升清降浊胶囊通过调节肠道菌群对db/db糖尿病肾病小鼠肾脏的保护作用及其机制探讨

Discussion of Shengqing Jiangzhuo Capsules on Renal Protection and Mechanism in db/db Mice with Diabetic Nephropathy by Regulating Intestinal Flora

目的通过整合代谢组学和肠道微生物群分析,探究升清降浊胶囊对db/db糖尿病肾病(DN)小鼠肾脏保护作用的机制。方法将12只模型复制成功的8周龄db/db糖尿病肾病小鼠随机分为升清降浊胶囊治疗组、生理盐水模型组,每组6只。以8周龄的野生小鼠db/m为空白对照组。自各组小鼠8周龄末开始,升清降浊胶囊治疗组连续给予升清降浊胶囊混悬液灌胃,生理盐水模型组及空白对照组给予生理盐水灌胃,连续灌胃8周。灌胃第0周及第8周,监测体质量、每日食物摄入量、每日水摄入量。取材后,PAS染色检测小鼠肾脏病理形态学变化,采用相关试剂盒检测尿白蛋白/肌酐比值(UACR),采用相关仪器检测空腹血糖,采用生化分析仪检测血清肌酐(SCr)、总胆固醇(TC)和甘油三酯(TG)。使用LC-MS/MS对小鼠血清样本进行代谢分析,同时,取粪便样品行16s rRNA高通量测序,观察肠道菌群组成。结果与生理盐水模型组比,升清降浊胶囊治疗组明显抑制空腹血糖和UACR的增加(P<0.05),并减轻db/db小鼠肾脏损伤。血清代谢组学分析在升清降浊胶囊治疗组和生理盐水模型组之间发现22个具有明显差异的代谢物,其中棕榈酰乙醇酰胺(PEA)和油酸酰胺在升清降浊胶囊治疗组中明显升高(P<0.05)。肠道菌群分析显示,升清降浊胶囊治疗组和生理盐水模型组在菌群的属和种的层面上都有不同的代谢情况。进一步的分析表明,在升清降浊胶囊治疗组,棕榈酰乙醇酰胺和油酰胺与几个乳酸杆菌物种存在相互作用。其中植物乳杆菌(Lactobacillus plantarum)与棕榈酰乙醇酰胺呈正相关,而格氏乳杆菌(Lactobacillus gasseri)和罗伊氏杆菌(Lactobacillus reuteri)与油酸酰胺呈负相关。结论升清降浊胶囊对db/db糖尿病肾病小鼠具有肾脏保护作用。升清降浊胶囊治疗可以改变DN小鼠的代谢情况及肠道菌群的组成。几种乳酸菌与棕榈酰乙醇酰胺和油酸酰胺的相互作用可能介导升清降浊胶囊对DN的治疗作用。

Objective To study the mechanism of Shengqing Jiangzhuo(SQJZ)Capsules protecting kidney in db/db mice with diabetic nephropathy by integrating metabolomics and gut microbiota analysis. Methods Twelve 8-week-old db/db mice with diabetic nephropathy were randomly divided into 2 groups:SQJZ group and normal saline group(6 rats in each group). 8-week-old db/m wild-type mice were used as the blank control group. At the post-modeling eighth week,SQJZ group was treated with SQJZ,normal saline group and blank control group were respectively administered with normal saline. Each group was gavaged for 8 weeks. Body mass,daily food and water intake were measured daily after intragastic administration from 0 week to 8 week. Then pathomorphologial changes in mice kidney were detected by PAS staining,urinary albumin/creatinine ratio(UACR) was measured by relevant kit,and fasting blood glucose was also observed. Serum creatinine(SCR),total cholesterol(TC) and triglyceride(TG) were determined by biochemical analyzer. Metabolic profiling of the serum samples of the mice was performed using LC-MS/MS,and the gut microbiota composition was assessed through 16 s rRNA high-throughput sequencing of fecal samples. Results Compared to normal saline group, the increasement of fasting blood glucose and UACR was inhibited(P<0.05),and renal damage in db/db mice was alleviated in SQJZ group. Twenty-two metabolic factors were identified, significant difference was found among SQJZ group, normal saline group, and blank group.Palmitoylethanolamide(PEA) and oleamide were both upregulated in SQJZ group(P<0.05). The analysis of intestinal microflora revealed different metabolic profiles at both the genus and species levels between SQJZ group and normal saline group were found. Further analysis showed that PEA and oleamide were correlated with several Lactobacillus species in SQJZ group. Lactobacillus plantarum was positively related to PEA,Lactobacillus gasseri and Lactobacillus reuteri were negatively related to oleamide. Conclusion Our results suggested that SQJZ Capsules have renal protection in db/db mice with diabetic nephropathy. SQJZ Capsules could alter metabolic profiles and gut microflora in diabetic nephropathy mice. Coordination of several Lactobacilli species with PEA and oleamide might mediate the treatment of diabetic nephropathy with SQJZ Capsules.