高脂血症性急性胰腺炎致胰岛β细胞焦亡的实验研究

Study on pancreatic β-cell pyroptosis caused by hyperlipidemic acute pancreatitis

目的:探讨高脂血症性急性胰腺炎(HTG-AP)脂毒性对胰岛β细胞的影响和发生机制。方法:棕榈酸(PA)的浓度分别为0、0.15、0.3、0.4和0.5 mmol/L,分别于0、8、16、24、36 h处理小鼠胰岛素瘤细胞(MIN6)。应用Western blot检测GSDMD和GSDME确定最佳刺激浓度和最佳刺激时间,根据此浓度和时间处理MIN6细胞,运用Western blot、CCK8、LDH释放实验、CCK-8法细胞活力测定、ELISA、PI染色和透射电镜检测细胞焦亡的情况。通过si RNA和质粒敲除和过表达NR4A1后用PA处理,运用Western blot、IL-1β ELISA和透射电镜检测细胞焦亡的情况。结果:与对照组相比,GSDMD和GSDME均随着PA浓度和时间的增高表达逐渐增高。选择0.4 mmol/L、36 h作为最佳刺激浓度和时间处理MIN6细胞,处理后GSDMD、GSDMD-N、GSDME、NLRP3、cleaved-Caspase1、cleaved-Caspase3表达及LDH释放和IL-1β较对照组均有升高。CCK-8实验PA组的细胞活力低于对照组。PI染色可见PA组出现红色荧光,电镜可见PA组细胞内出现空泡、细胞膜连续性中断。NR4A1敲除后经PA处理可见GSDMD、GSDMD-N、NLRP3、cleaved-Caspase1、cleaved-Caspase3表达均高于PA组,而过表达NR4A1可挽回这种趋势。ELISA结果显示NR4A1敲除组IL-1β释放要高于PA组,而过表达组则低于PA组。透射电镜结果显示NR4A1敲除组焦亡程度大于PA组,而过表达组则低于PA组。结论:PA可诱导MIN6细胞产生GSDMD介导的焦亡,NR4A1可能参与这一过程。

Objective:To investigate the effects of lipotoxicity on pancreatic β cells pyroptosis in hyperlipidemic acute pancreatitis(HTG-AP).Methods:The MIN6 cells were stimulated by Palmitic acid with different concentrations(0,0.2,0.3,0.4,0.5 mmol/L) and different duration(0,8,16,24,36 h).The best concentration and duration were determined by Western blot detection of GSDMD and GSDME.MIN6 cells were treated according to this concentration and duration,then Western blot,CCK8,LDH release assay,CCK-8 assay,ELISA,PI staining and transmission electron microscope were used to detect pyroptosis.After MIN6 cells were treated with si RNA and plasmid to knockout and overexpress NR4A1,the cells were stimulated by PA,and the pyroptosis were detected by Western blot,IL-1 β ELISA and transmission electron microscope.Results:Compared with the control group,the expression of GSDMD and GSDME increased gradually with the increase of PA concentration and duration.0.4 mol/L and 36 h were selected as the best concentration and duration to treat MIN6 cells.After treatment,compared with the control group,the expression of GSDMD,GSDME,NLRP3,Caspase1,Caspase3,cleaved-Caspase1,cleaved-Caspase3,LDH release and IL-1 β increased.The cell viability of PA group was lower than that of control group.PI staining showed red fluorescence in PA group.Electron microscope showed that there were vacuoles and pores in the cell membrane in the PA group.After NR4A1 was knocked down and were stimulated by PA,GSDMD,GSDME,Cleaved Caspase1,and Cleaved Caspase3 expressions were higher than those in the PA group.The results of IL-1 β ELISA showed that the release of IL-1 β in NR4A1knockout group was higher than that in PA group,while that in overexpression group was lower than that in PA group.The results of transmission electron microscope showed that the degree of pyroptosis in NR4A1 knockout group was higher than that in PA group,while that in overexpression group was lower than that in PA group.Conclusion:Palmitic acid can induce GSDMD and GSDME mediated pyroptosis in MIN6 cells,and NR4A1 may be involved in this process.