METTL3 Aggravates Atherosclerosis Via Modulating Ox-LDL Induced Macrophage Pyroptosis And Inflammation Responses

Atherosclerosis（AS）is a chronic inflammatory and progressive vascular inflammatory disease caused by inflammatory cell infiltration and lipid deposition,with high morbidity and mortality in the clinic.In recent years,the role of METTL3（methyltransferase-like protein 3）and m6A（N6-methyladenosine）modification in AS has aroused much concern.The research shows that METTL3-mediated m6A modification can promote the occurrence of AS by inducing the inflammatory response of endothelial cells,promoting the polarization of M1 macrophages,and promoting ox-LDL-mediated inflammatory response and angiogenesis.METTL3,as a key enzyme in catalytic methylation modification,can be widely expressed in AS,and METTL3knockdown can reduce lipid levels and inhibit plaque formation.So far,the specific mechanism by which METTL3 occurs and develops in macrophage pyroptosis induced by ox-LDL remains unclear.In this study,ox-LDL was used to induce mouse monocyte-macrophage leukemia cells（RAW264.7）,and METTL3 knockdown and overexpression intervention was carried out in vivo and in vitro,to explore the involvement of METTL3 in the occurrence and development of AS by promoting ox-LDL induced pyroptosis and inflammation of macrophagesObjective:To explore the effect of METTL3 on ox-LDL-induced macrophages pyroptosis in vitro and the impact of METTL3 on atherosclerosis in vivo.Method:1.RAW264.7 cells were incubated with ox-LDL（50μg/ml）for various periods,and the total cholesterol kit was used to evaluate the intracellular cholesterol content.The m RNA and protein expression levels of METTL3,NLRP3,NF-κB p65,GSDMD-N,Caspase-1,IL-1β,and IL-18 related genes were determined utilizing quantitative fluorescence PCR and Western blot.The METTL3 was then knocked down and overexpressed in cultured cells to detect the methylation level of total RNA m6A.m RNA and protein expression levels of METTL3 and pyroptosis related genes were detected after ox-LDL induction and METTL3 intervention by RT-PCR and Western blot;Its LDH cytotoxicity assay and Calcein AM/PI staining were utilized in order to assess cell pyroptosis;The enzyme-linked immunosorbent assay was applied to evaluate inflammatory cytokines the levels of IL-1β,IL-10,IL-18,TNF-α,and IL-6.2.Ten apo E^-/-normal diet mice were selected as control,50 apo E^-/-mice were selected to feed high fat diet for 12 weeks to establish high fat model,and then 40 apo E^-/-were randomly selected for follow-up experiment,with 10 mice in each group.The test subjects received a caudal vein injection.The following experimental groups were established:METTL3 overexpressed negative control lentivirus（OE-Control）;METTL3 overexpressed lentivirus（OE-METTL3）;METTL3 knockdown control lentivirus（METTL3-sh NC）;and METTL3 knockdown lentivirus（LV3-METTL3）.The injection cycle was 2 times/week for four weeks while high-fat feeding was maintained,and the remaining ten animals were fed a high-fat diet.Western blot was used to detect the knockdown and overexpression effects of METTL3,and the expression of METTL3 in each group.Plaque progression was evaluated by aorta gross oil red O staining,frozen sections of aortic sinus oil red O staining and HE staining.After the intervention,the biochemical levels of TG,TC,LDL-C,and HDL-C in serum of the 6groups of mice were detected by the kit.Western blot was applied to calculate the protein expression levels of pyroptosis-related genes in the aorta.Inflammatory cytokines expression of serum was detected by enzyme-linked immunosorbent assay,to further confirm the effect of METTL3 on pyroptosis of atherosclerosis.Results:1.Intracellular cholesterol content was significantly higher in RAW264.7 cells after ox-LDL（50μg/ml）incubation（P<0.05）;m RNA and protein expression levels of NLRP3,IL-1β,NF-κB p65,GSDMD-N,IL-18,and Caspase-1 were increased in contrast to the control group（P<0.05）;Furthermore,METTL3 and RNA m6A expression levels in RAW264.7 cells were dramatically upregulated after ox-LDL（50μg/ml）induction（P<0.05）.2.METTL3 knockdown and overexpression were used to confirm that METTL3 can promote macrophage pyroptosis and the expression of inflammatory factors using fluorescence quantitative PCR,Western blot,LDH cytotoxicity detection,Calcein AM/PI staining,and ELISA.3.The expression of METTL3 was significantly increased after high fat diet,and the increase was most obvious after METTL3 overexpression.Aorta gross oil red O staining and aortic sinus frozen section oil red O staining and HE staining showed that the plaques were the most obvious after METTL3 overexpression,with a large number of foam cells clustered and inflammatory cells infiltrated.4.Mice fed a high fat diet had higher levels of TC,LDL-C,and TG and lower levels of HDL-C than mice fed a normal diet;The levels of TC,TG,and LDL-C in the METTL3 overexpression group were significantly higher than in the apo E^-/-mice fed high fat（P<0.05）,and the levels of HDL-C in the METTL3knockdown group were significantly decreased（P<0.05）;While especially in comparison to the METTL3 overexpression negative control group,the levels of TG,TC,and LDL-C were significantly increased in the METTL3 overexpression（P<0.05）;While especially in comparison to the METTL3 knockdown control group,the levels of TG,TC,and LDL-C were markedly reduced（P<0.05）;nevertheless the level of HDL-C was remarkably increased（P<0.05）.5.Overexpression of METTL3 increased the expression of pyroptosis genes.6.The levels of IL-18,IL-6,and TNF-αwere significantly up-regulated（P<0.05）,the content of IL-1βwere increased,and the levels of IL-10 were significantly decreased in high-fat fed apo E^-/-mice（P<0.05）.The levels of IL-1β,IL-18,and TNF-αwere substantially increased in the METTL3overexpression group compared to the apo E^-/-mice fed high fat（P<0.05）.The levels of IL-1β,IL-6,IL-18,and TNF-αwere significantly higher in the METTL3overexpression group compared to the METTL3 overexpression negative control group（P<0.05）.When compared to the METTL3 knockdown control group,the levels of IL-1β,IL-6,IL-18,and TNF-αwere remarkably declined（P<0.05）,whereas the content of IL-10 was significantly enhanced（P<0.05）.Conclusion:1.Ox-LDL can aggravate macrophages pyroptosis and increase both the expression of METTL3 and the RNA m6A methylation level,whereas METTL3 levels can influence ox-LDL induced pyroptosis and the inflammatory factors expression of in macrophages.2.METTL3 can up-regulates the blood lipid level of atherosclerotic mice in vivo,promote the expression of pyroptosis and inflammatory factors,and aggravate plaque formation.