗粒对肝衰竭IETM大鼠TLR4 mRNA,TNF-α及肝细胞凋亡的影响  

Effect of Wenyang Jiedu Huayu Granule on TLR4 mRNA,TNF-α and Hepatocyte Apoptosis in IETM Rats with Hepatic Failure

作　　者：杜珊[1] 王杰 陈斌[3] 周为[3] 李武[3] 苏煦初[3] 彭杰[3] 周月 DU Shan;WANG Jie;CHEN Bin;ZHOU Wei;LI Wu;SU Xu-chu;PENG Jie;ZHOU Yue(Hunan University of Chinese Medicine,Changsha 410208,China;Jinhua Hospital of Traditional Chinese Medicine,Jinhua 321017,China;The First Affiliated Hospital of Hunan University of Chinese Medicine,Changsha 410007,China)

机构地区：[1]湖南中医药大学,长沙410208 [2]金华市中医医院,浙江金华321017 [3]湖南中医药大学第一附属医院,长沙410007

出　　处：《中国实验方剂学杂志》2020年第2期26-32,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医临床研究基地业务建设科研专项(JDZX2012062);湖南省中医药科研计划项目(201205);湖南省科技厅科技计划一般项目(2013SK3105);湖南省高层次卫生人才“225”工程培养项目

摘　　要：目的:研究Toll样受体4(TLR4) mRNA及其下游炎性因子肿瘤坏死因子-α(TNF-α)与肝衰竭肠源性内毒素血症(IETM)大鼠肝细胞凋亡的关系,并探索温阳解毒化瘀颗粒对内毒素介导的肝细胞凋亡的调控机制。方法:SPF级雄性SD大鼠85只,随机分为正常组,模型组,TLR4单克隆抗体组和温阳解毒化瘀颗粒组(8.925 g·kg^-1)。采用D-半乳糖胺(D-Gal)腹腔注射建立肝衰竭IETM模型,TLR4单克隆抗体组和温阳解毒化瘀颗粒组在造模前5 d给予温阳解毒化瘀颗粒溶液灌胃,正常组、模型组以等容积蒸馏水代替,直至处死。各组分别在24,48,72 h随机处死大鼠并采集标本。检测24,48,72 h各组时间点血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST)水平,苏木素-伊红(HE)染色观察肝组织病理变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测肝组织TLR4 mRNA表达,酶联免疫吸附测定法(ELISA)检测肝组织TNF-α表达,流式细胞仪检测肝细胞凋亡率。结果:与正常组比较,模型组ALT,AST升高,肝组织病理损伤程度明显加重,TLR4mRNA,TNF-α表达均增高(P<0.05,P<0.01),肝细胞凋亡率明显上升(P<0.05,P<0.01);与模型组比较,温阳解毒化瘀颗粒组ALT,AST显著降低(P<0.01),肝组织病理损伤程度明显减轻(P<0.05),TLR4 mRNA,TNF-α表达显著下降(P<0.01),肝细胞凋亡率亦显著降低(P<0.01)。结论:TLR4 mRNA,TNF-α在肝衰竭时与肝细胞凋亡呈正相关,温阳解毒化瘀颗粒能够改善肝衰竭IETM大鼠肝功能,减轻肝细胞损伤,减少肝细胞凋亡,其机制可能与其下调肝脏TLR4 mRNA表达,抑制TNF-α释放,降低肝细胞凋亡率有关。Objective: To investigate the relationship between Toll-like receptor 4(TLR4) mRNA and its downstream inflammatory factor-tumor necrosis factor-α(TNF-α) and hepatocyte apoptosis in mice with intestinal endotoxemia(IETM) of hepatic failure,and explore the regulatory mechanism of Wenyang Jiedu Huayu granule on endotoxin-induced hepatocyte apoptosis.Method: The 85 SPF male SD rats were randomly divided into normal group,model group,TLR4 monoclonal antibody group and Wenyang Jiedu Huayu granule group.D-galactosamine(D-Gal) intraperitoneal injection was performed to establish the IETM model of hepatic failure.The TLR4 monoclonal antibody group and the Wenyang Jiedu Huayu granule group were given Wenyang Jiedu Huayu granule solution by gavage 5 days before the modeling.The normal group and the model group were given isovolumetric distilled water.Each group was given by gavage until sacrifice.Rats in each group were randomly sacrificed at 24,48,72 h,respectively,and samples were collected.The levels of serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST) were detected at 24,48,72 h.The pathological changes of liver tissue were observed by hematoxylin and eosin(HE) staining.The expression of TLR4 in liver tissue was detected by realtime fluorescence quantitative PCR(Real-time PCR).The expression of TNF-α in liver tissue was detected by enzyme-linked immunosorbent assay(ELISA).The apoptotic rate of hepatocytes was detected by flow cytometry.Result: Compared with the normal group,ALT and AST were increased in model group,while the pathological injury degree of liver tissue was significantly increased.The expressions of TLR4 mRNA and TNF-α were increased(P<0.05,P<0.01),whereas the apoptosis rate of liver cells was significantly increased(P<0.05,P<0.01).Compared with the model group,ALT and AST were decreased in Wenyang Jiedu Huayu granule group(P<0.01),and the degree of pathological injury of liver tissue was significantly reduced(P<0.05).The expressions of TLR4 mRNA and TNF-α were significa

关 键 词：肝衰竭 温阳解毒化瘀颗粒 肠源性内毒素血症 TOLL样受体4 肿瘤坏死因子-α 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]