含Src同源2结构域蛋白酪氨酸磷酸酶2变构抑制剂缓解放射性肺炎的作用效应与机制研究

Effect and mechanism of allosteric inhibitor of Src homology 2 domain-containing protein tyrosine phosphatase 2 in relieving radiation-induced pneumonitis

目的探索含Src同源2结构域蛋白酪氨酸磷酸酶2(Src homology 2 domain-containing protein tyrosine phosphatase 2,SHP2)变构抑制剂对放射性肺炎的缓解作用及其可能的机制。方法以50 Gy的剂量进行双肺辐照建立辐射诱导放射性肺炎小鼠模型,分别提取SHP2变构位点抑制剂SHP099辐照组(辐照并予以SHP099灌胃)、辐照组(辐照未灌胃)、SHP099未辐照组(未辐照并予以SHP099灌胃)和对照组(未辐照且未灌胃)小鼠的肺组织制作HE切片。通过实时荧光定量聚合酶链反应(real time quantitative polymerase chain reaction,RT-qPCR)检测4组小鼠肺组织内炎性反应因子诱生型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)的mRNA水平。RT-qPCR检测小鼠单核巨噬细胞白血病细胞RAW264.7和骨髓原代巨噬细胞(bone marrow derived macrophage,BMDM)中iNOS、TNF-α和IL-6的表达情况。收集BMDM培养上清采用酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)检测TNF-α和IL-6的分泌情况。通过流式细胞术分析辐照后不同培养时间后RAW264.7和BMDM细胞产生活性氧(reactive oxygen species,ROS)的水平。采用RT-qPCR检测10 Gy辐照后24 h RAW264.7细胞还原型烟酰胺腺嘌呤二核苷酸磷酸(reduced nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶(NADPH oxidase,NOX)各亚基和同系物表达水平变化。采用蛋白质印迹法检测RAW264.7细胞中NOX4表达水平。结果通过SHP099灌胃辐射小鼠模型发现,SHP099预处理的辐照小鼠肺部损伤减弱,肺组织内炎性反应因子iNOS、TNF-α和IL-6的mRNA表达均下调(均P<0.05)。10 mmol/L SHP099预处理24 h后,RAW264.7和BMDM细胞因10 Gy辐照引起的上升的炎性反应因子iNOS、TNF-α和IL-6 mRNA表达均下降(均P<0.05)。收集BMDM细胞的培养上清显示,SHP099预处理也可减少辐照后TNF-α和IL-6蛋白的分泌(均P<0.05)。SHP099预处理也可降低10 Gy辐照后30 min引起的RAW264.7和BMDM细胞升高的ROS水平(均P<0.05)。采用RT-qPCR检测RAW264.7细胞10 Gy辐照后24 h NOX各个亚基和同系物的表达变化情况显示,p22^(phox)、p40^(phox)、Rac1、NOX2、NOX3、NOX4和NOX5表达均上调(均P<0.05),其中NOX4上调最多;而SHP099预处理可减少辐照后RAW264.7细胞的NOX4蛋白表达(P<0.01)。结论SHP2变构抑制剂可以缓解辐照诱导的小鼠肺部炎性反应。SHP2变构抑制剂可能是通过抑制巨噬细胞中NOX4的表达降低ROS产生,从而减少炎性反应因子分泌。

Objective To study the mitigation effect of the allosteric inhibitor of Src homology 2 domain-containing protein tyrosine phosphatase 2(SHP2)on radiation-induced pneumonitis and its possible mechanism.Methods A mouse model of radiation-induced pneumonitis was established by 50 Gy whole lung irradiation,lung tissues from four mouse groups including SHP099 group irradiated with SHP099 intragastric administration,irradiated group irradiated without intragastric administration,SHP099 unirradiated group unirradiated with SHP099 intragastric administration,and control group unirradiated without intragastric administration,were extracted,and HE staining was conducted.Real time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA levels of inducible nitric oxide synthase(iNOS),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in the lung tissues of the four groups.The expressions of iNOS,TNF-αand IL-6 in mouse mononuclear macrophage RAW264.7 cells and primary bone marrow derived macrophage(BMDM)cells were detected by RT-qPCR.BMDM supernatant was collected to detect the secretion of TNF-αand IL-6 using enzyme-linked immunosorbent assay(ELISA).The level of reactive oxygen species(ROS)from RAW264.7 and BMDM cells at different culture time after irradiation was analyzed by flow cytometry.Twenty-four hours after irradiation,the expressions of the subunits and homologues of reduced nicotinamide adenine dinucleotide phosphate(NADPH)oxidase(NOX)were detected by RT-qPCR.NOX4 expression in RAW264.7 cells after irradiation was detected by Western blotting.Results Lung inflammation was significantly relieved in irradiated mice by SHP099 administration,and the mRNA expressions of inflammatory cytokines including iNOS,TNF-α,and IL-6 in the lung tissues were down-regulated(all P<0.05).The increased mRNA expressions of iNOS,TNF-α,and IL-6,induced by 10 Gy irradation,in RAW264.7 and BMDM cells all decreased after 24 h pretreatment with 10 mmol/L SHP099(all P<0.05).The SHP099 pretreatment reduced the secretion of TNF-αand IL-6 in BMDM cells induced by irradiation(both P<0.05).The SHP099 pretreatment decreased the upregulated ROS levels in RAW264.7 and BMDM cells 30 min after 10 Gy irradiation(both P<0.05).RT-qPCR showed that the expressions of NOX subunits and homologues including p22^(phox),p40^(phox),Rac1,NOX2,NOX3,NOX4,and NOX5 all increased in RAW264.7 cells 24 h after irradiation(all P<0.05),with NOX4 being the most,and the SHP099 pretreatment reduced the expression of NOX4 in RAW264.7 cells after irradiation(P<0.01).Conclusions SHP2 allosteric inhibitor can alleviate radiation-induced pneumonitis in mice,possibly by reducing ROS production through inhibiting NOX4 expression in macrophages,thereby reducing inflammatory cytokine secretion.