Circulating MtDNA Mediates CGAS-STING Pathway To Aggravate Intestinal Barrier Damage In Ischemic Enteropathy

The first part:Correlation between circulating mitochondrial DNA release and intestinal ischemia-reperfusionBackground: The restoration of tissue blood flow and reoxygenation is often accompanied by manifestations of tissue damage and a severe inflammatory response,which is known as ischemia-reperfusion injury(ischemia-reperfusion).The gastrointestinal tract,because of its blood supply pattern and tissue characteristics,is the organ most vulnerable to hypoxic-ischemia and reperfusion injury.Intestinal ischemia-reperfusion injury is a pathophysiological process that can lead to systemic inflammatory response syndrome and multi-organ dysfunction.Ischemia/reperfusion(I/R)injury and related therapies have become an increasingly important topic in clinical medicine.Mitochondrial DNA(mitochondrial DNA,mt DNA),as DAMPs released from mitochondria,is one of the key aspects of cellular stress following trauma,inflammation and systemic inflammatory response syndrome(SIRS).Recent studies have shown that mitochondrial DNA is associated with ischemia-reperfusion injury.The role of mitochondrial DNA release in intestinal ischemia-reperfusion injury is still not well defined.Methods: An ischemia-reperfusion model was established by reperfusion of blood flow in mice with different reperfusion time points(45 min ischemia time, reperfusion 0h,1h,3h,6h,12 h,24h,48h)after superior mesenteric artery(SMA)clamping.The changes in intestinal mucosal barrier damage and inflammatory response were observed at different reperfusion time points,and the damage to mitochondrial structures in intestinal epithelial cells was assessed to detect changes in the concentration of free mitochondrial DNA in the circulation after ischemiareperfusion.Mucosal barrier damage was assessed by gross examination and HE staining with pathology scoring,inflammatory factors related to intestinal inflammatory response such as interferon γ(IFNγ)and interleukin 6(IL6)were detected by real-time fluorescence quantitative PCR,mitochondrial structural damage was assessed by transmission electron microscopy,and mitochondrial DNA such as Cyt B,COX3,and ND1 were measured by double antibody sandwich enzyme-linked immunosorbent assay related factors.Results: Reperfusion was associated with damage to the intestinal mucosal barrier,there was a reperfusion time peak in the inflammatory response after ischemia-reperfusion,there was significant mitochondrial damage in the intestinal epithelium after ischemia-reperfusion,and there was a clear correlation between circulating mitochondrial DNA and ischemia-reperfusion treatment.After ischemiareperfusion treatment,intestinal injury was significantly aggravated,accompanied by a progressive increase in injury with prolonged reperfusion time,inflammatory cell infiltration,and increased systemic inflammatory response caused by intestinal injury,which reached a peak within 1 h after reperfusion and was also significantly higher than normal afterwards.Mitochondrial structure was significantly damaged after ischemia-reperfusion,with mitochondrial vacuolization,destruction of spinal membrane structure,fracture,and loss of spinal membrane,and mitochondrial DNA level was significantly increased after ischemia-reperfusion injury and was at a higher level within 48 h.The second part:cGAS-STING pathway Pro-inflammatory pathway relevance of mt DNA released in ischemia-reperfusionBackground: The c GAS-STING signaling pathway is a newly discovered natural immune pathway that exerts anti-microbial infection effects by recognizing exogenous nucleic acids(derived from bacteria,viruses)and endogenous nucleic acids(e.g.mitochondrial DNA),activating pathogen-associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs),and inducing the release of type I interferons and other inflammatory factors.There are still few studies related to the link between STING signaling pathway and ischemia-reperfusion injury,and the potential of STING as a new natural immune pathway in clinical therapy remains to be explored.Methods: In this study,we examined the dynamic distribution of STING pathway-related signals in cells and validated the activation of STING pathway after ischemia-reperfusion in a hypoxic reoxygenation cell model.By confocal microscopy,we observed the localization of STING and IRF3 in cells in immunofluorescence experiments and their localization changes before and after hypoxia.An ischemia-reperfusion model was constructed,and the phosphorylation of STING,IRF3 and TBK1 was assessed by Western blot.Results: IRF3 significantly aggregated in the perinuclear and cytoplasmic areas after ischemia-reperfusion;STING significantly aggregated in the nucleus after ischemia-reperfusion.c GAS-STING pathway phosphorylated IRF3 and TBK1 at elevated levels after ischemia-reperfusion,with the most pronounced phosphorylation at one hour of reperfusion,and continued to maintain high levels.This indicates that the STING pathway is continuously activated after ischemia-reperfusion.