The Mechanism Of Preoperative Fasting Improve Intestinal Microbiota Dysbiosis Against Intestinal Ischemia/Reperfusion Injury

BackgroundGut-derived sepsis(GDS)is a special type of sepsis,with concealed occurrence,difficult diagnosis,and high mortality.Intestinal ischemia/reperfusion(I/R)injury is a common perioperative clinical and acute serious event,associated with trauma,severe infection,shock,small intestine transplantation,and cardiac bypass surgery.Intestinal I/R had caused the destruction of the intestinal barrier,the translocation of endotoxin,and microbiota dysbiosis,which were the key causes of GDS.Preoperative fasting(PF)is an important preparation to ensure safety in surgical patients.However,there is currently a lack of preoperative fasting strategies for patients with a high risk of intestinal I/R injury.Researchers had found the important impacts of fasting on the composition and abundance of gut microbiota.However,the effects of PF on microbiota dysbiosis induced by intestinal I/R have remained unclear.ObjectiveTo explore the optimal time of PF to protect against intestinal I/R injury and the effects and mechanisms of PF on gut microbiota and their metabolites after intestinal I/R injury in mice.Methods1.The intestinal I/R model was established by clamping the superior mesenteric artery(SMA)for 45 min and then reperfusion for 120 min in C57BL/6J mice.In the Sham group,the SMAs in mice were only isolated without clamped.The mice were randomly received with Ad libitum(AL)or PF for 6 h,12 h,or 24 h treatments.The pathological lesions of the intestinal section,intestinal permeability function,cell apoptosis level,intestinal and serum inflammatory cytokines,and mortality after intestinal I/R were observed.2.C57BL/6J mice were received with either AL or PF for 24 h treatment before I/R,and we detected and analyzed the gut microbiome of cecal contents in the AL or PF group with 16S rDNA sequencing.Besides,we recruited 18 healthy volunteers and analyzed their fecal microbiota before and after fasting(21.65±0.30 h)with 16S rDNA sequencing technology.We established the pseudo-germfree mice by administering intragastrically with combined antibiotics(Neomycin,200 mg/kg;Metronidazole,200 mg/kg;Ampicillin,200 mg/kg;Vancomycin,100 mg/kg)and mice were randomly divided into AL or PF recipient groups.The feces in mice or human beings after AL or PF were collected and dissolved in PBS(0.125 g/mL)to obtain the bacteria solutions,which were transplanted into the recipient mice respectively.The pathological lesions of the intestinal section,intestinal permeability function,cell apoptosis level,intestinal and serum inflammatory cytokines,and mortality after intestinal I/R were observed.3.C57BL/6J mice were received with either AL or PF for 24 h treatment,we detected and analyzed the microbiota metabolites of cecal contents in the AL or PF group with non-targeted metabolites sequencing.Petroselinic acid(PSA),a significantly different metabolite,was detected and verified by using LC/MS.Mice were randomly divided into Control,I/R,and PSA+I/R groups.The PSA+I/R mice were administrated intraperitoneally with 100 mg/kg PSA at 1 h before ischemia and the start of reperfusion,and the control and I/R mice were treated with vehicle.The pathological lesions of the intestinal section,intestinal permeability unction,cell apoptosis level,intestinal and serum inflammatory cytokines,and mortality after intestinal I/R were observed.4.We used intestinal epithelial cell 6(IEC6)to build the oxygen-glucose deprivation and reoxygenation(OGD/R)model.The cells were cultured in the ulbecco’s modified eagle medium(DMEM)without fetal bovine serum(FBS)and glucose and incubated under the hypoxia environment(1%O2,5%CO2,94%N2)for 12 h,and then replaced with high-glucose DMEM containing 10%FBS and incubated under the normoxia environment(5%CO2,21%O2)for 4 h.The cells were divided into five groups in this experiment,including NC,OGD/R,PSA+OGD/R,PSA+Compound C+ OGD/R,and PSA+ MHY1485+OGD/R groups.The NC group was treated with 0.1%DMSO without OGD/R.The OGD/R group was treated with 0.1%DMSO for 1 h before OGD/R.The PSA+OGD/R group was treated with 10 μM PSA for 1 h before OGD/R.The PSA+Compound C+OGD/R group was treated with 10 μM AMPK inhibitor for 4 h and 10 μM PSA for 1h before OGD/R.The PSA+MHY1485+OGD/R group was treated with 5 μM mTOR agonist for 2 h and 10 μM PSA for 1 h before OGD/R.The levels of cell apoptosis and the expressions of the AMPK-mTOR pathway were detected after OGD/R.Results1.PF for 24 h conferred protection against intestinal I/R injury,manifested as villi and crypt shedding reduced,cell apoptosis inhibited,mucosal permeability decreased,inflammatory response lessened,and survival rate increased.2.The abundance and composition of gut microbiota had been changed and the diversity has been decreased after intestinal I/R.PF for 24 h reshaped the intestinal microecology of mice and human beings and improved the microbiota dysbiosis induced by intestinal I/R.3.Fecal microbiota transplantation(FMT)has delivered the protection effects of PF on intestinal I/R.The recipients with FMT from PF mice or PF human beings have shown more resistance to intestinal I/R injury,manifested as villi and crypt shedding reduced,cell apoptosis inhibited,mucosal permeability decreased,inflammatory response lessened,and survival rate increased.4.The composition and abundance of microbiota metabolites have been changed and the PSA level was increased after PF.The PSA has alleviated the intestinal I/R injury and inhibited cell apoptosis by activating the AMPK-mTOR-P70S6K pathway after OGD/R.ConclusionPreoperative fasting for 24 h reduced the tissue damage and the mortality after intestinal I/R injury in mice.Gut microbiota played a key role in the protection of PF against intestinal I/R injury,and bacterial metabolite PSA restrained cell apoptosis by up-regulating the AMPK-mTOR-P70S6K pathway.