The Role Of Intestinal Bacteria Derived Extracellular Superoxide In Gut Barrier Disruption And Fatty Liver Progression

The human microbiome acts as a complex ecosystem that symbiotics with the host.Numerous studies have revealed a correlation between its dysbiosis and intestinal or systemic diseases of the host.As more and more research moves from correlation to causation,there is a deeper understanding of the interaction of the gut microbiome with host in health and diseases.The gut is the biggest barrier between the host and its environment,not only absorbing the dietary nutrients needed,but also responsible for protecting the host from various toxins and microorganisms.The proper functioning depends on the integrity of the intestinal barrier.A large number of previous studies have shown that the gut microbiome is susceptible to dysbiosis due to genetic and environmental factors.Although intrinsic mechanisms may vary,the associations between gut microbiome dysbiosis and diseases lays a commonality of intestinal barrier dysfunction.Normally,the redox signaling of the intestinal mucosa is in a complex state of dynamic equilibrium.On the one hand,immune cells limit microbial infection by producing reactive oxygen species,and on the other hand,they need to be precisely regulated to avoid collateral damage caused by excessive reactive oxygen species.The origin and the importance of intestinal oxidative stress are largely unknown.To understand the reactive oxygen species derived from the gut microbiota has great significance in studying gut barrier function.Methods:A high-fat feeding-induced intestinal microbiome dysbiosis model was established to evaluate the oxidative stress levels,intestinal barrier function and fatty liver phenotype.By using fecal transplantation to germ-free mice,the level of oxidative stress of different intestinal microbiomes was evaluated.Through in vitro detection and in vivo verification,the ability of human intestinal symbiotic bacteria to generate extracellular superoxide anions was explored,Two Enterococcus with different superoxide-producing ability were selected to mono-colonization to germ-free mice,and high-fat feeding for 8 weeks,the intestinal barrier function and fatty liver phenotype of the gnotobiotic mice were evaluated,and the effect of bacterial-derived superoxide anions on host health was discussed.Results:1.The dysbiotic microbiome induced by high fat feeding showed a time-related effect of intervention.Compared with the initial microbiota,the structure of the fecal microbiota has changed significantly from the beginning of high-fat diet feeding.As the time of high-fat feeding increases,the microbiota continues to change,shown as right-to-left movements on the PCo1 axis,which gradually stabilizes until the week eight.To explore the characteristics of the microbiota at week 8,the a diversity of the HFD-fed intestinal microbiota was significantly reduced,and from the β diversity analysis,the HFD-fed intestinal microbiota was significantly distinguished from that of ND-fed mice.By using LeFSe analysis,the fecal microbiota of HFD-fed mice was enriched with Bacteroides,Oscillospira,Desulfovibrionales and Proteobacteria significantly.Mice fed with HFD for 8 weeks has elevated oxidative stress in the intestine and accompanied by an upregulation of the permeability of the intestinal barrier and mild fatty liver phenotype.Oral gavage with a stable Ms-SOD,which keeps its activity in intestine,significantly reduces intestinal oxidative stress,restores the expression of intestinal barrier tight junction proteins,and amends fatty liver phenotype.2.By transplantation HFD-fed or ND-fed fecal microbiome to the germ-free mice,it was verified that the elevated oxidative stress in the intestine induced by HFD-fed was mediated by the fecal microbiome.Transplantation of different microbiome affects different gene expression in mice distal illum,especially those genes related to oxidative stress and mitochondrial related stress.By performing Mito Stress test,verified that extracellular superoxide anions can disrupt mitochondrial function in intestinal epithelial cells.3.The ability of 18 strains of symbiotic bacteria isolated from the intestine to produce extracellular superoxide was detected in vitro experiments.The results suggested a taxonomically diverse bacteria from human intestine are a vast source of extracellular superoxide.Two strains of enterococcus with different ability of superoxide production were verified in SPF mice.Colonizing germ-free mice with these two enterococci demonstrates that bacterial originated superoxide plays an essential role for gut barrier disruption and fatty liver phenotypes.Oral gavage with a stable Ms-SOD,which keeps its activity in intestine,significantly amends the gut barrier damage and ameliorates the liver steatosis phenotypes by dismutation superoxide.At last,by analyzing the metagenomic data from a published cohort,we found that fecal microbiota from moderate to severe NAFLD patients have increased expression of reactive oxygen species-producing related genes and confirmed that with clinical samples that the fecal microbiota of patients with moderate to severe fatty liver disease has a higher ability to produce extracellular superoxide.Conclusion:This study reveals that bacterial extracellular superoxide is one of the important origins of oxidative stress in gut,which,by disrupting gut barrier function,plays a shared key role in fatty liver diseases.