Effect and mechanism of allosteric inhibitor of Src homology 2 domain-containing protein tyrosine phosphatase 2 in relieving radiation-induced pneumonitis
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-α and 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-α and 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 irradi-ation was detected by Western blotting.Results Lung inflammation was significantly relieved in irradiated mice by SHP099 administra-tion,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-α and 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 p22phox,p40phox,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).Con-clusions 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.
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