The Role And Mechanisms Of Gut Microbiota Metabolite Succinate On Acute Lung Injury Induced By Intestinal Ischemia-Reperfusion

Background and objectiveIntestinal ischemia/reperfusion(I/R)injury is a fatal condition that frequently occurs after severe trauma,infection,shock,acute mesenteric ischemia and cardiopulmonary bypass(CPB)surgery.Injuries occur in the intestinal mucosa and intestinal barrier,resulting in intestinal bacteria and endotoxin translocation and subsequently resulting in multiple organ dysfunction or failure.Of the remote organ injuries,acute lung injury(ALI)is extremely common following intestinal I/R,and becomes a leading cause of mortality.Currently,the underlying mechanisms of ALI have not been fully elucidated,and specific therapeutic approaches targeting for gut-derived ALI are not available.Therefore,it is desirable to address the issues involved in ALI of critically ill patients.The gut microbiota and its metabolic byproducts play crucial role in modulating immune function and lung disease pathogenesis,and act as important modulators of termed the gut-lung axis.Recently,increasing attention has been paid to that succinate is an important metabolite of the gut microbiota.However,the role of succinate in intestinal I/R-induced ALI remains unclear.The present study was designed to explore the role and mechanisms of gut microbiota-derived succinate in the progression of intestinal I/R-induced ALI,and aimed to reveal the pathogenesis of ALI and to develop novel therapeutic strategy for gut-derived ALI.Methods1.Investigate the relationship between accumulating lung succinate and specific intestinal flora during intestinal I/R.(1)An ALI model induced by intestinal I/R was established in mice.Succinate levels in the lung,blood,intestine and cecal contents were measured at different reperfusion time points during intestinal I/R.(2)16S rRNA sequencing was performed to analyze the diversity and abundance of intestinal flora,and to screen specific intestinal flora involved in succinate metabolism.Subsequently,the correlation between lung succinate and the screened intestinal flora was analyze.2.Verification the contribution of gut microbiota in the source of lung succinate during intestinal I/R.(1)We perform antibiotics(ABX)pre-treatment and fecal microbiota transplantation(FMT)experiments to prove that gut microbiota is a major source of lung succinate.(2)An in vivo fluorescein labeling experiment was conducted to provide evidence of succinate transferred from the intestine to the lungs during intestinal I/R.3.Elaboration of the role of succinate-induced alveolar macrophage(AM)polarization in the development of intestinal I/R-induced ALI.(1)The effect of succinate on AM polarization and/or ALI after intestinal I/R was determined in mice and AMs or co-culture of alveolar epithelial cells with AMs in vitro.(2)The dependent role of AMs was elucidated by depleting AMs with liposome-encapsulated clodronate.4.Exploration of the mechanisms involved in succinate-induced AM polarization and ALI after intestinal I/R.(1)The critical role of succinate receptor 1(SUCNR1)was determined by SUCNR1-antibody neutralization and Sucnrl gene silence experiments in vitro and in vivo.(2)Specific inhibitors were used to elucidated the role of downstream PI3K/AKT/HIF-1α pathway in vitro.5.Confirmation of the correlation between succinate and postoperative lung injury in patients underwent CPB surgery.Clinical data and blood samples of patients underwent CPB surgery were collected to verify the correlation between succinate and lung injury after CPB surgery.Results(1)An increase in the abundance of succinate-producing bacteria and a decrease in the abundance of succinate-consuming bacteria were found in the gut(but not the lung)in mice underwent intestinal I/R.Succinate accumulated in lungs after intestinal I/R,and this was positively correlated with the ratio of succinate-producing and succinate-consuming bacteria in the gut.(2)Lung succinate accumulation,lung inflammation and injury were absent in germ-free mice and was reversed by gut microbiota depletion with antibiotics.However,germ-free mice that received faces from I/R mice presented an increase in succinate levels,pulmonary inflammation and lung injury.In addition,mice with fluorescein labeling presented higher levels of green fluorescence signal intensity in the gut and lung after intestinal I/R,indicating succinate may deliver from the gut to the lung during intestinal I/R.Collectively,gut microbiota is probably a major source of succinate.(3)Succinate promoted AM polarization toward M1-like phenotype,and exacerbated alveolar epithelial cell apoptosis and intestinal I/R-induced ALI.However,depletion of AMs attenuated lung inflammation and injury in mice,indicating AMs are essential for the detrimental effect of succinate.(4)Knockdown of Sucnrl or blockage of SUCNR1 in vitro and in vivo reversed the effects of succinate.Succinate aggravated intestinal I/R-induced ALI by modulating the AM polarization,at least partially,via the SUCNR1 and the the phosphoinositide 3-kinase-AKT(PI3K)/AKT/hypoxia-inducible factor(HIF)-1αpathway.(5)Plasma succinate levels increased after CPB,and significantly correlated with intestinal I/R-related lung injury after CPB.ConclusionGut microbiota-derived succinate exacerbates intestinal I/R-induced ALI through M1-like polarization of AMs,identifying succinate as a novel target for gut-derived ALI in critically ill patients.In future,novel therapeutic approach for gut-derived ALI by modulating the gut microbioa involved in succinate metabolism should be developed.