Transcriptome sequencing for exploring the mechanism of islet cell protection by anthocyanin.

We applied GSIS, immunofluorescence staining, WB, and flow cytometry to observe the protective effects of C3G on palmitate-induced beta cell apoptosis and insulin deficiency, after which we explored the mechanism using RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR), and finally validated the mechanism using Western Blot (WB) and small interfering RNA (siRNA).The results of GSIS on both INS-1E cells and mouse primary islets plus a series of experiments related to cell apoptosis showed that C3G could restore the damage caused by lipotoxicity through promoting insulin secretion and alleviating cell apoptosis. RNA-seq and qPCR results indicated that the protective effect of C3G was most likely mediated by repressing ER stress pathways, especially the PERK pathway, while partly due to elevated expression of insulin secretion-related genes. Finally, the results of knockdown of the pro-apoptotic marker CHOP in PERK pathway further confirmed the above findings.Conclusion: C3G may act though reducing ER stress to exert its protective impact on pancreatic beta cells under lipotoxicity. This research provides favorable evidence for the therapeutic application of C3G in T2DM.我们采用葡萄糖刺激胰岛素分泌实验（GSIS）、免疫荧光染色、蛋白质印迹（WB）以及流式细胞术，观察C3G对棕榈酸诱导的β细胞凋亡与胰岛素分泌缺陷的保护作用；随后通过RNA测序（RNA-seq）与实时定量聚合酶链反应（qPCR）解析其作用机制，并通过蛋白质印迹（WB）与小干扰RNA（siRNA）对该机制进行验证。针对INS-1E细胞与小鼠原代胰岛开展的GSIS实验及一系列细胞凋亡相关实验结果显示，C3G可通过促进胰岛素分泌、缓解细胞凋亡，逆转脂毒性造成的细胞损伤。RNA-seq与qPCR结果表明，C3G的保护作用极有可能通过抑制内质网应激通路，尤其是蛋白激酶R样内质网激酶（PERK）通路实现，同时部分归因于胰岛素分泌相关基因表达的上调。最后，靶向敲低PERK通路中促凋亡标志物CCAAT增强子结合蛋白同源蛋白（CHOP）的实验结果进一步验证了上述结论。研究表明，在脂毒性条件下，C3G或可通过减轻内质网应激发挥对胰腺β细胞的保护作用。本研究为C3G在2型糖尿病（T2DM）中的临床治疗应用提供了有力依据。