Mechanism of Action of Bushen Huoxue Prescription(补肾活血方)on DR Mice Based on Gut Microbiota-Bile Acid Metabolism-Chronic Inflammation
Objective:To explore the mechanism of action of Bushen Huoxue Prescription(补肾活血方)on diabetic retinopathy(DR)based on gut microbiota-bile acid metabolism-chronic inflammation.Methods:The 18-week-old db/db mice were used as the spontaneous DR model and were randomly divided into model group,Qiming(芪明)Granules 2.19 g/kg group(positive control)and Bushen Huoxue Prescription 11.36 g/kg group,and db/m mice were set as normal control group.Mice in the treatment group were intervened for 8 weeks.Luminex tech-nology was used to detect expressions of retinal intercellular adhesion molecule-1(ICAM-1),interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α)and IL-17A,to analyze the degree of chronic inflammation in the retina.Retinal digest preparations were used to analyze the retinal vascular status.Immunofluorescence was used to detect the fluorescence intensity of vascular endothelial growth factor-A(VEGF-A)and angiopoietin-2(Ang-2).Luminex technology was used to detect VEGF-A and Ang-2 expression to analyze the degree of retinal mi-croangiopathy.HE staining was used to observe changes in retinal morphology,structure and thickness.Immunofluorescence was used to de-tect the fluorescence intensity of glial fibrillary acidic protein(GFAP),and TUNEL staining was used to detect the apoptosis of retinal gangli-on cells to analyze the extent of retinal neuropathy.Metabolomics combined with 16S rRNA sequencing was used to analyze the gut microbiota composition as well as bile acids metabolism.Correlation analysis was conducted to study the association of retinal inflammatory factors,gut microbiota,and bile acids.Results:Compared with the normal control group,the fasting blood glucose and body weight was significantly in-creased in mice of model group(P＜0.01),and the contents of inflammatory factors IL-17A,IL-6,and ICAM-1 were significantly increased in retinal tissues(P＜0.01 or P＜0.05).The retinal microangiopathy related indicators were significantly increased(P＜0.01).Disorganized and sparse arrangement was observed in retinal ganglion cell layer,and the thickness of retinal outer plexiform layer,inner plexiform layer,and ganglion cell layer was significantly decreased(P＜0.01).The number of apoptotic ganglion cells and GFAP expression was significantly in-creased(P＜0.01 or P＜0.05).The abundance of Bacteroidetes was significantly reduced(P＜0.01),and the abundance of Firmicutes and Lactobacillus were significantly increased(P＜0.01).The serum levels of cholic acid,lithocholic acid,and taurodeoxycholic acid were signifi-cantly increased(P＜0.01).Compared with the model group,the expressions of IL-1β,IL-17A,IL-6 and TNF-α in the retina of the Bushen Huoxue Prescription 11.36 g/kg group were significantly reduced(P＜0.01 or P＜0.05),and the chronic inflammation in the retina was alle-viated.The indicators related to microangiopathy in the retina were significantly reduced(P＜0.01 or P＜0.05).The thickness of the outer plexiform layer,inner plexiform layer and ganglion cell layer was significantly increased(P＜0.01).The number of apoptotic ganglion cells and GFAP expression was significantly decreased(P＜0.01),and retinal neuropathy was improved.The abundance of the Firmicutes and Lactobacillus was significantly decreased(P＜0.01),and the abundance of the Lachnospiraceae was significantly increased(P＜0.01),indi-eating that the gut microbiota was remodeled.The serum levels of cholic acid and lithocholic acid were significantly decreased(P＜0.01),and bile acids were differentially metabolized.Chronic inflammatory factors in the retina were associated with gut microbes and bile acid me-tabolism.The retinal IL-6 expression was significantly positively correlated with Clostridiaceae(P＜0.05),and TNF-α expression was signifi-cantly positively correlated with Clostridiaceae(P＜0.01).IL-17A expression was significantly negatively correlated with Lachnospiraceae(P＜0.05),while IL-17A,IL-6,TNF-α,and ICAM-1 expression was significantly positively correlated with cholic acid and lithocholic acid(P＜0.01 or P＜0.05).Conclusion:Bushen Huoxue Prescription may improve retinal microangiopathy and neuropathy in DR mice by affecting bile acid metabolism,regulating gut microbiota and inhibiting retinal chronic inflammation.
万方数据
维普
