Intestinal Microbiota Impact Sepsis Associated Encephalopathy Via The Vagus Nerve

Part Ⅰ The Characteristics of Intestinal Microbiota and its Effect on Brain Function in rats and Patients with SepsisObjective To reveal the feature of intestinal microbiota in rats and patients with sepsis,and the effects on the brain function.Methods 1.A prospective control study was conducted.The adult sepsis patients with or without SAE admitted to Emergency Department of Hebei General Hospital from June 2016 to October 2017 were enrolled,10 healthy subjects served as controls.Fecal samples were collected at 7d and analysed by 16SrDNA high-throughput sequencing.The results of EEG were recorded and analysised.2.Male rats were randomly divided into two groups: CON group(n=20),lipopolysaccharide(LPS)group(n=40).According to the results of the behavioral test,electroencephalogram(EEG)and somatosensory evoked potential(SEP),the rats in LPS group were divided into sepsis(SEP)group,and sepsis associated encephalopathy(SAE)group.Fecal samples were collected at 7d and analysed by 16 S rDNA high-throughput sequencing.Brain function was evaluated by behavioral test and EEG.The levels of TNF-α,IL-10,IL-6 in hippocampus were detected by ELISA.Results Significant imbalance of microbiota composition,characterized by a profound increase of commensals in the Proteobacteria phylum and depletion of commensals in the Firmicutes,Bacteroidetes phylum,was observed in SAE group compared to CON group.Furthermore,at the level of genus,enriched in Alistipes,Staphylococcus aureus,Pseudomonas and depleted of Faecalibacterium,Bifidobac-terium,Lactobacillus was observed in SAE group compared to CON group.CON and SEP groups demonstrated a better spatial memory and attenuated levels of IL-6 and TNF-α.Summary 1.Significant imbalance of microbiota composition,features of increase in the Alistipes and depletion in the Faecalibacterium was observed in SAE rats;2.Deterioration of brain function,and significant imbalance of microbiota both were observed in SAE rats.Part Ⅱ Intestinal Microbiota Impact Sepsis Associated Encephalopathy via the Vagus NerveObjective The pathogenesis of sepsis associated encephalopathy(SAE)remains poorly understood.Vagus nerve plays an important role in gut-microbiota-brain axis.This study aimed to investigate whether vague nerve is a key mediator of the impact of intestinal microbiota on SAE.Methods Male rats were randomly divided into four groups(n=20): SHAM(SHA)group,lipopolysaccharide(LPS)group,fecal microbiota transplantation(FMT)+LPS group,and vagotomy(VGX)+LPS+FMT group.LPS+FMT and LPS+FMT+VGX groups received nasogastric infusion of feces from healthy donor three times a day.Fecal samples were collected every two days to monitor changes in microbiota composition by 16 S rDNA analysis.Brain function was evaluated by behavioral tests and EEG.The levels of TNF-α,IL-1β,IL-6,IL-10 in hippocampus were detected by ELISA.The expression of Iba-1 in hippocampus was assessed by immunohistochemistry and Western blot analysis.Results Significant modification of microbiota composition,characterized by a profound increase of commensals in the Firmicutes phylum and depletion of opportunistic organisms in the Proteobacteria phylum,was observed in FMT groups compared to LPS group.Furthermore,we identified a reconstituted bacterial community enriched in Firmicutes and depleted of Proteobacteria.In both FMT groups the diversity of the fecal microbiota and the microbiota composition were similar to SHA group.LPS rats treated with FMT demonstrated a better spatial memory and less EEG abnormalities,significantly attenuated levels of IL-1β,IL-6,TNF-α,and decreased number of Iba-1 positive microglia in the hippocampus,but these beneficial effects of FMT were reversed by VGX.Summary FMT can change intestinal microbiota in rats with sepsis,and vagus nerve is a key mediator between intestinal microbiota and SAE.These findings suggest that FMT and vagus nerve are potential therapy targets for treating SAE.Part Ⅲ Vagus Nerve Impact Sepsis Associated Encephalopathy via the level of SIRT6 in the HippocampusObjective This study aimed to investigate whether SIRT6 is a key mediator of the impact of vagus nerve on SAE.Methods Male rats were randomly divided into: SHA group,LPS group,LPS+VNS group,LPS+VGX,LPS+ICA group.Brain function was evaluated by behavioral tests.The expression of Iba-1,SIRT6,α7n ACh R were tested by Western blot.The expression of SIRT6 in hippocampus was tested by immunehistochemistry.The levels of TNF-α,IL-10,IL-6 in hippocampus were detected by ELISA.Results LPS+VNS and LPS+ICA rats treated with vagus nerve stimulation and icariin demonstrated a better behavioral score,significantly attenuated levels of IL-6,TNF-α,and decreased number of Iba-1 positive microglia in the hippocampus,and increased number of SIRT6,α7n ACh R in the hippocampus.Summary Vagus nerve impact sepsis associated encephalopathy via the level of SIRT6 in the hippocampus.Conclusions 1.Significant imbalance of microbiota composition,features of increase in the Alistipes and depletion in the Faecalibacterium was observed in SAE rats;2.FMT can change intestinal microbiota in rats with sepsis,and vagus nerve is a key mediator between intestinal microbiota and SAE.These findings suggest that FMT and vagus nerve are potential therapy targets for treating SAE.3.Vagus nerve impact sepsis associated encephalopathy via the level of SIRT6 in the hippocampus.