PKCα对脂多糖诱导的C57BL/6J小鼠急性肺损伤中细胞自噬及焦亡的调节作用

Regulatory effect of PKCαon autophagy and pyroptosis in acute lung in⁃jury induced by lipopolysaccharide in C57BL/6J mice

目的:探讨蛋白激酶Cα(PKCα)在脂多糖(LPS)诱导的C57BL/6J小鼠急性肺损伤中的作用及PKCα对小鼠肺损伤过程中细胞自噬及焦亡的调控。方法:将雄性C57BL/6J小鼠随机分成空白组、DMSO组、DMSO+calphostin C组、LPS组和LPS+DMSO+calphostin C组,每组9只。按0.15 mg/kg剂量腹腔注射calphostin C预处理小鼠4 h以抑制PKCα的活化。将LPS以10 mg/kg的剂量经口气管插管滴入小鼠气道刺激24 h后收取支气管肺泡灌洗液(BALF)及肺组织标本。BALF检测蛋白浓度及炎症因子白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)分泌水平;肺组织HE染色后进行肺损伤病理学评分;通过Western blot及免疫组化染色检测肺组织自噬相关蛋白及NLRP3炎症小体相关蛋白的表达。体外实验中,用calphostin C(100 nmol/L)预处理RAW264.7细胞2 h后予LPS(1 mg/L)刺激6 h及24 h,分别提取细胞蛋白及RNA,Western blot检测自噬相关蛋白(LC3B和p62)及细胞焦亡相关蛋白(NLRP3、cleaved caspase-1、caspase-1和ASC)表达水平,RT-qPCR检测NLRP3炎症小体相关mRNA水平。结果:在LPS诱导的小鼠急性肺损伤中,PKCα磷酸化水平上调(P<0.05);抑制PKCα后LPS诱导的肺损伤病理学改变及肺内炎症反应显著减轻(P<0.01),细胞自噬及焦亡水平显著下调(P<0.01)。在RAW264.7细胞中,抑制PKCα也可部分逆转LPS诱导的细胞自噬及焦亡活性(P<0.05)。结论:抑制PKCα对LPS诱导的急性肺损伤C57BL/6J小鼠发挥保护作用,其保护机制涉及PKCα-自噬/焦亡信号通路。

AIM:To investigate the role of protein kinase Cα(PKCα)in lipopolysaccharide(LPS)-induced acute lung injury in C57BL/6J mice and the regulatory effect of PKCαon autophagy and pyroptosis during lung injury.MEHTODS:Male C57BL/6J mice were randomly divided into control group,DMSO group,DMSO+calphostin C group,LPS group,and LPS+DMSO+calphostin C group.The mice were pretreated with 0.15 mg/kg calphostin C for 4 h to inhibit PKCαactivation.LPS was injected into the airway by endotracheal intubation at a dose of 10 mg/kg for 24 h,and then the bronchoalveolar lavage fluid(BALF)and lung tissue samples were collected.BALF was used to detect protein concentra-tion and secretion of inflammatory factors.Lung injury scoring was performed by HE staining.The expression of autopha-gy-related proteins LC3B and p62,and NLRP3 inflammasome-related proteins were determined by Western blot and immu-nohistochemistry.In vitro,RAW264.7 cells were pretreated with calphostin C at 100 nmol/L for 2 h and then stimulated with LPS at 1 mg/L for 6 h and 24 h.Protein and RNA were extracted from the macrophages.The expression levels of au-tophagy-related proteins LC3B and p62,and pyroptosis-related proteins NLRP3,cleaved caspase-1,caspase-1 and ASC were determined by Western blot,and the mRNA levels of NLRP3 inflammasome-related molecules were detected by RT-qPCR.RESULTS:Phosphorylation of PKCαwas up-regulated in LPS-induced acute lung injury in mice.After inhibiting PKCα,LPS-induced lung injury and lung inflammation were significantly attenuated,and autophagy and pyroptosis levels were markedly down-regulated.In RAW264.7 cells,PKCαinhibition also obviously reversed LPS-induced autophagy and pyroptosis.CONCLUSION:Inhibition of PKCαplays a protective role in C57BL/6J mice with LPS-induced acute lung injury,and the protective mechanism involves the PKCα-autophagy/pyroptosis signaling pathway.