1,25(OH)2D3通过细胞焦亡经典途径促进Min6细胞胰岛素分泌的机制研究
首发时间:2022-06-14
摘要:目的探究1,25(OH)2D3对DM胰腺细胞焦亡的作用机制研究。 方法不同浓度1,25(OH)2D3(5×10-6、5×10-7、5×10-8、5×10-9 mmol/L)干预Min6细胞24 h后检测细胞活性;GSDMD siRNA转染前后Min6 细胞(NC组和siRNA组)分别经STZ处理后(STZ组和siRNA-STZ组)建立糖尿病细胞模型,再以1,25(OH)2D3干预各实验组(NC-VD组、STZ-VD组、siRNA-VD组、siRNA-STZ-VD组);ELISA试剂盒检测各组细胞上清胰岛素分泌情况;乳酸脱氢酶试剂盒检测各组细胞上清LDH释放情况;ELISA检测各组细胞上清炎症因子IL-18、L-1β释放情况;Western Blot检测各组细胞焦亡相关蛋白(NLRP3、NF-κB、caspase-1、GSDMD)表达情况。 结果 STZ处理后造成细胞功能损伤,胰岛素分泌含量显著性下降(p<0.001),且上调NLRP3、NF-κB、caspase-1、GSDMD蛋白表达(p<0.01),促进炎症因子IL-18、IL-1β以及LDH的释放,诱导细胞焦亡的发生(p<0.01);1,25(OH)2D3干预后提高胰岛素分泌(p<0.001),下调细胞NLRP3、NF-κB、caspase-1、GSDMD蛋白表达(p<0.001),降低炎症因子IL-18、IL-1β以及LDH的释放(p<0.01);抑制GSDMD蛋白的表达能够下调相关蛋白的表达水平,阻碍细胞质膜上的孔洞形成,抑制细胞焦亡的发生,降低炎症级联反应的扩大。 结论 STZ能造成胰腺细胞损伤,诱导细胞焦亡的发生,而1,25(OH)2D3可有效缓解STZ造成的细胞损伤,抑制细胞焦亡的发生。
关键词: 1,25(OH)2D3 糖尿病 细胞焦亡 炎症 Min6细胞
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1,25(OH)2D3 promotes insulin secretion in Min6 cells through the canonical pyroptosis pathway
Abstract:In this paper we explore the mechanism of 1,25(OH)2D3 on the pyroptosis of T2DM pancreatic cells. Different concentrations(5×10-6,5×10-7,5×10-8,5×10-9 mmol/L) of 1,25(OH)2D3 interfered with Min6 cells for 24 hours and the cell viability was detected. Before and after GSDMD siRNA transfection(NC group and siRNA group), Min6 cells were treated with STZ(STZ group and siRNA-STZ group) to establish diabetic cell models, and then 1,25(OH)2D3 was used to intervene in each experimental group(NC-VD group, STZ-VD group, siRNA-VD group, siRNA-STZ-VD group). ELISA kit was used to detect the secretion of insulin from cell supernatant in each group; Lactate dehydrogenase kit was used to detect the release of LDH in the cell supernatant of each group; ELISA kit was used to detect the release of inflammatory factors IL-18 and IL-1β in the cell supernatant of each group; Western Blot was used to detect the expression of pyroptosis-related proteins(NLRP3, NF-κB, caspase-1, GSDMD). Results: STZ treatment caused cell function damage, and insulin secretion content decreased significantly(p<0.001). The protein expression of NLRP3, NF-κB, caspase-1 and GSDMD were upregulated(p<0.01),the expression of the inflammatory factors IL-18、IL-1β were upregulated and the release of LDH was upregulated; 1,25(OH)2D3 intervention can reduce cell damage and increase insulin secretion of the damaged cells(p<0.001), downregulated the protein expression of NLRP3, NF-κB, caspase-1, and GSDMD(p<0.001), reduce the release of inflammatory factors IL-18、IL-1β, and LDH(p<0.01). Inhibiting the expression of GSDMD protein can downregulate the expression level of related proteins, hinder the formation of pores in the plasma membrane of the cell, inhibit the occurrence of cell pyrolysis, and reduce the expansion of the inflammatory cascade. Conclusion: STZ can cause pancreatic cell damage and induce pyroptosis, while 1,25(OH)2D3 intervention can alleviate the cell damage caused by STZ and inhibit the occurrence of pyroptosis.
Keywords: 1,25(OH)2D3 Diabetes Pyroptosis Inflammation Min6 cell
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1,25(OH)2D3通过细胞焦亡经典途径促进Min6细胞胰岛素分泌的机制研究
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