The Effect And Mechanism Of RNA N6-methyladenosine Modification In Lps-induced Pulmonary Inflammation And The Effect Of Vitamin D₃ On Its Mitigation

There are many complex pathogenic factors such as bacterial infection and ammonia stimulation in farms,which induce inflammation in livestock and poultry.The deterioration of lung inflammation without timely treatment results in seriously threatens health and quality of livestock and poultry.Currently,therapeutic strategies for lung inflammation focus on anti-infective therapy and neutralizing inflammatory mediators.N6-methyladenosine（m6A）of m RNAs modification plays an essential role in the progress of immunity and inflammation,but the mechanism of action remains controversial.The role of m6A modification in lung inflammation,especially the regulatory mechanism in epithelial cell damage caused by pneumonia,remains to be investigated.In addition,as a feed additive-vitamin D₃（VD₃）supplementation increase the immune function of animals and efficiently alleviate pneumonia.Yet the role of VD₃ on RNA epigenetic modification during lung inflammation remain unclear.Therefore,in this study,by using overexpression and knockdown,applicating immunohistochemistry,Wright’s Giemsa staining,Dot blot,immunofluorescence staining,protein solid-phase antibody chip analysis,RT-q PCR,Western blot,RNA-seq,m6A-seq,RIP-q PCR,Ch Ip-q PCR,half-life analysis,dual-luciferase reporter analysis and other technical methods,aimed to explore the role and potential regulation mechanism of m6A modification in the development of lung inflammation at the individual and cellular levels,as well as the protective effect of vitamin D₃ supplementation on lung inflammation and injury.The main findings of the study are as follows:1.There is accompanied by down-regulation of METTL3 and RNA m6A modification levels in LPS-induced broiler and mouse lung inflammation models.The broiler pneumonia model was established by intraperitoneal injection of 1 mg/kg LPS into AA broilers three times.HE staining showed that the infiltration of inflammatory cells in the lung tissue of broilers was increased and the pro-inflammatory cytokines TNFαand IL-1βwere significantly up-regulated in the lung tissue of LPS-induced broiler pneumonia model（P<0.05）.These results indicated that the pneumonia model was successfully constructed.Further,in broiler pneumonia model,m6A modification levels and METTL3 expression were significantly reduced（P<0.05）.Other key m6A-catalytic enzymes METTL14,ALKBH5 and FTO were not significantly changed in acute lung injury.Furthermore,immunohistochemistry results indicated that METTL3 was localized in lung airway epithelial cells.2.TNFαand IL-1βco-regulate the dynamic METTL3 expression and m6A levels.In vitro,primary human peripheral blood mononuclear cells（PBMCs）and pulmonary epithelial cells（BEAS-2B cells）were co-cultured in trans-wells.The cytokine TNFαrelease from LPS-stimulated PBMCs significantly down-regulated METTL3 and m6A levels,whereas IL-1βsignificantly increased METTL3 and m6A modification levels.BEAS-2B cells were stimulated with different ratios of IL-1βand TNFα,and the m6A levels and METTL3expression changed dynamically.Further,TNFαmitigated METTL3 and m6A modification through SOCS3/STAT3 signaling pathway in lung epithelial cells.STAT3 functions as a transcription factor for METTL3.3.METTL3 targets GATA6.In TNFα-stimulated BEAS-2B cells,24 target genes were significant changes in both m6A and RNA sequencing.Further,in TNFαstimulated lung epithelial cells,METTL3 overexpression significantly alleviated the TNFα-induced the increase of GATA6 expression.RIP assay showed that METTL3 targets the 3’UTR region of GATA6 m RNA to promote GATA6 m RNA degradation in YTHDF2 dependent manner,a m6A recognition protein.Further,knockdown of GATA6 expression significantly inhibited NF-κB signaling pathway and the release of pro-inflammatory factors stimulation by TNF-αin pulmonary epithelial cells,thereby ameliorating pulmonary epithelial injury.4.METTL3 overexpression ameliorates LPS-induced lung inflammation.In lung epithelial cells BEAS-2B,knockdown or overexpression of METTL3 significantly inhibited or upregulated m6A modification levels in total m RNA（P<0.05）.METTL3 knockdown promoted pro-inflammatory cytokine expression release from epithelial cells,whereas METTL3 overexpression inhibited the expression of proinflammatory cytokines such as IL-6and TNFαin BEAS-2B cells.Similarly,METTL3 overexpression in vivo suppressed LPS-induced pro-inflammatory cytokines such as IL-6,IL-1β,and TGF-βrelease in lung tissue and bronchoalveolar lavage fluid companied with decreased GATA6 expression level,thereby ameliorating LPS-induced lung inflammation and damage.5.VD₃ treatment alleviates LPS-induced lung inflammation.In the LPS-induced lung inflammation models,SOCS3 expression level was significantly increased（P<0.05）while STAT3 and METTL3 expression level were significantly up-regulated（P<0.05）.Single treatment of VD₃ up-regulated STAT3 and METTL3 protein expression levels by inhibiting SOCS3 expression and also significantly inhibited the LPS-induced infiltration of inflammatory cells and increased pro-inflammatory cytokines levels in the lung tissue,thereby alleviating pneumonia.While,long-term vitamin D₃ treatment had no effect.In conclusion,our study clarifies the signaling pathway that regulates the changes of METTL3 expression and m6A levels in lung inflammation.The findings further elucidate METTL3-mediated m6A modification alleviates lung inflammation and injury by targeting GATA6 to inhibit the of pro-inflammatory cytokines release from lung epithelial cells.It was found that single treatment of VD₃ ameliorated lung inflammation and injury by regulating METTL3.These results provide a scientific basis for the prevention of pulmonary inflammatory diseases in the process of livestock and poultry breeding.