热打击诱导血管内皮细胞NOD样受体蛋白3信号通路激活可被丙酮酸乙酯抑制

Activation of NOD like receptor protein 3 signaling pathway in vascular endothelial cells induced by heat stress can be inhibited by ethyl pyruvate

目的探讨热打击所致血管内皮细胞中NOD样受体蛋白3(NLRP3)信号通路激活能否被丙酮酸乙酯(EP)抑制。方法体外培养人脐静脉内皮细胞(HUVEC),取对数生长期细胞进行实验。分别设置不同温度梯度(39、41、43℃热打击4 h)及不同时间梯度(43℃分别持续热打击2、3、4 h)的热打击条件,并将HUVEC分别置入相应条件的细胞培养箱内实施热打击,然后选取43℃持续热打击4 h为最终实验条件,作为热打击组;在实施热打击时加入10 mmol/L的EP进行干预;同时设置常温对照组,将细胞同步置于37℃细胞培养箱中培养。采用蛋白质免疫印迹试验(Western blotting)检测HUVEC中NLRP3的蛋白表达及天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)的活性;采用酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素(IL-18、IL-1β)的释放水平。结果经不同条件热打击后,HUVEC中NLRP3的蛋白表达水平随着热打击温度不断升高或热打击时间不断延长而逐渐升高,以43℃热打击4 h时升高最为显著,与常温对照组比较差异均有统计学意义〔NLRP3蛋白表达(NLRP3/GAPDH):1.54±0.08比0.97±0.17,P<0.05〕;而在EP干预后,HUVEC中NLRP3的表达水平及caspase-1的活化水平均较热打击组明显下降〔NLRP3蛋白表达(NLRP3/GAPDH):1.15±0.07比1.57±0.09,caspase-1活性:40.87±6.54比59.75±9.92,均P<0.05〕,且细胞上清液中IL-18及IL-1β的释放也较热打击组明显下降〔IL-18(ng/L):1.09±0.08比1.41±0.13,IL-1β(ng/L):1.38±0.10比2.02±0.10,均P<0.05〕。结论热打击所致血管内皮细胞中NLRP3信号通路激活可以明显被EP抑制;EP的干预有助于减少热打击所诱导的血管内皮细胞中炎性因子的释放。

Objective To investigate whether the activation of NOD like receptor protein 3(NLRP3)signaling pathway in vascular endothelial cells induced by heat stress(HS)could be inhibited by ethyl pyruvate(EP).Methods Human umbilical vein endothelial cells(HUVEC)were cultured in vitro,and the cells in logarithmic growth phase were taken to carry out experiment.Different temperatures gradients(39,41,43℃for 4-hour HS)and different duration gradients(43℃continuously HS respectively for 2,3,4 hours)were set up respectively.For HS group,HUVECs were placed in incubators with corresponding different conditions to carry out HS,then 43℃persisting for 4-hour HS(43℃,4 hours)was selected as the final experimental condition;during HS,EP 10 mmol/L was added.For control group,the cells were synchronously cultured in 37℃cell incubator.The protein expression of NLRP3 in HUVEC and activity of aspartate specific cysteine protease 1(caspase-1)were detected with Western blotting;enzyme linked immunosorbent assay(ELISA)was used to detect the released levels of interleukins(IL-18 and IL-1β)in the cell culture supernatant fluid after HS.Results After HS,the protein expression level of NLRP3 in HUVEC was increased with the increasing of HS temperature or extension of exposure duration in HS condition,and reached to the highest in 43℃for 4 hours.Compared with control group,there was significant difference[NLRP3 protein expression(NLRP3/GAPDH):1.54±0.08 vs.0.97±0.17,P<0.05];after EP intervention,the expression of NLRP3 and the activation of caspase-1 in HUVEC were significantly lower than those in HS group[NLRP3 protein expression(NLRP3/GAPDH):1.15±0.07 vs.1.57±0.09,caspase-1 activity:40.87±6.54 vs.59.75±9.92,both P<0.05],moreover,the released levels of IL-18 and IL-1βin cell supernatant were also significantly decreasing than those in HS group[IL-18(ng/L):1.09±0.08 vs.1.41±0.13,IL-1β(ng/L):1.38±0.10 vs.2.02±0.10,both P<0.05].Conclusion The activation of NLRP3 signaling pathway in vascular endothelial cells induced by HS could be significantly inhibited by EP,which helps to reduce the release of inflammatory cytokines from vascular endothelial cells induced by HS.