丹参酚酸B和山楂黄酮合用对游离脂肪酸诱导的大鼠肝细胞脂质沉积和凋亡的作用  

Effects of salvianolic acid B and hawthorn flavone on lipid deposition and apoptosis of hepatocytes in rats induced by free fatty acids

作　　者：薛冬英[1] 袭渤人 张洁[1] 叶军[1] 

机构地区：[1]上海中医药大学附属普陀医院感染科,200062

出　　处：《肝脏》2016年第3期183-190,共8页Chinese Hepatology

基　　金：上海市普陀区科委课题(2010PTKW004)

摘　　要：目的研究丹参酚酸B和山楂黄酮对游离脂肪酸诱导大鼠肝细胞脂质沉积和凋亡的作用及其机制。方法采用肝脏原位胶原酶灌注法分离大鼠原代肝细胞,油酸:棕榈酸=2:1造模。原代肝细胞和HepG2均分为正常对照组、模型组、模型+丹参酚酸B组、模型+山楂黄酮组、模型+丹参酚酸B+山楂黄酮组、模型+SP600125组、丹参酚酸B组、山楂黄酮组、丹参酚酸B+山楂黄酮组、SP600125组、DMSO对照组。游离脂肪酸刺激原代肝细胞的浓度为250μmol/L、刺激HepG2的浓度为500μmol/L;丹参酚酸B的浓度为1μmol/L;山楂黄酮的浓度为5μg/mL;SP600125的浓度为10μmol/L。观察1油红O染色:丹参酚酸B和山楂黄酮对游离脂肪酸刺激的肝细胞内脂滴变化的影响;2ELISA法:丹参酚酸B和山楂黄酮对游离脂肪酸诱导的肝细胞凋亡的影响;3高内涵筛选Hoechst33258染色:丹参酚酸B和山楂黄酮对游离脂肪酸诱导的肝细胞凋亡的影响;4Western blot:丹参酚酸B和山楂黄酮对游离脂肪酸刺激的肝细胞内p-JNK表达的影响。结果 1油红O染色显示:与正常对照组比较,游离脂肪酸作用于原代肝细胞、HepG2细胞24 h后,细胞内脂滴含量显著增加(P均<0.01)。丹参酚酸和山楂黄酮可显著减少游离脂肪酸诱导的原代肝细胞、HepG2细胞内脂滴的含量(P<0.01,P<0.05)。2ELISA检测结果:与正常对照组相比游离脂肪酸组原代肝细胞及HepG2细胞吸光值[Absorbance(A405 nm-A490nm)]显著增高,细胞凋亡明显(P均<0.01);丹参酚酸B和山楂黄酮显著抑制游离脂肪酸诱导的原代肝细胞、HepG2细胞凋亡(P均<0.01)。3高内涵筛选Hoechst33258染色:与正常对照组比较,游离脂肪酸组细胞核平均荧光强度值及凋亡率明显增高(P均<0.01)。丹参酚酸B和山楂黄酮显著降低游离脂肪酸刺激的原代肝细胞细胞核平均荧光强度值及凋亡率(P均<0.01)。HepG2细胞检测结果与原代肝细胞趋势一致。4Western blot结果:与正常Objective To investigate the effects of salvianolic acid B and hawthorn flavone on free fatty acids（FFA）-induced lipid deposition and apoptosis of hepatocytes in rats,and its potential mechanism.Methods HepG2 cells and primary cultured hepatocytes isolated from male SD rats by collagenase perfusion were subjected for this study.Oleic acid and palmitic acid（O∶P=2∶1）were used to induce nonalcoholic steatohepatitis（NASH）in primary cultured hepatocytes and HepG2 cells,respectively.The cell models were divided into 11 groups,including normal control group,FFA group,FFA ＋ salvianolic acid B group,FFA＋ hawthorn flavone group,FFA ＋ salvianolic acid B ＋ hawthorn flavone group,FFA＋ SP600125 group,salvianolic acid B group,hawthorn flavone group,hawthorn flavone＋ salvianolic acid B group,SP600125 group and DMSO control group,respectively.Correspondingly,the cell models of NASH in primary cultured hepatocytes and HepG2 cells were stimulated with different doses of FFA（250μmol/L and 500μmol/L）,salvianolic acid B（10-6mol/L）,hawthorn flavone（5μg/mL）and SP600125（10μmol/L）,respectively.In order to evaluate the therapy effects of salvianolic acid B and hawthorn flavone against NASH,the following tests were performed：Oil Red O staining for lipid deposition,and enzyme-linked immune-sorbent assay（ELISA）,chromatin dye and Hoechst 33258 staining for apoptosis.In addition,it was speculated that the therapy effect was associated with c-Jun N-terminal kinase（c-JNK）,which was verified by western blot to assess the expression of phosphorylated-JNK（p-JNK）.Results After 24 h of incubation with FFA alone,lipids in primary cultured hepatocytes and HepG2 cells were significantly increased（P〈0.01）.Moreover,salvianolic acid B,hawthorn flavoneor and SP600125 sharply reduced lipids deposition,respectively（P〈0.01,P〈0.05）.The intracellular lipids in DMSO control group had no significant difference than that in normal control group.ELISA showed that FFA induced apoptosis in prim

关 键 词：肝细胞 丹参酚酸B 山楂黄酮 游离脂肪酸 肝细胞凋亡 C-JUN氨基末端激酶 

分 类 号：R285.5[医药卫生—中药学]