Gut Microbiota Influence POD Induced By Cardiopulmonary Bypass In Rats Through The Enteric Glial Cell

ObjeciveTo explore whether gut microbiota disorder after cardiopulmonary bypass(CPB)can promote postoperative delirium(POD)by regulating the expression of enteric glial cells(EGC),establishing a rat model of gut microbiota humanization by means of metagenomic sequencing and fecal microbiota transplantation.MethodsApproved by the Ethics Committee of the General Hospital of the Northern Thearter Command,this study collected 10 feces of healthy volunteers who met the inclusion and exclusion criteria and 10 feces of patients with POD after CPB cardiac surgery in our hospital from July 2020 to December 2020.16 S ribosomal RNA(16S r RNA)gene sequencing analysis was performed to detect differences and fecal suspension was prepared,which was stored in a freezer at-8 ℃.A total of 30 SD male rats of SPF grade,weighing approximately 400 g,were provided by the Experiment Animal Center of the General Hospital of the Northern Thearter Command.The rat were randomly assigned to three groups:pseudo-grem-free rat group(S group),pseudo-grem-free rat transplantation with healthy human fecal suspension group(NP group)and pseudo-grem-free rat transplantation with POD patient fecal suspension group(P group),with 10 rats in each group.The rats in each group were trained in Morris water maze test for seven days and four times a day,and those with low intelligence or unable to swim were eliminated to ensure that there was no statistical difference in cognitive function of rats in all groups.Group S,group NP and group P were treated with antibiotic cocktail made up of ampicillin(250mg/kg),neomycin sulfate(250mg/kg)and metronidazole(250mg/kg),twice a day,2ml each time for 14 consecutive days;Group S was regarded as successful models after 14 days of intragastric administration;Group NP and group P received fecal microbiota transplantation from healthy people and POD patients on the 15 th day,once a day for 14 consecutive days.After modeling,Morris water maze test was performed on all groups of rats,and super MAZE animal behavior recording and analysis system was used to collect trace images and data;the feces were collected by sterilized centrifuge tube and were stored in a freezer at-80°C until analysis;metagenomics sequencing detect differences in gut microbiota composition.Rats were killed by spinal dislocation method,venous blood was taken aseptically,serum was retained after centrifugation,and the contents of serum inflammatory factors IL-1,IL-6 and TNF were measured by enzyme-linked immunosorbent assay(Elisa).Carefully peel off the left cerebral hemisphere and several segments of colon tissue with a length of 2cm,preserve them in 4% paraformaldehyde solution.Morphological changes of hippocampal CA1 and CA3 areas,colonic mucosa,submucosa and intestinal myenteric ganglion plexus were observe under an optical microscope.The right hippocampus and colon tissues were stored in a freezer at-80 ℃.The levels of glial fibrillary acidic protein(GFAP),brain derived neurotrophic factor(BDNF),amyloid precursor protein(APP)and phosphorylated Tau protein in hippocampus and GFAP and calcium binding protein S100 in colon were detected by Western-blot.The positive expression of BDNF and GFAP in hippocampus was observed by immunohistochemistry.The positive expression of GFAP in colon tissue was observed by immunofluorescence.Data are presented as the mean ± standard deviation(x ± s)of the mean(SEM).Analysis was performed using Graph Pad Prism 8.0.Comparisons among groups were performed using one-way or two-way analysis of variance(ANOVA)followed by Fisher’s exact tests.A correlation analysis was conducted using Spearson correlation coefficient.The diagnostic cutoff values,sensitivity,specificity,and accuracy were determined by ROC curve analysis.P values<0.05 were considered statistically significant.Results1.Comparison of gut microbiota composition between patients with POD and healthy volunteersThe alpha diversity of gut microbiota between patients with POD and healthy volunteers showed that there was no significant difference among the groups(P>0.05);the beta diversity showed that the samples of each group were clustered and distinguished well,and there were significant differences in gut microbiota structure and relative abundance between the two groups(P<0.05).Compared with healthy volunteers,the relative abundance of Bacteroidetes,Proteobacteria,Prevotella and Enterobacteriaceae in POD patients increased significantly,while the relative abundance of Firmicutes,Actinobacteria and Bifidobacterium decreased significantly(P<0.05).2.Comparison of gut microbiota composition among S group,NP group and P groupThe results of cluster analysis showed that the gut microbiota of the three groups of rats aggregated and partitioned well after fecal microbiota transplantation.There were significant differences in the distribution and relative abundance of gut microbiota among the groups from the six classification levels of phylum,class,order,family,genus and species(P<0.05).Compared with group S,the relative abundance of Proteobacteria,Klebsiella,Escherichia and Enterococcus in group P and NP decreased significantly(P<0.05),but there was no statistical difference between group P and NP(P>0.05);compared with group S,the relative abundance of Firmicutes,Actinobacteria and Lactobacillus in group NP was significantly higher(P<0.05),but there was no statistical difference between group P and group S(P>0.05);compared with group S,the relative abundance of Bacteroidetes,Proteobacteria and Prevotella in group P was significantly higher(P>0.05),while there was no statistical difference between group NP and group S(P>0.05).3.Changes of various indexes of colon in rats3.1 HE stainingIn group S,there were different degrees of injury,villi shortened,nerve fibers vacuolated and disordered;In group P,the intestinal mucosa was damaged and shortened,the morphology of EGC was abnormal,the number increased,and the arrangement was irregular;in NP group,the