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

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；Westernblot、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-δ siRNA +FFA组相比，PKC-δ siRNA+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，改善HepG2功能状态，有效地缓解1 mmol/L游离脂肪酸所致的内质网应激，其作用机制在一定程度上与下调PKC-δ表达有关。

Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in aHepG2 cell model of nonalcoholic fatty liver disease

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) andexplore the possible mechanism. Methods FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2∶1. HepG2cells were treated with the sera from rats fed with low- , moderate-or high-dose HGT (HGT sera) or sera of rats fed withfenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O stainingwas 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 acommercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. Theprotein/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). Thechanges in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transienttransfection of PKC-δ siRNA for PKC-δ knockdown. Results Compared with the control cells, the cells treated with FFAsshowed significantly increased levels of TG, AST, and ALT (P<0.05). Compared with FFAs- treated cells, the cells pretreatedwith HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P<0.05), reduced accumulationof the lipid droplets (P<0.05), and lowered protein ormRNA 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 reducedprotein expressions of GRP78, p-PERK, CASPASE-12and CHOP in the cells with FFA- induced hepaticsteatosis (P<0.001); treatment with high-dose HGTserum more significantly reduced the expressions ofGRP78 (P<0.001) and P-PERK (P<0.01) in FFAs-induced cells with PKC-δ knockdown. Conclusion HGT serum can effectively prevent FFAs-induced steatosis in HepG2cells by alleviating ER stress, in which PKC-δ may act as an important target.