高糖高脂环境下生长激素释放肽对胰腺β细胞自噬的影响

摘要

目的:探讨高糖高脂(HGHL)环境下生长激素释放肽(ghrelin)对胰腺β细胞自噬的影响.方法:将胰岛NIT-1细胞分为对照组(葡萄糖5.6mmol/L)、ghrelin组(葡萄糖5.6mmol/L+100nmol/L ghrelin)、HGHL组(葡萄糖33.3mmol/L+0.5 mol/L棕榈酸)、HGHL+ghrelin组(葡萄糖33.3mmol/L+0.5mol/L棕榈酸+100nmol/L ghrelin).采用CCK-8法检测NIT-1细胞活性.采用单丹磺酰戊二胺(MDC)染色和微管相关蛋白1轻链3(LC3)B免疫荧光染色检测细胞自噬活性.结果:HGHL+ghrelin组细胞活力、Beclin 1蛋白水平、LC3Ⅱ/LC3Ⅰ的比值、Atg6和LC3B基因相对表达量均显著高于HGHL组(均P<0.05).MDC染色和免疫荧光染色显示,HGHL+ghrelin组细胞内自噬泡数量和阳性LC3B斑点数目增加.结论: Ghrelin能保护胰岛β细胞免受HGHL损伤,可能与自噬能力的增强有关.%Objective:To investigate the effect of ghrelin on the autophagy of islet β cells under high glucose and high lipid (HGHL) environment.Methods:Islet NIT-1cells were cultured and divided into four groups:control group(glucose 5.6 mmol/L) ,ghrelin group(glucose 5.6mmol/L+100nmol/L ghrelin) ,HGHL group(glucose 33.3mmol/L+0.5mmol/L palmitic acid) ,HGHL+ghrelin group (glucose 33.3mmol/L+0.5mmol/L PA+100nmol/L ghrelin).The cell counting kit (CCK-8) was used to assess the NIT-1cells viability. Monodansylcadaverine(MDC) staining and microtubule-associated protein 1light chain 3 (LC3) B immunofluorescence staining were employed to detect the autophagy.Western blotting was performed to determine the protein expression of Beclin 1and the ratio of LC3 Ⅱ to LC3Ⅰ,and real-time fluorescence quantitative PCR(qPCR) was tomeasure the mRNA expression of Atg6 and LC3B.Results:The cell viability,the protein level of Beclin 1,the ratio of LC3 Ⅱ/LC3 Ⅰ,and the mRNA expression levels of Atg 6 and LC3B in HGHL+ghrelin groupweresignificantly increased compared with HGHL group (P<0.05).MDC staining and immunofluorescence staining also showed increased deposition of autophagic vesicles and autophagic protein LC3B in HGHL+ghrelin group. Conclusion:Ghrelin could protect islet β cells from HGHL injury, which might be related to the enhancement of autophagy.