摘要
目的:探讨程序性死亡受体配体1(PD-L1)介导淋巴细胞yes相关蛋白(YAP)磷酸化在病毒性急性肺损伤中的作用.方法:将SPF级C57BL/6J小鼠随机分为对照组、根据小鼠气管内滴注聚肌苷—聚胞苷酸(即Poly I:C)的时间分为4h、8 h、1d、3d、7d组,在这五组中选取炎症损伤最严重的组作为Poly I:C组;利用PD-1/PD-L1抑制剂BMS-1 10mg/kg腹腔注射预处理后气管内滴注Poly I:C,作为Poly I:C+ BMS-1组.野生型小鼠Poly I:C组和PD-L1敲基因小鼠Poly I:C组处理同Poly I:C组;野生型小鼠对照组和PD-L1敲基因小鼠对照组腹腔注射麻醉后气管内给予等量生理盐水.相应时间点将小鼠安乐死收集标本.采用苏木精—伊红(HE)染色法评估小鼠肺组织损伤程度;通过检测支气管肺泡灌洗液(BALF)中总蛋白浓度、总细胞数以及酶联免疫吸附法(ELISA)检测BALF中肿瘤坏死因子(TNF)-α水平评估炎症情况;蛋白免疫印迹(western blotting,WB)检测程序性死亡受体1(PD-1)、PD-L1、YAP、p-YAP蛋白表达.结果:与对照组比较,8 h、1 d、3 d、7 d组肺组织病理学损伤评分及BALF总蛋白浓度显著升高,8 h、1 d、3 d组BALF中总细胞数明显增多,4 h、8 h、1 d、3 d组BALF中TNF-α水平显著上调,差异均有统计学意义(P＜0.05);其中1d组小鼠肺组织病理学损伤评分、BALF中总蛋白浓度、BALF总细胞数以及BALF中TNF-α水平均高于4h、8h、3d、7 d组.与对照组比较,1 d、3 d、7 d组小鼠肺组织中PD-1和PD-L1蛋白水平均明显升高(P＜0.05).Poly I:C组小鼠肺组织病理损伤评分和BALF中TNF-α水平明显高于对照组,与Poly I:C组相比,Poly I:C+ BMS-1组小鼠肺组织病理损伤评分和BALF中TNF-α水平显著降低(P＜0.05).与对照组相比,Poly I:C组和Poly I:C+BMS-1组小鼠淋巴细胞中YAP蛋白表达明显下调,p-YAP蛋白表达显著上调(P＜0.05);与Poly I:C组相比,Poly I:C+BMS-1组小鼠淋巴细胞中YAP蛋白表达上升,p-YAP蛋白表达下降(P＜0.05).与对照组相比,Poly I:C组小鼠淋巴细胞中YAP蛋白表达明显下调而p-YAP蛋白表达显著上调,PD-L1基因敲除后Poly I:C组小鼠淋巴细胞中YAP蛋白表达有所上升而p-YAP蛋白表达明显下降,差异均有统计学意义(P＜0.05).结论:病毒性急性肺损伤小鼠肺组织中高表达PD-L1蛋白,PD-L1可能通过激活淋巴细胞的YAP磷酸化加重病毒性急性肺损伤.
Abstract
Objective:To investigate the role of programmed death receptor ligand 1(PD-L1)in mediating the phosphorylation of lymphocyte yes-associated protein(YAP)in viral acute lung injury.Methods:SPF-grade C57BL/6J mice were randomly divided into control groups,and divided into groups of 4 h,8 h,1 d,3 d,and 7 d according to the time of intratracheal drip of polyinosinic-polycytidylic acid(Poly I:C).The group with the most severe inflammatory injury among these five groups was selected as the Poly I:C group;PD-1/PD-L1 inhibitor BMS-1 10mg/kg intraperitoneal injection pretreated with intratracheal drip of Poly I:C,as Poly I:C+ BMS-1 group.The Poly I:C group of wild-type mice and the Poly I:C group of PD-L1 knockout mice were treated as the Poly I:C group;the control group of wild-type mice and the control group of PD-L1 knockout mice were anesthetized by intraperitoneal injection and given an equal amount of saline intratracheally.Mice were eutha-nized at the corresponding time points and specimens were collected.Hematoxylin-eosin(HE)staining was used to assess the degree of lung tissue injury in mice;inflammation was assessed by measuring the total protein con-centration,total cell number in bronchoalveolar lavage fluid(BALF),and tumor necrosis factor(TNF)-α levels in BALF using enzyme-linked immunosorbent assay(ELISA).Western blotting was used to detect the expression of PD-1,PD-L1,YAP,and p-YAP proteins.Results:Compared with the control group,the lung histopathological in-jury score and total protein concentration in BALF were significantly increased in the 8 h,1 d,3 d,and 7 d groups,the total cell number in BALF was significantly increased in the 8 h,1 d,and 3 d groups,the TNF-α level in BALF was significantly up-regulated in the 4 h,8 h,1 d,and 3 d groups,and the differences were statistically significant(P＜0.05);among them,the lung histopathological injury score,total protein concentration in BALF,total cell number in BALF and TNF-α level in BALF in the 1d group were higher than those in the 4 h,8 h,3 d and 7 d groups.Compared with the control group,the levels of PD-1 and PD-L1 proteins in the lung tissues of mice in the 1 d,3 d,and 7 d groups were significantly increased(P＜0.05).The scores of pathological injury in the lung tissues of mice in the Poly I:C group and the levels of TNF-α in BALF were significantly higher than those in the control group.Compared with the Poly I:C group,the lung histopathological injury score and the lev-els of TNF-α in BALF were significantly lower than those in the Poly I:C+BMS-1 group(P＜0.05).Compared with the control group,YAP protein expression was significantly down-regulated and p-YAP protein expression was significantly up-regulated in the lymphocytes of mice in the Poly I:C and Poly I:C+BMS-1 groups(P＜0.05).Compared with the Poly I:C group,YAP protein expression in the lymphocytes of mice in the Poly I:C+ BMS-1 group was increased and p-YAP protein expression was decreased(P＜0.05).Compared with the control group,YAP protein expression was significantly down-regulated,and p-YAP protein expression was significantly up-regulated in the lymphocytes of mice in the Poly I:C group;YAP protein expression in the lymphocytes of mice in the Poly I:C group was increased,while p-YAP protein expression was significantly decreased after the knockdown of the PD-L1 gene,and the differences were all statistically significant(P＜0.05).Conclusion:PD-L1 protein is highly expressed in the lung tissues of mice with viral acute lung injury,and PD-L1 may aggravate viral acute lung injury by activating YAP phosphorylation in lymphocytes.
基金项目
国家自然科学基金(82060024)
广西医学高层次骨干人才"139"计划(G202002015)
广西麻醉学临床医学研究中心建设项目(桂科AD22035214)
维普
万方数据