The Role Of IRG1/Itaconate In Intestinal Ischemia-Reperfusion Injury

Backgound and objective:Intestinal ischemia-reperfusion(I/R)injury has been a problem for clinical surgeons,because it can not only lead to injury of the small intestine,but also trigger systemic inflammatory response,multiple organ failure,and even death in severe cases.Intestinal I/R injury is a multifactorial regulatory process involving inflammatory response,oxidative stress,bacterial translocation and mucosal barrier dysfunction.Immune response gene 1(IRG1)is an important regulator of cellular immunity and metabolism,and itaconate isan IRG1-mediated decarboxylation of cis-aconitate.IRG1/itaconate has attracted much attention because of its considerable and antioxidant effects,but its role in intestinal I/R has not been reported.For this experiment we use 4-octyl itaconate(4-OI),a cell-permeable itaconate derivative.4-OI and itaconate have similar thiol reactivity and are considered to be a suitable cell-permeable substitute for itaconate.In this study,the mechanism of IRG1/itaconate in intestinal ischemia-reperfusion injury are studied by inducing intestinal I/R model in mice in vivo and establishing hypoxia/reoxygenation(H/R)injury model of Caco-2 cells in vitro toinvestigate the mechanism of IRG1/itaconate in intestinal ischemia-reperfusion injury.Methods:1.Effect of 4-OI on I/R injury in mice: Twenty healthy male C57-BL mice were randomly divided into sham operation group(Sham group),intestinal ischemia-reperfusion group(I/R group),sham operation+4-OI group(Sham+4-OI)and 4-OI treatment group(I/R+4-OI).The intestinal I/R model was constructed by the Medison method.The superior mesenteric artery was clipped for 45 minutes,and released and reperfused for 4 hours.In the 4-OI group,the corresponding dose was injected intraperitoneally 2 hours before surgery.By observing the histopathological changes of the small intestine,detecting intestinal barrier-related proteins:zonula occludens 1(ZO-1),occluding protein(occluding),tight junction protein 1(claudin-1)and the expression levels of inflammatory mediators: interleukin 1-β(IL-1β),interleukin 6(IL-6),and tumor necrosis factor-α(TNF-α)in serum and intestinal oxidative stress indicators: glutathione(GSH),malondialdehyde(MDA),superoxide dismutase(SOD)and apoptosis-related indicators caspase-3,caspase-7were observed.2.Effect of 4-OI on H/R damage of Caco-2 cells: The cells were randomly divided into 5 groups: normal control group(control group);H/R group;Control+4-OI group;H/R+4-OI group group;si-IRG1+H/R group.The drug was administered 2 hours before modeling,followed by hypoxia for 6 hours and reoxygenation for 6 hours to model,and the activity of the cells was detected by CCK8 assay and apoptosis was measured by microplate reader.Results:1.Compared with the Sham group,the histopathology of the small intestine of the mice in the I/R group showed that the arrangement of the small intestinal villi was disordered,the top of the villi fell off,the capillaries were congested,and the subepithelial space did not show significant widening;the protein expression of intestinal barrier indicators ZO-1,occludin,and claudin-1 was significantly decreased,and the serum IL-1β,IL-6,and TNF-α levels were significantly decreased;the levels of MDA in the small intestine tissues were significantly increased,and the levels of GSH and SOD were significantly decreased;and the caspase-3 and caspase-7 protein levels in the small intestine tissues were significantly increased.2.Compared with the I/Rgroup,the I/R+4-OI group: small intestinal histopathology showed that the small intestinal villi were arranged slightly neatly,the congestion was relieved,and the subepithelial dilatation was mild;the intestinal barrier indexes ZO-1,occludin,and claudin-1 proteins expression increased compared with before,and the serum IL-1β,IL-6,and TNF-α concentrations were significantly decreased;the level of MDA in the small intestinal tissue decreased,and the levels of GSH and SOD increased;the protein expression of caspase-3 and caspase-7 in the small intestinal tissue decreased significantly.3.Compared with the control group,the cell activity of the H/R group was significantly decreased,and the level of apoptosis was significantly increased;after pretreatment with4-OI,the cell activity increased,and the level of apoptosis was significantly decreased.Conclusion:1.Intestinal ischemia-reperfusion can lead to disruption of intestinal mucosal integrity,increased apoptosis,and massive release of systemic inflammatory cytokines.2.Pretreatment with 4-OI can reduce the damage of intestinal mucosal barrier,reduce the level of oxidative stress and apoptosis in the intestine,and improve the inflammatory response in mice.3.IRG1/itaconate can protect intestinal ischemia-reperfusion injury through anti-inflammatory and anti-oxidative stress effects.