Cx43调控NLRP3炎性小体参与α1-AR激活诱导的心脏急性交感应激

Cx43 regulates NLRP3 inflammasome and participates inα1-AR activation induced cardiac acute sympathetic stress

目的观察缝隙连接蛋白43(Cx43)在苯肾上腺素(PE)过度激活心肌细胞α1-肾上腺素受体(α1-AR)诱导急性交感应激中的作用。方法将H9C2大鼠心肌细胞随机分为对照组(control组)、PE单独处理组、Gap26(Cx43特异性抑制剂)干预组、Gap26单独处理组,其中PE单独处理组给予50μmol/L PE作用15 min,Gap26干预组先给予0.5μmol/L Gap26预处理30 min,再给予50μmol/L PE作用15 min。Western blot法及qRT-PCR法检测心肌细胞Cx43、NLRP3炎性小体、白介素-1β(IL-1β)、半胱天冬氨酸蛋白酶-1(Caspase-1)、白介素-18(IL-18)蛋白及mRNA表达水平,免疫荧光法观察心肌细胞Cx43的表达和共定位,ELISA法检测心肌细胞炎症因子IL-1β、IL-18的表达。结果与control组相比,PE单独处理组Cx43及NLRP3、Caspase-1、IL-18蛋白及mRNA水平均升高;与PE单独处理组比,Gap26干预后Cx43及NLRP3、Caspase-1、IL-18的蛋白及mRNA水平均降低,但仍高于control组;同样,免疫荧光显示PE单独处理组心肌细胞Cx43蛋白表达增加,而Gap26干预组的Cx43表达均较PE单独处理组下调;ELISA结果显示PE单独处理组心肌细胞因子IL-1β、IL-18的表达明显上调,Gap26干预组则显著下调上述细胞因子的表达。结论Cx43可通过调控NLRP3炎性小体参与α1-AR激活诱导的心脏急性交感应激。

Objective To investigate the role of connexin 43(Cx43)in acute sympathetic stress induced by phenylephrine(PE)overactivation ofα1-adrenergic receptor(α1-AR)in cardiomyocytes.Methods Cardiomyocytes of H9C2 rats were randomly divided into control group,PE alone treatment group,Gap26(Cx43 specific inhibitor)intervention group and Gap26 alone treatment group.PE alone treatment group was treated with 50μmol/L PE for 15 min.The Gap26 intervention group was pretreated with 0.5μmol/L Gap26 for 30 min,and then treated with 50μmol/L PE for 15 min.The protein and mRNA expression levels of Cx43,NLRP3 inflammasome,interleukin-1β(IL-1β),Caspase-1,interleukin-18(IL-18)were detected by Western blot and qRT-PCR.The expression and co-location of Cx43 in cardiomyocytes were observed by immunofluorescence assay,and the expression of inflammatory cytokines IL-1βand IL-18 in cardiomyocytes was detected by ELISA.Results Compared with control group,the protein and mRNA levels of Cx43,NLRP3,Caspase-1 and IL-18 in PE group increased.Compared with PE alone treatment group,the protein and mRNA levels of Cx43,NLRP3,Caspase-1 and IL-18 decreased after Gap26 intervention,but they were still higher than those of control group.Similarly,immunofluorescence showed that Cx43 protein expression increased in PE alone group,while Cx43 expression was down-regulated in Gap26 intervention group compared with PE alone group.ELISA results showed that the expression of IL-1βand IL-18 was significantly up-regulated in PE alone group,but down-regulated in Gap26 intervention group.Conclusion Cx43 is involved inα1-AR activation induced cardiac acute sympathetic stress by regulating NLRP3 inflammasome.