低氧、高糖环境对肾脏足细胞焦亡相关机制的研究

Study on the mechanism of a pyroptosis of renal podocytes in hypoxia and high glucose environment

目的探讨低氧、高糖单因素和低氧高糖复合因素对大鼠肾小球足细胞焦亡的影响。方法体外培养大鼠肾小球足细胞,随机分为对照组、高糖组、低氧组及低氧高糖组,EdU法检测各组细胞增殖情况,透射电镜观察细胞核与线粒体形态及大小的变化,Western blot法检测焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、消皮素D(GSDMD)及炎症因子白细胞介素-1β前体(Pro-IL-1β)、白细胞介素(IL)-1β、IL-18的表达水平,分析低氧和高糖环境对大鼠肾小球足细胞焦亡的影响。结果EdU结果显示低氧和高糖抑制了大鼠肾小球足细胞的增殖能力(P<0.05)。透射电镜结果提示低氧和高糖条件促进大鼠肾小球足细胞焦亡的发生。Western blot显示低氧和高糖条件促进大鼠肾小球足细胞焦亡,使焦亡相关蛋白NLRP3、Caspase-1、GSDMD的表达增加,其中低氧高糖组焦亡蛋白的表达量增加最为显著(P<0.05)。同时也促使炎症因子Pro-IL-1β、IL-1β、IL-18的表达增加(P<0.05)。结论低氧、高糖条件可诱导大鼠肾小球足细胞发生焦亡,其中的机制之一可能是通过影响NLRP3-Caspase-1-GSDMD及其下游炎症因子而发挥作用。

Objective To investigate the effects of hypoxia,high glucose single factor and hypoxia high glucose compound factors on the pyroptosis of rat glomerular podocytes.Methods Rat glomerular podocytes were cultured in vitro and randomly divided into control group,high glucose group,hypoxia group and hypoxia high glucose group,EdU method was used to detect the cell proliferation,transmission electron microscope was used to observe the morphology and size changes of nucleus and mitochondria,and Western blot was used to detect pyroptosis related proteins nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),Cysteinyl aspartate specific proteinase-1(Caspase-1),gasdermin(GSDMD)and inflammatory factor pro-interleukin-1β(Pro-IL-1β),interleukin(IL)-1β,IL-18.The effect of hypoxia and high glucose on the pyroptosis of rat glomerular podocytes was analyzed.Results EdU results showed that hypoxia and high glucose inhibited the proliferation ability of rat glomerular podocytes(P<0.05).The results of transmission electron microscopy suggested that hypoxia and high glucose promoted the occurrence of pyroptosis of rat glomerular podocytes.Western blot showed that hypoxia and high glucose promoted pyroptosis of rat glomerular podocytes,and increased the expression of pyroptosis related proteins NLRP3,Caspase-1 and GSDMD,among which the expression of pyroptosis protein increased most significantly in hypoxia and high glucose group(P<0.05).At the same time,it also increased the expression of pro-inflammatory factor Pro-IL-1β,IL-1βand IL-18(P<0.05).Conclusion hypoxia and high glucose can induce pyroptosis of rat glomerular podocytes,one of the mechanisms may be through affecting NLRP3-Caspase-1-GSDMD and its downstream inflammatory factors.