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

作　　者：刘庆泽 魏方勇 王佳[1] 黎晓冬 谢学军[2] 谢孟君 张梅[1] LIU Qingze;WEI Fangyong;WANG Jia;LI Xiaodong;XIE Xuejun;XIE Mengjun;ZHANG Mei(School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137;Hospital of Chengdu University of Traditional Chinese Medicine,Chengdu 610072)

机构地区：[1]成都中医药大学药学院,成都611137 [2]成都中医药大学附属医院,成都610072

出　　处：《中药药理与临床》2024年第8期10-19,共10页Pharmacology and Clinics of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(编号:82174273、81774202);成都中医药大学“杏林学者”学科人才科研提升计划苗圃人才专项(编号:MPRC2022019)。

摘　　要：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�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 observe

关 键 词：补肾活血方 糖尿病视网膜病变 慢性炎症 肠道菌群 胆汁酸 

分 类 号：R285.5[医药卫生—中药学]