扶正化纤方调控PI3K/AKT/BAD信号通路对肝纤维化小鼠的作用及机制  

Effect and Mechanism of Fuzheng Huaxian Prescription on Hepatic Fibrosis Mice by Regulating PI3K/AKT/BAD Signaling Pathway

作　　者：孙竞然 卢秉久[1] 郑佳连[1] 孙晓宁 肖璐嘉 SUN Jingran;LU Bingjiu;ZHENG Jialian;SUN Xiaoning;XIAO Lujia(Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032 Liaoning,China;Department of Cardiology,Guang′anmen Hospital,China Academy of Chinese Medical Sciences,Beijing 100032,China;Liaoning University of Traditional Chinese Medicine,Shenyang 110032 Liaoning,China)

机构地区：[1]辽宁中医药大学附属医院,辽宁沈阳110032 [2]中国中医科学院广安门医院心内科,北京100032 [3]辽宁中医药大学,辽宁沈阳110032

出　　处：《中药新药与临床药理》2024年第11期1652-1660,共9页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：沈阳市科学技术计划公共卫生研发专项(21-173-9-37);国家中医药管理局项目(卢秉久全国名老中医药专家传承工作室建设项目,2022-304)。

摘　　要：目的基于PI3K/AKT/BAD信号通路探讨扶正化纤方对肝纤维化小鼠的作用及机制。方法采用腹腔注射20%四氯化碳-橄榄油溶液0.2 mL,每周3次,连续4周,复制肝纤维化小鼠模型。将C57B/L雄性小鼠随机分为正常组、模型组、安络化纤丸组(0.78 g·kg^(-1))以及扶正化纤方低、中、高剂量组(3.1、6.2、12.4 g·kg^(-1)),每组6只。灌胃给药(10 mL·kg^(-1)),每日1次,持续4周。使用小动物超声仪检测肝脏弹性值;HE、Masson、天狼星红染色法观察小鼠肝脏组织病理变化;ELISA法检测血清透明质酸(HA)、层黏蛋白(LN)、Ⅲ型前胶原(P-ⅢC)及Ⅳ型胶原(Ⅳ-C)水平;qRT-PCR及Western Blot法检测肝脏组织中AKT、BAD、Bax、Bcl-2 mRNA及蛋白表达;免疫荧光法检测肝脏组织中α-SMA表达水平。结果与空白组比较,模型组小鼠的体质量、肝质量明显降低(P<0.05,P<0.01);肝脏弹性值显著升高(P<0.01);胶原纤维面积占比显著升高(P<0.01);血清HA、LN、P-ⅢC、Ⅳ-C水平均显著升高(P<0.01);肝脏组织α-SMA阳性表达面积占比显著升高(P<0.01),AKT、Bcl-2 mRNA及蛋白表达显著上调(P<0.01),BAD、Bax mRNA及蛋白表达显著下调(P<0.01)。与模型组比较,各给药组小鼠的体质量、肝质量均明显升高(P<0.05,P<0.01);肝脏弹性值均显著降低(P<0.01);胶原纤维面积占比均显著降低(P<0.05,P<0.01);血清HA、LN、P-ⅢC、IV-C水平均显著下降(P<0.01);肝脏组织α-SMA阳性表达面积占比显著降低(P<0.01),Bcl-2 mRNA及蛋白表达显著下调(P<0.01),AKT蛋白表达显著下调(P<0.01),BAD、Bax mRNA表达显著上调(P<0.01)。扶正化纤方中、高剂量组小鼠肝脏组织中AKT mRNA表达显著下调(P<0.01),Bax蛋白表达显著上调(P<0.01);扶正化纤方低、高剂量组小鼠肝脏组织中BAD蛋白表达显著上调(P<0.01)。结论扶正化纤方可能通过PI3K/AKT/BAD信号通路调控下游Bcl-2、Bax蛋白表达,促进活化肝星状细胞(HSC)凋亡,减少细胞外基质(ECM)及胶原纤维�Objective To investigate the effect and mechanism of Fuzheng Huaxian Prescription on hepatic fibrosis mice based on PI3K/AKT/BAD signaling pathway.Methods A mouse model of hepatic fibrosis was established by intraperitoneal injection of 0.2 mL CCl4 solution(20%in olive oil)three times per week for four consecutive weeks.C57B/L male mice were randomly divided into normal group,model group,Anluo Huaxian Pills group(0.78 g·kg^(-1)),low-,medium-,and high-dose Fuzheng Huaxian Prescription groups(3.1,6.2,12.4 g·kg^(-1),respectively),with six mice in each group.The corresponding drug(10 mL·kg^(-1))was administered by gavage once a day for four weeks.A small-animal ultrasound device was used to measure the value of liver elasticity.Hematoxylin and eosin(HE)staining,Masson’s trichrome staining and Sirius red staining were used to observe pathological changes in liver tissues.Enzymelinked immunosorbent assay(ELISA)was used to detect the serum levels of hyaluronic acid(HA),laminin(LN),procollagen III(P-IIIC),and collagen IV(IV-C).Real-time quantitative polymerase chain reaction(qRT-PCR)and Western Blot were used to measure the mRNA and protein expression levels of protein kinase B(AKT),Bcl-2-associated agonist of cell death(BAD),the anti-apoptotic protein Bcl-2,and the pro-apoptotic protein Bax in liver tissues.Immunofluorescence was used to detectα-SMA expression in liver tissues.Results Compared with the normal group,the body mass and liver mass of rats in the model group were significantly reduced(P<0.05,P<0.01).There was significant increase in the value of liver elasticity(P<0.01),the proportion of collagen fiber area(P<0.01),serum levels of HA,LN,P-IIIC,and IV-C(P<0.01).The proportion ofα-SMA positive expression area in liver tissue significantly increased(P<0.01).The mRNA and protein expressions of AKT and Bcl-2 were significantly up-regulated(P<0.01),while those of BAD and Bax were significantly down-regulated(P<0.01).Compared with the model group,the treatment groups exhibited significant increase in the bod

关 键 词：肝纤维化 扶正化纤方 肝星状细胞 细胞凋亡 PI3K/AKT/BAD信号通路 小鼠 

分 类 号：R285.5[医药卫生—中药学]