护肝清脂片药物血清对非酒精性脂肪肝细胞模型内质网应激的影响  

Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease

作　　者：杨妙婷 陈芝娟 肖淳欣 唐外姣[1] 周本杰[1,3] YANG Miaoting;CHEN Zhijuan;XIAO Chunxin;TANG Waijiao;ZHOU Beijie(Department of Pharmacy,Zhujiang Hospital,Southern Medical University,Guangzhou 510282,China;Shenzhen Beimei Pharmaceutical Co.,Ltd,Shenzhen 518057,China;Department of Pharmacy,Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen 518017,China)

机构地区：[1]南方医科大学珠江医院药学部,广东广州510282 [2]深圳市贝美药业有限公司,广东深圳518057 [3]中山大学附属第七医院药学部,广东深圳518017

出　　处：《南方医科大学学报》2018年第11期1277-1287,共11页Journal of Southern Medical University

基　　金：国家自然科学基金(81274160);广州市科技计划项目(201604020033)~~

摘　　要：目的探讨护肝清脂片药物血清对混合脂肪酸诱导的非酒精性脂肪肝HepG2细胞模型内质网应激(ER stress)的影响及其可能机制。方法油酸和棕榈酸以2∶1比例混合制备成1 mmol/L游离脂肪酸(FFA)混合液,诱导HepG2细胞24 h发生脂肪变性,建立NAFLD体外模型,将HepG2细胞分为对照组(CON)、FFA组、护肝清脂片低剂量组(HG-L)、护肝清脂片中剂量组(HGM)、护肝清脂片高剂量组(HG-H)及非诺贝特阳性对照组(FF),在加入1 mmol/L FFA前分别加入各组对应的药物血清作用24 h,CON组及FFA组则加入大鼠空白血清作为对照。油红O染色观察细胞内脂滴分布;试剂盒检测细胞内甘油三酯(TG)含量及培养上清液谷草转氨酶(AST/GOT)、谷丙转氨酶(ALT/GPT)的水平;透射电镜观察细胞内质网形态结构的改变;Western blot、qRT-PCR技术检测ERS相关信号分子GRP78、PERK、p-PERK、ATF6、ATF4、XBP-1、CASPASE-12、CHOP、PKC-δ、p-PKC-δ蛋白及(或)mRNA的表达情况。利用siRNA瞬时转染技术沉默PKC-δ在HepG2细胞中的表达后观察GRP78、PERK、p-PERK、CASPASE-12和CHOP蛋白表达变化。结果与CON组相比,FF组HepG2细胞油红染色可见红色脂滴密布细胞质,TG、AST、ALT含量升高(P<0.001);与FF组相比,HG-L、HG-M、HG-H及FF均在不同程度上降低TG、AST、ALT含量,减少细胞内脂滴堆积,差异有统计学意义(P<0.05),并能够在蛋白及mRNA水平上有效地下调ERS相关信号分子GRP78、p-PERK、ATF6、ATF4、CHOP、CASPASE-12、XBP-1、PKC-δ、p-PKC-δ的表达,差异有统计学意义(P<0.05)。在1 mmol/L FFA共同作用下,PKC-δsiRNA转染组GRP78、p-PERK、CHOP、CASPASE-12蛋白表达均较对照组明显下调,差异具有统计学意义(P<0.001)。然而,与PKC-δsiRNA+FFA组相比,PKC-δsiRNA+HG-H组GRP78和P-PERK表达下调更显著(P<0.001,0.01),CHOP和CASPASE-12则无统计学差异(P>0.05)。结论护肝清脂片药物血清可以有效地防治混合脂肪酸诱导的NAFLD细胞模型脂肪变性,�Objective To investigate the effects of sera from rats fed with Huganqingzhi tablets(HGT) on endoplasmic reticulum(ER) stress in a steatotic hepatocyte model of free fatty acids(FFAs)-induced nonalcoholic fatty liver disease(NAFLD) and explore the possible mechanism. Methods FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2∶1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT(HGT sera) or sera of rats fed with fenofibrate(fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride(TG),lactated hydrogenase(LDH), aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12,CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR(qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. Results Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT(P<0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels(P<0.05), reduced accumulation of the lipid droplets(P<0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6,ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ(P<0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis(P<0.001);treatment with high-dose HGT serum more significantly r

关 键 词：护肝清脂药物血清 非酒精性脂肪肝 内质网应激 脂肪变性 蛋白激酶C-δ 

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