基于肠道菌群-代谢途径探讨左归降糖舒心方干预糖尿病动脉粥样硬化作用及机制

Effect and Mechanism of Zuogui Jiangtang Shuxin Decoction on Diabetic Atherosclerosis Based on Intestinal Flora-Metabolic Pathway

目的观察左归降糖舒心方对链脲佐菌素(STZ)联合高脂饮食诱导的ApoE^(-/-)小鼠糖尿病动脉粥样硬化的影响,探讨其调控肠道菌群及糖脂代谢干预糖尿病动脉粥样硬化的作用机制。方法以8周龄雄性ApoE^(-/-)小鼠为研究对象,采用腹腔注射STZ(40 mg/kg)联合高脂饮食构建糖尿病动脉粥样硬化小鼠模型,将小鼠随机分为模型组、左归降糖舒心方组和二甲双胍组,每组5只,另将5只C57BL/6小鼠作为空白对照组。左归降糖舒心方组予左归降糖舒心方药液28.86 g/kg灌胃,二甲双胍组予二甲双胍溶液0.065 g/kg灌胃,模型组和空白对照组予等体积蒸馏水灌胃,连续8周。检测小鼠空腹血糖(FBG),全自动生化分析仪检测血清三酰甘油(TG)和总胆固醇(TC)含量,HE染色观察胰腺、主动脉及结肠组织形态。收集肠道内容物,采用16S rRNA扩增子测序检测肠道菌群变化。结果与空白对照组比较,模型组小鼠FBG、TG、TC含量明显升高(P<0.01);胰腺小叶结构紊乱,小叶间隔不清晰,大量腺泡萎缩;主动脉内膜增生,出现明显粥样斑块,管腔狭窄;结肠组织结构模糊,肠上皮黏膜糜烂,并伴有炎性细胞浸润。与模型组比较,左归降糖舒心方组和二甲双胍组小鼠FBG、TG、TC含量明显降低(P<0.01);胰腺组织结构改善,动脉粥样斑块和脂质沉积减少,结肠黏膜上皮修复,炎性细胞浸润减轻。Alpha和Beta多样性分析显示,各组样本能够显著分离,模型组肠道菌群物种总数和丰度较低;与空白对照组比较,模型组小鼠厚壁菌门、脱硫杆菌门、梭菌纲、脱硫弧菌纲、毛螺菌科相对丰度升高,拟杆菌门、拟杆菌纲、Muribaculaceae科、Muribaculaceaes属相对丰度降低(P<0.01);与模型组比较,左归降糖舒心方组小鼠厚壁菌门、脱硫杆菌门、梭菌纲、脱硫弧菌纲、毛螺菌科相对丰度降低,拟杆菌门、拟杆菌纲、Muribaculaceae科、Muribaculaceaes属相对丰度升高(P<0.05,P<0.01)。结论左归降糖舒心方可调节肠道微生态,抑制糖尿病动脉粥样硬化斑块形成,作用机制可能与调节肠道菌群结构、改善糖脂代谢、保护胰腺组织、修复肠黏膜屏障相关。

Objective To observe the effect of Zuogui Jiangtang Shuxin Decoction on diabetes atherosclerosis in ApoE^(-/-)mice induced by streptozotocin combined with high-fat diet;To explore its mechanism of diabetes atherosclerosis by regulating intestinal flora and glucose and lipid metabolism.Methods Diabetic atherosclerosis model was constructed by intraperitoneal streptozotocin 40 mg/kg combined with a high-fat diet in 8-week-old male ApoE^(-/-)mice,which were randomly divided into model group,Zuogui Jiangtang Shuxin Decoction group and metformin group,with 5 mice in each group,another 5 C57BL/6 mice were set as the blank control group.Zuogui Jiangtang Shuxin Decoction group were given Zuogui Jiangtang Shuxin Decoction 28.86 g/kg for gavage,the metformin group was given metformin 0.065 g/kg for gavage,and the model group and blank control group were given same volume of distilled water for gavage for consecutive 8 weeks.Fasting blood glucose(FBG)was detected,automatic biochemical analyzer was used to detect serum triglycerides(TG)and total cholesterol(TC)contents,morphology of pancreas,aorta and colonic tissue were observed by HE staining.Intestinal contents were collected to detect the changes of intestinal flora by sequencing with 16S rRNA amplicon.Results Compared with the blank control group,the FBG,TG,TC content in the model group significantly increased(P<0.01);the pancreatic lobules had an unclear lobe structure and a large number of atrophied glandular follicles;the intimamedia of the aorta was hyperplastic,exhibiting visible atheromatous plaques and lumen stenosis;the structure of colonic tissue was unclear,and the intestinal mucosa in certain parts was vesiculously eroded,accompanied by inflammatory cells infiltration.Compared with the model group,the FBG,TG,TC content in Zuogui Jiangtang Shuxin Decoction group and metformin group significantly decreased(P<0.01);the pancreatic tissue structure was improved,atherosclerotic plaque and lipid deposition reduced,the colonic mucosal epithelium repaired,and inflammatory cell infiltration reduced.Alpha and Beta diversity analyses showed that the groups of samples were able to separate significantly,with the total number and abundance of intestinal flora species being lower in the model group.Compared with the blank control group,the relative abundance of the model group increased in Firmicutes,Desulfobacterota,Clostridia,Desulfovibrionia and Lachnospiraceae,decreased in Bacteroidota,Bacteroidia,Muribaculaceae and Muribaculaceaes(P<0.01).Compared with the model group,the relative abundance of Zuogui Jiangtang Shuxin Decoction group decreased in Firmicutes,Desulfobacterota,Clostridia,Desulfovibrionia and Lachnospiraceae,and increased in Bacteroidota,Bacteroidia,Muribaculaceae and Muribaculaceaes(P<0.05,P<0.01).Conclusion Zuogui Jiangtang Shuxin Decoction can regulate the intestinal microecology,thereby inhibiting the formation of diabetic atherosclerotic plaque.The mechanism may be related to its regulation of intestinal flora structure,improvement of glucose and lipid metabolism,protection of pancreatic tissues,and repair of the intestinal mucosal barrier.