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目的探讨PACS-2(phosphofurin acidic cluster sorting protein-2)及其所调控的葡糖调节蛋白78(glucoseregulating protein,GRP78)和细胞凋亡标志蛋白Bax、Caspase-3在非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)中的表达变化及意义。方法应用软脂酸诱导HepG2细胞脂肪变建立非酒精性脂肪性肝病体外模型,并按0、4、8、12、24 h时相点收获细胞,通过油红O(Oil red)染色检测细胞脂肪变程度。同时应用高脂饮食喂SD大鼠建立NAFLD模型,并按时相点分为4、8、12、16、20周组,以普通饮食喂养为对照组。通过q-PCR及Western blot检测PACS-2及内质网应激标志蛋白GRP78和Bax、Caspase-3的表达。结果软脂酸成功诱导HepG2细胞脂肪变性,应用高脂饮食喂养SD大鼠成功建立NAFLD大鼠模型。与对照组相比,PACS-2 mRNA相对表达量在HepG2细胞脂肪变模型早期(4、8 h)下降,脂肪变模型晚期(12、24 h)升高;GRP78在8 h组开始上升后,24 h组达到高峰(P<0.01);Bax、Caspase-3的表达均在12、24 h组显著上升(P<0.01)。在蛋白水平上,PACS-2、GRP78、Bax、Caspase-3的表达与mRNA水平表达基本一致(P<0.01)。NAFLD大鼠模型蛋白表达上,与对照组相比,PACS-2同样在早期(4、8周)下降,晚期(16、20周)上升(P<0.01);GRP78在表达在4周上调后,20周达到高峰(P<0.01)。Bax、Caspase-3的表达均在晚期显著上升(P<0.01)。结论 PACS-2早期的低表达可能参与了NAFLD过程中的内质网应激,晚期升高可能与肝细胞凋亡所致的肝损伤密切相关。
Objective To investigate the role of PACS-2 and its regulation of glucose regulatory protein 78 (GRP78) and apoptosis markers Bax and Caspase-3 in non-alcoholic fatty liver disease -alcoholic fatty liver disease (NAFLD)) and its significance. Methods The fatty acids in HepG2 cells were induced by palmitate to establish an in vitro model of non-alcoholic fatty liver disease. The cells were harvested at 0, 4, 8, 12 and 24 h, and the cell fat was detected by Oil red staining Variable degree. At the same time, high fat diet was fed to rats with NAFLD to establish NAFLD model, which were divided into 4, 8, 12, 16 and 20 weeks according to the time point, and fed with normal diet as the control group. The expression of PACS-2 and endoplasmic reticulum stress markers GRP78, Bax and Caspase-3 were detected by q-PCR and Western blot. Results Palmitate successfully induced steatosis of HepG2 cells. The NAFLD rat model was successfully established by feeding high fat diet to SD rats. Compared with the control group, the relative expression of PACS-2 mRNA in HepG2 cells steatosis model early (4,8 h) decreased, late steatosis model (12,24 h) increased; GRP78 in the 8 h group began to rise, 24 h group reached the peak (P <0.01). The expression of Bax and Caspase-3 in 12 and 24 h groups increased significantly (P <0.01). At the protein level, the expressions of PACS-2, GRP78, Bax and Caspase-3 were basically consistent with the mRNA levels (P <0.01). Compared with the control group, PACS-2 also decreased in early (4th and 8th week) and late phase (16th and 20th week) (P <0.01) in NAFLD rat model. Compared with control group, PACS- , Peaked at 20 weeks (P <0.01). The expression of Bax and Caspase-3 were significantly increased in the late stage (P <0.01). Conclusion The early low expression of PACS-2 may be involved in endoplasmic reticulum stress during NAFLD. The late increase may be closely related to the liver injury induced by hepatocyte apoptosis.