| Objective:1.To explore the relationship between Diabetic retinopathy(DR)and gut microbiota dysbiosis.2.To observe the effects of sodium butyrate,a metabolite of gut microbiota,on vital signs and the severity of DR in diabetic mice.3.To analyze the effects of sodium butyrate on intestinal barrier function,gut microbiota and systemic short-chain fatty acids(SCFA)level in diabetic mice.Methods:1.Fecal samples of 25 patients respectively from diabetes mellitus with and without retinopathy and healthy subjects were collected for 16 S rRNA sequencing.Bioinformatics analysis was performed on the sequencing data to screen out the gut microbiota that was significantly expressed in DR individuals.2.A disease classification model was constructed by random forest analysis,and the accuracy of the bacterial community biomarker classification model was evaluated by the subject operating characteristic curve.Two-factor correlation network was used to analyze the correlation between bacterial markers and fasting blood glucose and the duration of diabetes.3.C57BL/6J mice were treated with streptozotocin to establish a diabetic animal model,which was divided into diabetes group,diabetes combined with sodium butyrate intervention group and normal control group,with 5-7 mice in each group.The weight and blood glucose levels were recorded every 4 weeks,and the 24-hour food and water consumption were recorded weekly.Optical coherence tomography(OCT),Hematoxylin-eosin staining(HE),whole retinal staining,and Electroretinogram(ERG)were used to detect the changes in retinal morphology,vascular status,microglial cell activation,and retinal function in diabetic mice treated with sodium butyrate for 12 weeks.4.Morphological changes in the small intestine of mice were observed by HE staining,tight junction protein expression was assessed by immunohistochemistry(IHC)staining,and SCFA content in plasma was assessed by LC-MS sequencing.Fecal samples of mice were collected for16 S rRNA sequencing to clarify the change characteristics of gut microbiota,and microbiota with significant differences in expression were screened out.Correlation analysis was conducted on SCFA and distinct gut microbiota to clarify the correlation between microbiota and SCFA changes.5.The test results of each group were expressed as mean ± standard error.Chi-square test was used for categorical variables and one-way ANOVA was used for continuous variables.The non-normal data were analyzed by Kruskal-Wallis H test,FDR multiple test correction,and Spearman correlation analysis.P < 0.05 indicated that the difference was statistically significant.Results:1.The alpha and beta diversity of gut microbiota in diabetic subjects were significantly different from that of healthy people(P < 0.05).A total of 16 phyla have been identified in all samples.Among them,Firmutes,Actinobacteriota,Proteobacteria,and Bacteroidota are the top four phyla.Bacteroidetes,the gram-negative bacteria related to endotoxin,have increased in the DR subjects,and the relative abundance of Firmicutes and Desulfobacterota is statistically different in the three groups(all P < 0.05).A total of 307 genera was identified,of which Blautia was the highest relative abundance genus and enriched in the diabetes group,while the lowest content was found in the control group.The SCFA-production bacteria,especially the butyrate-producing bacteria was decreased in the DR group.2.A total of 25 families were identified as microbial markers by random forest analysis,and these microbial markers showed good diagnostic accuracy in distinguishing subjects from different groups.Using only Pasteurellaceae as a biomarker predictor to distinguish the DM and DR samples,the AUC value was 0.74 with a 95% confidence interval of0.63-0.85.Among all the selected biomarkers,14 families were significantly associated with fasting blood glucose and clinical parameters of diabetes(correlation coefficient absolute value ≥ 0.1,P < 0.05).Only Eubacteriaceae and Eggerthellaceae family were correlated with fasting blood glucose level and the duration of diabetes.The Eubacteriaceae family was positively correlated with fasting blood glucose level while the Eggerthellaceae family was negatively correlated.The remaining families were only negatively associated with the duration of diabetes.3.Sodium butyrate had no significant effect on the body weight,but slightly reduced the food and water intake of diabetic mice.It can effectively reduce the blood glucose level of diabetic mice,especially after12 weeks of intervention(P < 0.05).4.OCT and HE staining results showed that sodium butyrate significantly increased the thickness of the whole,inner,and middle layers of the retina of diabetic mice,and mainly improved the number of cells in the ganglion cell layer,inner nuclear layer,and outer nuclear layer,which was close to that of normal mice,but the effect of sodium butyrate on the outer layer of the retina was not significant.The results of whole retinal staining showed that sodium butyrate could reduce the cell number,branch number,and branch length of activated microglia and restore the normal morphology and structure of microglia.ERG results showed that sodium butyrate improved decreased retinal function,shortened latency,and increased amplitude of EGR and OPs waves in diabetic mice.5.HE and IHC results showed that sodium butyrate could improve the loose structure of the small intestine and enhance the expression of ZO-1and Occludin tight junction protein in diabetic mice.The results of LC-MS showed that the composition of SCFA in the circulation of diabetic mice was changed,and sodium butyrate could improve the abnormal SCFA and increase the levels of butyric acid,4-methylvaleric acid,and caproic acid in the circulatory system.6.16 S rRNA sequencing results showed that sodium butyrate could improve the diversity and composition of gut microbiota in diabetic mice,helping the disordered microbiota return to normal.Increasing the positive correlation microbiota of norank＿f＿Muribaculaceae,while decreasing the negative correlation microbiota of Lactobacillus and Lachnospiraceae＿NK4A136＿group.Conclusion:1.The gut microbiota in DR individuals was changed,with an increase in endotoxin-related gram-negative bacteria and a decrease in SCFA-producing bacteria,especially butyrate-producing bacteria.A disease classification model based on gut microbial biomarkers was established and validated to identify the correlation between key microbiota with fasting blood glucose and the duration of diabetes.2.Sodium butyrate can effectively improve the level of blood glucose and the reduction of retinal thickness,restore the number,morphology,and structure of retinal microglia,and improve the function of retinal ERG in diabetic mice.3.Sodium butyrate can enhance intestinal barrier function,delay the gut microbiota disorder,and regulate the levels of butyric acid,4-methylvaleric acid,and caproic acid in plasma.Increasing the microbiota positively associated with SCFA and decreasing the microbiota negatively associated with SCFA. |
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