疏肝健脾活血方含药血清对肝纤维化模型大鼠肝窦内皮细胞失窗孔化的影响  

Effects of Serum Containing Shugan Jianpi Huoxue Decoction(疏肝健脾活血方) on Defenestration of Sinusoidal Endothelial in Hepatic Fibrosis Model Rats

作　　者：夏雪皎[1] 黄棪 鲁军 杨东升 周凝[1] 张俊杰[1] XIA Xuejiao;HUANG Yan;LU Jun;YANG Dongsheng;ZHOU Ning;ZHANG Junjie(Zheiiang Chinese Medical University,Hangzhou 310052)

机构地区：[1]浙江中医药大学

出　　处：《中医杂志》2018年第23期2037-2042,共6页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81473647)

摘　　要：目的探讨疏肝健脾活血方治疗肝纤维化的可能作用机制。方法 10只SD大鼠以10 ml/kg疏肝健脾方浓缩液(浓度2. 7 g/ml)灌胃,每日1次,连续3天后制备含药血清。中药血清设5%、10%、15%、20%、25%共5个浓度,采用MTT法筛选实验药物浓度。采用四氯化碳(CCl4)腹腔注射建立肝纤维化大鼠模型后分离肝纤维化肝窦内皮细胞(SEC),同时集正常大鼠肝星状细胞(HSC)和SEC。实验分为正常组、模型组、DAPT组、NF组、中药组、中药加DAPT组、中药加NF组,共7组。各组均加入HSC,正常组加入正常大鼠SEC,其余组加入肝纤维化大鼠SEC,常规培养24 h。吸出上清液,DAPT组、中药加DAPT组加入0. 2μmol/L DAPT (Notch通路阻断剂),NF组、中药加NF组加入1 gsu/ml rh NF-κB (Notch通路激动剂)。前4组加入含10%FBS DMEM/12培养基,后3组加入含预筛选浓度中药血清、10%FBS DMEM/12培养基,培养72 h后MTT法检测各组HSC活力,Western Blot法检测SEC中CD31、vWF蛋白表达情况。结果筛选出10%浓度的中药血清对细胞增殖有抑制作用。与正常组比较,造模2、4、8周模型组HSC活力上升,造模4、8周CD31蛋白表达升高(P <0. 05或P <0. 01);与模型组比较,NF组HSC活力上升,DAPT组和中药组HSC活力、CD31、vWF蛋白表达下降(P <0. 05或P <0. 01);与NF组比较,中药加NF组HSC活力、CD31、vWF蛋白表达下降(P <0. 05或P <0. 01);与DAPT组比较,中药加DAPT组HSC活力、CD31、vWF蛋白表达均下降(P <0. 05或P <0. 01)。结论疏肝健脾活血方可通过干预Notch通路,抑制HSC活力及SEC失窗孔化,可能是其治疗肝纤维化的作用机制之一。Objective To explore the possible mechanism of Shugan Jianpi Huoxue Decoction(疏肝健脾活血方)(STHD) in treating hepatic fibrosis.Methods Ten SD rats were orally administered with 10 ml/kg Shugan Jianpi Huoxue Decoction(concentration 2.7 g/ml) once a day for 3 consecutive days to prepare drug-containing serum.The serum of traditional Chinese medicine is set at 5 concentrations of 5%,10%,15%,20% and 25%.The concentration of the experimental drug was screened by MTT method,and the liver fibrosis sinusoidal endothelial cells(SEC)were isolated by intraperitoneal injection of carbon tetrachloride(CCl4).The normal rat hepatic stellate cells(HSC)and SEC were collected.In this experiment,the rats were divided into 7 groups including normal group,model group,DAPT group,NF group,Chinese medicine group,Chinese medicine plus DAPT group and Chinese medicine plus NF group.HSC was added to each group,and SEC of normal rats was added to the normal group,and SEC of liver fibrosis rats were added to the other groups for 24 hours.The supernatant was aspirated,and the DAPT group,the Chinese medicine plus DAPT group were added with Notch pathway blocker DAPT 0.2 μmol/L,and the NF group and the Chinese medicine plus NF group were added with Notch pathway agonist rhNF-κB 1 gsu/ml.The first4 groups were added with 10% FBS DMEM/12 medium,and the last 3 groups were added with pre-screened concentration Chinese medicine serum,10% FBS DMEM/12 medium.After 72 hours of culture,the MTC activity of each group was detected by MTT method,and the expression of CD31 and vWF proteins in SEC was detected by Western Blot method.Results The screening out 10% concentration of traditional Chinese medicine in serum had an inhibitory effect on cell proliferation.Compared with the normal group,the HSC activity of the model group was increased after 2,4,and 8 weeks,and the CD31 protein expression was increased after 4 and 8 weeks since model establishment(P<0.05 or P<0.01).Compared with the model group,the HSC activity of the NF group was increased.T

关 键 词：肝纤维化 疏肝健脾活血方 肝星状细胞 肝窦内皮细胞 失窗孔化 NOTCH通路 

分 类 号：R285.5[医药卫生—中药学] R-332[医药卫生—中医学]