琥珀酸通过活性氧途径诱导人脐静脉内皮细胞焦亡

Succinate induces pyroptosis of human umbilical vein endothelial cells via reactive oxygen species pathway

目的探讨琥珀酸对人脐静脉内皮细胞(HUVEC)焦亡的影响及其调控机制。方法用琥珀酸类似物琥珀酸二乙酯(DS)处理HUVEC 24 h,比色法检测细胞内琥珀酸含量,Western blot检测细胞焦亡相关蛋白半胱氨酸天冬氨酸特异性蛋白酶1(Caspase-1)、白细胞介素1β(IL-1β)、IL-18、NOD样受体蛋白3(NLRP3)、消皮素D N端(GSDMD-N)的含量;ATP测定试剂盒以及活性氧(ROS)荧光探针分别检测琥珀酸对HUVEC的ATP以及ROS生成的影响。ROS清除剂N-乙酰半胱氨酸(NAC)检测ROS在琥珀酸诱导HUVEC焦亡中的作用。琥珀酸氧化抑制剂丙二酸二甲酯(DMM)检测琥珀酸氧化代谢对ROS产生的影响。结果DS促HUVEC内琥珀酸蓄积,上调焦亡相关蛋白Caspase-1、IL-1β、IL-18、GSDMD-N和NLRP3的表达,抑制ATP生成并上调ROS产生。NAC抑制琥珀酸诱导的ROS生成,并下调上述焦亡相关蛋白的表达。DMM下调琥珀酸诱导的ROS产生以及HUVEC的焦亡。结论琥珀酸通过氧化代谢上调ROS生成,进而促进HUVEC焦亡。

Aim To investigate the effect of succinate on pyroptosis of human umbilical vein endothelial cells(HUVEC)and its regulatory mechanism.Methods HUVECs were treated with succinate analogue diethyl succinate(DS)for 24 h,and the content of succinate was detected by colorimetry.Western blot was used to detect the expressions of pyroptosis-related protein cysteinyl aspartate specific proteinase 1(Caspase-1),interleukin-1β(IL-1β),IL-18,NOD-like receptor protein 3(NLRP3),gasdermin D N termine(GSDMD-N).The effects of succinate on ATP and reactive oxygen species(ROS)production of HUVEC were detected by ATP assay kit and ROS fluorescent probe.ROS scavenger N-acetylcysteine(NAC)was used to observe the role of ROS in HUVEC pyroptosis induced by succinate.Dimethyl malonate(DMM),a succinate oxidation inhibitor,was used to detect the effect of succinate oxidative metabolism on ROS production.Results DS promoted the accumulation of succinate in HUVEC,up-regulated the expressions of pyroptosis-related proteins Caspase-1,IL-1β,IL-18,GSDMD-N and NLRP3,inhibited ATP production and up-regulated ROS production.NAC inhibited the production of ROS induced by succinate and down-regulated the expressions of above pyroptosis-related proteins.DMM down-regulated succinate-induced ROS production and HUVEC pyroptosis.Conclusion Succinate up-regulates ROS production through oxidative metabolism,thus promoting HUVEC pyroptosis.