mucosal layer became thicker,the EGC morphology was regular and arranged evenly,and its protrusions were faintly seen to surround the ganglia to form a nerve plexus.3.2 Western-blot method was used to detect GFAP and S-100β proteinCompared with group S,the expression levels of GFAP and S100β protein in colon tissue of group P and group NP were significantly higher(P<0.05);compared with NP group,the expression of GFAP and S-100β protein in colon tissue of P group was significantly higher(P<0.5).3.3 Detection of GFAP by immunofluorescenceIn group S,the size and number of glial cells around colonic mucosa and submucosa neurons decreased,single cells could not be recognized,and the fluorescence was low;compared with group S,the fluorescence intensity of GFAP protein in group P and NP was significantly higher(P<0.05);compared with NP group,EGC cells in P group were enlarged,reactive proliferation and relative fluorescence intensity of GFAP protein were significantly increased(P<0.05).4.Comparison of serum inflammatory factors IL-1,IL-6 and TNF-αconcentrations in rats of each groupCompared with group S,the concentrations of serum IL-1,IL-6 and TNF-αin group P and NP were significantly lower(P<0.05);Plasma IL-1,IL-6 and TNF-α levels in NP group were significantly lower than those in P group(P<0.5).5.Changes of various indexes related to cognitive function in rats5.1 Morris water maze test Positioning Cruise ExperimentIn the positioning cruise experiment,the average swimming speed and average incubation period of rats in S group,P group and NP group before modeling were compared,and it was found that there was no statistical difference among the three groups(P>0.05).In terms of swimming speed,there was no statistical difference between the three groups after and before modeling(P>0.05),and there was no statistical difference among the three groups(P>0.05).In the comparison of the average incubation period,group S and group P were significantly longer after modeling than before modeling,and the difference was statistically significant(P<0.05),while group NP had no statistical difference before and after modeling(P>0.05);Compared with group S,the average incubation period of group NP after modeling was significantly shorter(P<0.05),but there was no significant difference between group P and group S(P>0.05);Compared with group P,the average incubation period of group NP was significantly shortened(P<0.05).5.2 Morris Water Maze Space Exploration ExperimentIn the space exploration experiment,the swimming distance in interest quadrant,the crossing times of escape platform and the residence time in interest quadrant were compared among the three groups before modeling,and it was found that there was no statistical difference among the three groups(P>0.05).After modeling,the swimming distance in the interest quadrant,the number of times of escaping platform crossing and the stay time in the interest quadrant in group S and group P were significantly lower than those before modeling,and the difference was statistically significant(P<0.05).There was no statistical difference in each index before and after modeling in group NP(P>0.05);Compared with the swimming distance in the interest quadrant,the number of times of escape platform crossing and the stay time in the interest quadrant after modeling in the S group,the swimming distance in the interest quadrant,the number of times of escape platform crossing and the stay time in the interest quadrant in the NP group after modeling were significantly increased(P<0.05).There was no statistical difference between the P group and the S group(P>0.05);Compared with the swimming distance in the interest quadrant,the number of times of escape platform crossing and the stay time in the interest quadrant after modeling in the P group,the indexes of the NP group after modeling were increased,and the difference was statistically significant(P<0.05).5.3 HE stainingIn group S,the morphology of cells was cone-shaped,the arrangement was loose and irregular,the staining color was obviously deepened,the gap between glial cells was widened,and the nerve fiber network was loose and spongy or vacuolated;In group P,the cytoplasmic staining of cells was uneven,and neurons with pyknosis and dense staining could be seen;In NP group,the cell arrangement was relatively regular,the cytoplasm stained evenly,and a large number of normal cells were seen.5.4 The expression of BDNF and GFAP in hippocampus was detected by immunohistochemistryCompared with S group,the positive expression of BDNF increased and the positive expression of GFAP decreased in P group and NP group.Compared with P group,BDNF positive expression increased and GFAP positive expression decreased in NP group.5.5 Western-blot method was used to detect GFAP,BDNF,APP,phosphorylated Tau proteinCompared with group S,the expression level of BDNF in hippocampus of group P and group NP increased significantly(P<0.05);the expression of BDNF in NP group was significantly higher than that in P group(P<0.05).Compared with group S,the expression levels of GFAP,APP and phosphorylated Tau protein in hippocampus of group P and group NP decreased significantly(P<0.05);compared with group P,the expression levels of GFAP,APP and phosphorylated Tau protein in hippocampus of group NP decreased significantly(P<0.05).Conclusion1.The gut microbiota composition of patients with POD after cardiac surgery under cardiopulmonary bypass has variation.The abundance of Bacteroides,Proteobacteria,Prevotella and Enterobacter increased significantly,while the relative abundance of Firmicutes,Actinobacteria and Bifidobacterium decreased significantly.2.The increase of abundance of Proteobacteria,Prevotella,Enterobacteriaceae and Bacteroides after cardiopulmonary bypass cardiac surgery can regulate the abnormal expression of EGC,destroy the intestinal barrier function,produce cascade amplified inflammatory reaction,lead to the accumulation of Aβ protein in plaque compacta and the high phosphorylation of Tau protein,thus promoting the occurrence of POD.