热打击通过活化NLRP3炎性小体增加肺毛细血管的通透性

Heat stress increases pulmonary capillary permeability via activating NLRP3 inflammasome

目的验证热打击通过活化NLRP3炎性小体增加肺毛细血管通透性的机制。方法C57BL/6小鼠和肺微血管内皮细胞,用42℃热打击。体内实验分组包括:对照组和热打击组,每组12只小鼠;体外实验分组包括:对照组、热打击组、热打击+2,2,6,6-四甲基哌啶氧化物(TEMPO)组、热打击+半胱氨酸的天冬氨酸蛋白水解酶(caspase)抑制剂(Z-VAD-FMK)组、热打击+白细胞介素-1受体拮抗剂(IL-1Ra)组,每组4例。检测肺组织和肺微血管内皮细胞活性氧(ROS)表达;Western blot和(或)免疫荧光检测caspase-1、白细胞介素-1β(IL-1β)、紧密连接蛋白ZO-1、occludin、claudin-5的表达;用伊文氏蓝检测小鼠肺毛细血管的通透性。结果体内实验结果显示,与对照组比较,热打击组小鼠肺组织伊文氏蓝浓度明显升高、ROS表达上调、NLRP3炎性小体活化、IL-1β表达上调和紧密连接蛋白的表达下调(P<0.01)。体外实验结果显示,用TEMPO清除ROS后,NLRP3炎性小体活化被抑制(P<0.01);用ZVAD-FMK抑制caspase-1作用后,IL-1β表达显著下调(P<0.01);用IL-1Ra阻断IL-1β作用后,紧密连接蛋白表达显著上调(P<0.01)。结论热打击可通过活化NLRP3炎性小体促进IL-1β的表达,进而下调紧密连接蛋白的表达,导致肺微血管通透性增加。

Objective To explore whether heat stress would increase pulmonary capillary permeability via activating the NLRP3(NLR family,pyrin domain-containing 3)inflammasome.Methods C57BL/6 mice and pulmonary microvascular endothelial cells(PMVECs)were underwent 42℃heat stress.The mice were randomly divided into two groups:control group and heat stress group,12 mice for each group.The PMVECs were randomly divided into five groups:control group,heat stress group,heat stress+TEMPO group,heat stress+Z-VAD-FMK group and heat stress+interleukin-1 receptor antagonist(IL-1Ra)group,4 cases for each group.Caspase-1,IL-1βand tight junction proteins(ZO-1,occludin,claudin-5)expressions were assessed by Western blot assay or double immunofluorescence.The permeability of blood-brain barrier was assessed by Evans blue staining.Results The concentrations of Evans blue in lung tissue were significantly increased in heat stress group compared with those of control group(P<0.01).The expression levels of ROS,caspase-1 and IL-1βwere significantly increased,and the expression levels of ZO-1,occludin and claudin-5 were significantly decreased,in heat stress group compared with those of control group(P<0.01).Scavenging of ROS decreased the expression levels of caspase-1(P<0.01).The pharmacological(Z-VAD-FMK)inhibition of NLRP3 inflammasome activation decreased the expression levels of IL-1β(P<0.01).The pharmacological(IL-1Ra)blocking of IL-1βreceptor increased the expression levels of tight junction protein(ZO-1:P<0.01,occludin:P<0.01,claudin-5:P<0.01).Conclusion Heat stress may increase pulmonary capillary permeability via the activation of NLRP3 inflammasome and the reducing of tight junction protein expression.