内质网应激对非酒精性脂肪性肝病细胞线粒体凋亡影响研究

Study the effect of endoplasmic reticulum stress on mitochondrial apoptosis in non-alcoholic fatty liver disease

目的探讨内质网应激对非酒精性脂肪性肝病(NAFLD)细胞模型中线粒体途径凋亡的影响。方法采用油酸诱导HepG2细胞建立非酒精性脂肪变性细胞模型,将实验细胞分为对照组、造模12 h组、造模16 h组和4-苯基丁酸(4-PBA,加入内质网抑制剂)组,BODIPY 493/503染色检测细胞内脂肪变性;采用Hoechst 33258染色观察各组细胞凋亡情况及Annexin V-FITC/PI双染法检测各组HepG2细胞凋亡率;采用JC-1法检测各组细胞线粒体膜电位(MMP)变化。采用免疫蛋白印迹(Western blot)检测内质网应激蛋白葡萄糖调节蛋白78(GRP78)及C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)及线粒体途径凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)的以及细胞质细胞色素C(CytC)的表达。结果与对照组相比,造模组细胞内脂质沉积明显增加;与造模16 h组相比,4-PBA组细胞内脂质沉积明显降低。Hoechst 33258染色及Annexin V-FITC/PI双染法检测提示与对照组相比,造模后细胞凋亡增加;与造模16 h组相比,4-PBA组细胞凋亡率明显降低。JC-1法检测显示与对照组相比,造模后细胞MMP下降,加用4-PBA后细胞MMP升高。Western blot检测显示造模后细胞Bax、Caspse-3、GRP78、CHOP、胞质CytC蛋白表达增加,而Bcl-2表达降低;加用4-PBA后细胞GRP78、CHOP、Bax、Caspse-3、胞质CytC蛋白表达降低,而Bcl-2表达增加。结论内质网应激可诱导NAFLD细胞线粒体途径凋亡。

Objective To investigate the effect of endoplasmic reticulum stress on mitochondrial apoptosis in a cell model of non-alcoholic fatty liver disease(NAFLD).Methods HepG2 cells were induced by oleic acid to establish a non-alcoholic steatosis cell model.The test cells were divided into a control group,a 12-hour modeling group,a 16-hour modeling group,and 4-Phenylbutyric acid(4-PBA,adding endoplasmic reticulum inhibit)group.BODIPY 493/503 staining was used to detect intracellular fatty degeneration;Hoechst 33258 staining was used to observe cell apoptosis in each group and Annexin V-FITC/PI double staining method was used to detect the apoptosis rate of HepG2 cells in each group;JC-1 method was used to detect mitochondrial membrane potential(MMP)changes in each group.Western blot was used to detect the expression of endoplasmic reticulum stress proteins glucose regulatory protein 78(GRP78)and C/EBP cyclic adenosine monophosphate response element binding transcription factor homologous protein(CHOP)and mitochondrial pathway apoptosis-related protein caspase-3(caspase-3),B lymphoma-2 gene(Bcl-2),Bcl-2 related X protein(Bax)and cytoplasmic cytochrome C(CytC).Results Compared with the control group,the intracellular lipid deposition of the model group was significantly increased.Compared with the 16 h group,the intracellular lipid deposition of the 4-PBA group was significantly reduced.Hoechst 33258 staining and Annexin V-FITC/PI double staining method showed that compared with the control group,apoptosis increased after modeling;Compared with the 16 h group,the apoptosis rate of 4-PBA group was significantly lower.JC-1 method showed that compared with the control group,cell MMP decreased after modeling,and cell MMP increased after adding 4-PBA.Western blot detection showed that the expression of Bax,Caspse-3,GRP78,CHOP,CytC protein in the cells increased,while the expression of Bcl-2 decreased after modeling;the cells GRP78,CHOP,Bax,Caspse-3,Caspse-3,CytC protein expression decreased in cytoplasm,while Bcl-2 protein expression increased with the 4-PBA group.Conclusion Endoplasmic reticulum stress can induce mitochondrial apoptosis of NAFLD cells.