氧糖剥夺/复氧所致细胞焦亡在肺微血管内皮细胞损伤中的作用

Role of pyroptosis induced by oxygen-glucose deprivation/reoxygenation in pulmonary microvascular endothelial cell injury

目的:探讨氧糖剥夺/复氧(OGD/R)是否导致人肺微血管内皮细胞(HPMVECs)焦亡及其在细胞损伤中的作用。方法:采用HPMVECs复制OGD/R细胞模型,模拟体外循环中HPMVECs缺血/再灌注过程。将细胞分为对照(control)组(正常培养)、OGD/R组(OGD 8 h+恢复12 h)及VX-765(caspase-1抑制剂)组(在OGD/R之前4 h给予50μmol/L VX-765处理,其余培养条件同OGD/R组)。采用RT-qPCR和Western blot法检测caspase-1、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)及含caspase募集结构域的凋亡相关斑点样蛋白(ASC)的mRNA及蛋白表达水平;采用ELISA法检测上清液中白细胞介素1β(IL-1β)及IL-18水平;采用乳酸脱氢酶(LDH)试剂盒检测上清液中LDH的水平。结果:与control组比较,OGD/R组caspase-1、NLRP3和ASC的mRNA和蛋白表达均升高(P<0.05),炎症因子IL-1β和IL-18分泌均增多(P<0.05);使用VX-765可逆转OGD/R造成的caspase-1、NLRP3和ASC mRNA和蛋白表达水平的上调及IL-1β和IL-18的分泌(P<0.05),同时下调OGD/R后的LDH水平(P<0.05)。结论:OGD/R可导致HPMVECs发生焦亡,抑制细胞焦亡可缓解OGD/R对HPMVECs的损伤。

AIM:To explore whether oxygen-glucose deprivation/reoxygenation(OGD/R)causes pyroptosis of human pulmonary microvascular endothelial cells(HPMVECs)and its role in cell damage.METHODS:The HPMVECs were used to replicate the OGD/R cell model for simulating the ischemia-reperfusion process of HPMVECs during cardiopulmonary bypass.The cells were divided into control group,OGD/R group and VX-765(a caspase-1 inhibitor)group.The cells in control group were cultured normally.The cells in OGD/R group were treated with OGD for 8 h and recovery for 12 h.The cells in VX-765 group were treated with VX-765 at 50μmol/L for 4 h before OGD/R,and the remaining culture conditions were the same as those in OGD/R group.The mRNA and protein expression levels of caspase-1,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC)were detected by RT-qPCR and Western blot.The levels of interleukin(IL)-1βand IL-18 in the supernatant were measured by ELISA.The lactate dehydrogenase(LDH)level in the supernatant was determined by LDH kit.RESULTS:Compared with control group,the mRNA and protein expression levels of caspase-1,NLRP3 and ASC,and the secretion of inflammatory cytokines IL-1βand IL-18 were all increased in OGD/R group(P<0.05),which were reversed by VX-765(P<0.05),and the LDH activity after OGD/R was decreased by VX-765 treatment(P<0.05).CONCLUSION:OGD/R causes pyroptosis of HPMVECs,and inhibition of pyroptosis alleviates the damage of HPMVECs after OGD/R.