Study On The Characteristics Of Gut Microbiome And Its Relationship With Cellular Immunity In Patients With Spinal Cord Injury

Objective: To explore the changes of gut microbiome in Chinese Han patients with spinal cord injury,compare the differences of T lymphocyte subsets,NK cells and intestinal flora between patients with spinal cord injury and healthy subjects,and explore the relationship between gut microbiomeand cellular immunity in patients with spinal cord injury.Methods: A case-control study was conducted.Twenty-five patients with spinal cord injury from January 2018 to December 2018 in the Department of Rehabilitation Medicine,Western War Zone General Hospital of the Chinese People's Liberation Army were selected as the case group and 25 healthy subjects as the control group.All the selected subjects signed the informed consent before the experiment and were approved by the Ethics Committee of the Western War Zone General Hospital.The general data of age,sex,ethnicity,complications and drug use were analyzed.ASIA score,NBD score,frequency of antibiotics and probiotics use and one-month dietary records were performed in the case group.According to the excluding criteria,23 cases were included in spinal cord injury group and 21 cases were included in healthy group.Fecal samples and fasting peripheral blood were collected from both groups in the morning.Firstly,DNA extraction and PCR amplification were performed on fecal samples.The second generation high-throughput sequencing of 16 SrDNA was used to detect and analyze the number of V4 region gene sequences of two groups of gut microbiome.OTU cluster analysis,Alpha diversity analysis,Beta diversity analysis,species composition analysis,species difference analysis and other bioinformatics methods were used to analyze the change characteristics of gut microbiome in the two groups.Find out the different species between the patients with spinal cord injury and healthy subjects;then,detect the number of CD3 + T cells,CD4 + T cells,CD8 + T cells,CD4 + / CD8 + ratio and NK cells reflecting the cellular immune status of patients with spinal cord injury by flow cytometry;finally,evaluate the correlation between bacteria and cellular immunity by calculating the spearson correlation coefficient.Result:1.Comparison of gut microbiome structure between two groups(1)Comparison of gut microbiome diversityAlpha diversity in spinal cord injury group was higher than that in healthy control group(P < 0.05).Beta diversity analysis showed that there were significant differences in gut microbiomestructure between patients with spinal cord injury and healthy subjects.(2)Comparison of gut microbiome abundance1)phylum level:The relative abundance of Firmicutes,Bacteroides and Proteobacteria was the highest in healthy subjects and spinal cord injury group,and was the absolute dominant flora of gut microbiome in the two groups.In spinal cord injury group,the relative abundance of Fusobacterium decreased significantly(P < 0.05),while the relative abundance of Synergistetes and Actinomycetes increased signify-cantly(P < 0.05).2)Family level:The relative abundance of Ruminococcaceae,Tannerellaceae,Muribaculaceae,Synergistaceae,Erysipelotrichaceae in the spinal cord injury group was higher than that in the healthy control group(P < 0.05)and the relative abundance of Fusobacteriaceae was lower than that in the healthy control group(P < 0.05).3)genera level:There were significant differences in the abundance of bacteria between the two groups;The relative abundance of Escherichia-Shigella,Lachnospiraceae UCG-008,Alistipes,Parabacteroides,[Ruminococcus] torques group in the spinal cord injury group was higher than that in the healthy control group(P < 0.05).while that of Fusobacterium decreased significantly(P < 0.05).(3)Species Difference Analysis1)Phylum level: Actinomycetes were the marker of significant increase in spinal cord injury group(P < 0.05).2)Class level: Actinobacteria were the markers of significant increase in spinal cord injury group(P < 0.05).3)Order level: Bifidobacteriales and Micrococcales were the markers of significant increase in spinal cord injury group(P < 0.05).4)Family level: Rikenellaceae,Bifidobacteriaceae and Tannerellaceae were the markers of significant increase in the spinal cord injury group(P < 0.05).5)Genus level: Significant increase in the number of important marker genera in spinal cord injury group(P < 0.05)were as follows:(1)UBA1819,Ruminiclostridium 9,Ruminococcaceae NK4A214 group,Ruminococcus 2,Ruminococceae UCG-005,Ruminiclostridium 5,Flavonifractor belonging to Ruminococceae;(2)Aglistes,dgA-11 group,Rikenaceae RC9 belonging to Tannerellaceae;(3)[Eubacterium] oxidoreducens group belonging to Lachnospiraceae;(4)Intestinibacter belonging to Peptostreptococcaceae;(5)Escherichia-Shigella belonging to Enterobacteriaceae;(6)Tannerellaceae belonging to Parabacteroides.However,the significant decrease in spinal cord injury group was attributed to Fusobacterium of Fusobacteriaceae(P < 0.05).2.Comparison of T lymphocyte subsets and NK cells between two groupsCD4 + cells and CD4 +/CD8 + in spinal cord injury group were significantly lower than those in healthy control group(P < 0.01);there was no significant difference in CD3 +,CD8 + cells and NK cells between the two groups(P > 0.05);Spearman correlation test showed that CD3 + T,CD4 + T lymphocyte(%)and CD4 +/CD8 + were positively correlated with the course of disease(month)(P < 0.05).3.Analysis of correlation between cellular immunity and gut microbiome in patients with spinal cord injurySpearman correlation test showed that there were significant correlations between five genera and cellular immune function,among which Lachnospiraceae UCG-008 was negatively correlated with CD3 +(P < 0.05).Lachnoclostridium 12 was negatively correlated with CD4+/CD8+(P < 0.05).Tyzzerella 3 was negatively correlated with NK cells(P < 0.05).[Eubacterium] eligens group was positively correlated with CD3 + and negatively correlated with NK cells(P < 0.05).Ruminococcaceae UCG-002 was negatively correlated with NK cells(P < 0.05).Conclusion: 1.There are significant differences between gut microbiome of spinal cord injury patients and healthy control group;spinal cord injury patients increase the relative abundance of bifidobacteria;at the same time,increase the relative abundance of Ruminococcaceae related to depression,increase the relative abundance of Ruminococcus closely related to central nervous system diseases,and increase the phase of enteropathogenic bacteria such as Escherichia-Shigella and Erysipelothrix.It also reduced the relative abundance of butyrate-producing bacteria and anti-inflammatory bacteria beneficial to the intestine,which may play a role in the recovery of spinal cord injury and the occurrence and development of complications.2.Compared with the healthy control group,the cellular immune function of patients with spinal cord injury was significantly reduced,and its cellular immune function gradually increased with the course of disease.3.Ruminococcaceae UCG-002 and other four bacteria in gut microbiome of patients with spinal cord injury are correlated with cellular immune function indicators,suggesting that the cellular immune function of patients with spinal cord injury may be improved through the regulation of gut microbiome in the future to prevent infection.