基于肠道菌群-胆汁酸代谢-慢性炎症探讨补肾活血方对糖尿病视网膜病变作用机制

Mechanism of Action of Bushen Huoxue Prescription(补肾活血方)on DR Mice Based on Gut Microbiota-Bile Acid Metabolism-Chronic Inflammation

目的:基于肠道菌群-胆汁酸代谢-慢性炎症探讨补肾活血方对糖尿病视网膜病变(Diabetic retinopathy,DR)的作用机制。方法:以18周龄db/db小鼠为自发性DR模型,随机分为模型对照组、芪明颗粒2.19 g/kg组、补肾活血方11.36 g/kg组,另设db/m小鼠为正常对照组,给药组干预8 w。Luminex技术检测视网膜细胞间粘附分子-1(ICAM-1)、白介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)、IL-17含量,以分析视网膜慢性炎症程度;视网膜消化铺片分析视网膜血管状况;免疫荧光检测血管内皮生长因子-A(VEGF-A)、血管生成素-2(Ang-2)荧光强度;Luminex技术检测VEGF-A、Ang-2的浓度,以分析视网膜微血管病变程度;HE染色观察视网膜形态结构和厚度变化;免疫荧光检测胶质纤维酸性蛋白(GFAP)荧光强度;TUNEL染色观察视网膜神经节细胞的凋亡,以分析视网膜神经病变程度;代谢组学联合16S rRNA测序分析胆汁酸代谢以及肠道菌群组成,相关性分析获得视网膜炎症因子与肠道菌群、胆汁酸的关联。结果:与正常对照组比较,模型对照组小鼠空腹血糖与体质量均显著升高(P<0.01),视网膜组织中IL-17A、IL-6和ICAM-1含量明显升高(P<0.05或P<0.01);视网膜微血管病变相关指标显著升高(P<0.01);视网膜神经节细胞层排列紊乱、稀疏,视网膜外从状层、内从状层、神经节细胞层厚度显著降低(P<0.01),神经节细胞凋亡数量、GFAP表达明显增加(P<0.05或P<0.01),肠道菌群拟杆菌门丰度显著降低,厚壁菌门、乳杆菌属丰度显著升高(P<0.01),血清中胆酸、石胆酸、牛磺去氧胆酸含量显著增加(P<0.01);与模型对照组比较,补肾活血方11.36 g/kg组视网膜中IL-1β、IL-17A、IL-6、TNF-α含量明显降低(P<0.05或P<0.01),视网膜慢性炎症缓解,视网膜微血管病变相关指标明显降低(P<0.05或P<0.01),视网膜外从状层、内从状层、神经节细胞层厚度显著增加(P<0.01),神经节细胞凋亡数量、GFAP表达显著降低(P<0.01),视网膜神经病变改善,厚壁菌门、乳杆菌属丰度显著降低,毛螺菌科丰度显著增加(P<0.01),血清中胆酸、石胆酸含量显著降低(P<0.01),胆汁酸出现差异代谢;视网膜慢性炎症因子与肠道菌群、胆汁酸代谢相关,其中视网膜IL-6表达与梭菌科显著正相关(P<0.05),TNF-α表达与梭菌科显著正相关(P<0.01),IL-17A表达与毛螺菌科显著负相关(P<0.05),IL-17A、IL-6、TNF-α、ICAM-1含量与胆酸、石胆酸明显正相关(P<0.05或P<0.01)。结论:补肾活血方可能通过调节肠道菌群,影响胆汁酸代谢,抑制视网膜慢性炎症,以改善DR小鼠视网膜微血管病变和神经病变。

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 technology 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 microangiopathy.HE staining was used to observe changes in retinal morphology,structure and thickness.Immunofluorescence was used to detect the fluorescence intensity of glial fibrillary acidic protein(GFAP),and TUNEL staining was used to detect the apoptosis of retinal ganglion 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 increased 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 increased(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 significantly increased(P<0.01).Compared with the model group,the expressions of IL-1β,IL-17A,IL-6 and TNF-αin 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 alleviated.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),indicating 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 metabolism.The retinal IL-6 expression was significantly positively correlated with Clostridiaceae(P<0.05),and TNF-αexpression was significantly 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.