The Effect Of CGAS-STING Pathway Mediates Macrophage Death And M1 Polarization On Atherosclerosis

Objective:Atherosclerosis（AS）is an important pathological basis leading to cardiovascular disease,and macrophages and inflammation play an important role in the occurrence and development of AS.Numerous studies have shown that the Cyclic GMP-AMP synthase（c GAS）-Stimulator of interferon genes（STING）signaling pathway mediates inflammatory responses in cardiovascular disease,while its role in AS has not been elucidated.Our study aimed to investigate the role of the c GAS-STING signaling pathway in palmitic acid（PA）-induced macrophage death and inflammation.Methods:1.PA induces c GAS-STING pathway activation and cell death in macrophages:Bone marrow-derived macrophages were extracted,and Western blot was used to detect c GAS,STING,phosphorylated Tank binding kinase 1（TBK1）,phosphorylation interferon regulatory factor 3（IRF3）,nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3（NLRP3）,cysteinyl aspartate specific proteinase 1（caspase1）,cysteinyl aspartate specific proteinase 3（caspase3）,cleaved-caspase3,B cell lymphoma-2（Bcl2）and Bcl-2associated X protein（Bax）protein expression after PA treatment;immunofluorescence observed the nuclear translocation of p-IRF3 in macrophages treated with PA;q RT-PCR detected inflammatory factor type1 interferonβ（IFN-β）,interleukin-6（IL-6）,interleukin-1β（IL-1β）,interleukin-18（IL-18）and tumor necrosis factor-α（TNF-α）m RNA expression after PA treatment.2.The effect of knockdown of c GAS or STING on macrophage death:using small interfering RNA to knock down the expression of c GAS or STING in macrophages,and detect the protein expression of NLRP3,caspase1,cleaved-caspase3,Bax and Bcl2 by western blot;q RT-PCR to detect the m RNA expression of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-α;TUNEL and caspase1 double-staining and flow cytometry to observe cell death;LDH release to detect cell membrane integrity.3.The effect of knockdown of c GAS or STING on macrophage polarization:western blot was used to detect the changes of macrophage M1 and M2 marker proteins,and q RT-PCR was used to detect the m RNA expression of macrophage M1 and M2 macrophage markers.4.Activated macrophage mitochondrial oxidative stress and endoplasmic reticulum stress after PA treatment:the changes of mitochondrial morphology of macrophages after PA treatment were observed by transmission electron microscope;the content of mitochondrial reactive oxygen species in macrophages after PA treatment was detected by flow cytometry;Immunofluorescence was used to observe the oxidative damage of DNA;q RT-PCR was used to detect the content of mitochondrial DNA in the cytoplasm after PA stimulation,western blot was used to detect the effect of mt DNA depleting agent ethidium bromide（Et Br）on the protein expressions of c GAS,STING and p-TBK1,q RT-PCR detected the m RNA expression of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-α;endoplasmic reticulum stress inhibitor4-hydroxybutyric acid（4-PBA）inhibited endoplasmic reticulum stress activation and then detected the expression of heavy chain binding protein（Bip）,C/EBP-homologous protein（CHOP）,STING and p-TBK1 proteins were detected by western blot,and the m RNA expressions of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αwere detected by q RT-PCR.5.Effects of inhibition of c GAS-STING signaling pathway activation on plaque stability in Apo E^-/-mice:32 male Apo E^-/-mice aged 6-8 weeks were randomly divided into four groups:normal diet group,high-fat diet group,high-fat diet given C176 treatment group and high-fat diet given RU.521 treatment group for 12 weeks.Serum was collected to detect blood lipid levels,and ELISA was used to detect the content of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αin serum;immunohistochemistry was used to observe the content ofα-SMA in the aortic root,and plaques were calculated stability index.Results:1.Compared with the CTRL group,the protein expressions of c GAS,p-TBK1,p-IRF3,NLRP3,caspase1,cleaved-caspase3,Bax and Bcl2 were significantly increased after PA treatment,but there was no significant change in the expression of STING protein,and the inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αm RNA expression was significantly increased.2.Compared with the PA group,after knockdown of c GAS and STING expression in macrophages with small interfering RNA,the protein expressions of NLRP3,caspase1,and cleaved-caspase3 in macrophages were significantly down-regulated,and the ratio of Bcl2/Bax was significantly increased;the m RNA expressions of IL-6,IL-1β,IL-18 and TNF-αwere significantly decreased;the co-localization of TUNLE and caspase1 was reduced,the apoptosis rate was significantly decreased,and the release of LDH was significantly decreased.3.Knockdown of c GAS and STING expression in macrophages could reverse the PA-induced up-regulation of macrophage M1 polarization marker i NOS protein expression but failed to reverse the PA-induced decrease in macrophage M2 polarization marker CD206 expression.Compared with the NC+PA group,knockdown of c GAS and STING in macrophages significantly decreased the m RNA expressions of M1polarization-related markers i NOS,CD86 and CXCL-10,and knockdown of STING reversed the PA-induced CD206 and Arg1 m RNA levels,but without improve the expression level of YM1 m RNA,whereas knockdown of c GAS reversed the PA-induced decrease in YM1 m RNA expression level without significant effects on the expression of CD206 and Arg1.4.Compared with the CTRL group,the mitochondrial morphology of macrophages treated with PA was abnormal,the mt ROS content was significantly increased,the DNA oxidative damage was obvious,and the mt DNA content in the cytoplasm was significantly up-regulated.Compared with the PA group,the protein expressions of c GAS and p-TBK1 were significantly down-regulated after Et Br treatment,the expression of STING protein had no significant change,and the m RNA expressions of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αwere significantly decreased.4-PBA pretreatment significantly reduced the expression of endoplasmic reticulum stress marker proteins Bip and CHOP,and significantly reduced the protein expression of p-TBK1,the m RNA expression of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αwas also significantly decreased.5.Inhibiting the activation of the c GAS-STING signaling pathway decreased the contents of total cholesterol,triglycerides and low-density lipoprotein cholesterol and the contents of inflammatory cytokine IFN-β,IL-6,IL-1β,IL-18 and TNF-αin serum of Apo E^-/-mice,and increased plaque stability in Apo E^-/-mice.Conclusion:The c GAS-STING signaling pathway is activated in macrophages,thereby increasing the pyroptosis,apoptosis and M1polarization of macrophages,and further aggravating the formation of unstable plaques in AS.