Caveolin-2通过Hippo信号通路调控LPS诱导的小鼠肺泡巨噬细胞凋亡

Caveolin-2 regulates LPS induced apoptosis of mouse alveolar macrophages through the Hippo signaling pathway

目的探讨Caveolin-2(Cav-2)调控Hippo信号通路在LPS诱导下对肺泡巨噬细胞(MH-S)凋亡的作用。方法使用LPS诱导建立MH-S细胞凋亡模型,质粒构建Cav-2低表达和过表达模型。CCK-8检测细胞活力。ELISA检测细胞培养上清液中TNF-α、IL-1β和IL-6含量。ROS检测细胞活性氧水平。qRT-PCR检测Cav-2、YAP、TNF-α、IL-1β及IL-6基因表达水平。Western blot检测Cav-2蛋白、Hippo信号通路相关蛋白和细胞凋亡相关蛋白的表达水平,细胞免疫荧光检测Cav-2、YAP蛋白的荧光表达水平。结果随着LPS作用浓度的升高,MH-S细胞活力下降,促凋亡蛋白Bak、Bax、cleaved-Caspase-3/Caspase-3表达水平升高,抗凋亡蛋白Bcl-2表达水平降低(P<0.05)。YAP的表达水平升高,p-YAP的表达水平降低(P<0.05)。Cav-2表达水平升高(P<0.05)。随着LPS作用时间的增加,YAP蛋白的表达水平升高,p-YAP蛋白的表达水平降低(P<0.05),Cav-2的表达水平升高(P<0.01)。与LPS组相比,Cav-2低表达组TNF-α、IL-1β和IL-6的基因及蛋白表达水平降低(P<0.05),细胞活性氧生成减少(P<0.05),YAP的mRNA、蛋白及荧光表达水平降低,p-YAP蛋白表达水平升高(P<0.05),促凋亡蛋白表达水平降低,抗凋亡蛋白表达水平升高(P<0.05);而Cav-2过表达组结果与上述结果相反。结论靶向抑制Cav-2的表达可以增加YAP的磷酸化,激活Hippo信号通路,减少ROS生成,抑制炎症因子释放,抑制细胞凋亡,Cav-2可能作为干预肺部炎性疾病的潜在治疗靶标。

Objective To explore the role of Caveolin-2(Cav-2)in regulating the Hippo signaling pathway in LPS induced apoptosis of alveolar macrophages(MH-S).Methods MH-S cell apoptosis model was established by using LPS induction,and the low expression and overexpression models of Cav-2 were constructed with plasmids.The cell viability was detected with CCK-8.ELISA was used to detect TNF-α,IL-1β,IL-6 content in cell culture supernatant.ROS was used to detect cellular reactive oxygen species levels.qRT-PCR was used to detect Cav-2,YAP,TNF-α,IL-1β,IL-6.Western blotting was used to detect the expression levels of Cav-2 protein,Hippo signaling pathway related protein,and apoptosis related protein.Cellular immunofluorescence was used to detect the fluorescence expression levels of Cav-2 and YAP proteins.Results As the concentration of LPS increased,the activity of MH-S cells decreased,the expression level of pro-apoptotic proteins Bak,Bax,and cleared Caspase 3/Caspase 3 increased,and the expression level of anti-apoptotic proteins Bcl-2 decreased(P<0.05).The expression level of YAP increased,while the expression level of p-YAP decreased(P<0.05)and the expression level of Cav-2 increased(P<0.05).As the duration of LPS treatment increases,the expression level of YAP protein increases.Texpression level of p-YAP protein decreased(P<0.05),and the expression level of Cav-2 increased(P<0.01).Compared with the LPS group,the gene and protein expression levels of TNF-α,IL-1β,IL-6 decreased in the low expression group of Cav-2(P<0.05),cell reactive oxygen species generation decreased(P<0.05),YAP mRNA,protein,and fluorescence expression levels decreased,p-YAP protein expression levels increased(P<0.05),pro-apoptotic protein expression levels decreased,and anti-apoptotic protein expression levels increased(P<0.05);The overexpression group of Cav2 showed the opposite results.Conclusion Targeted inhibition of Cav-2 expression can increase YAP phosphorylation,activate Hippo signaling pathway,reduce ROS generation,inhibit inflammatory factor release,and inhibit cell apoptosis.Cav-2 may serve as a potential therapeutic target for intervening in pulmonary inflammatory diseases.