基于肠道菌群探讨补脾益肾方改善5/6肾切除大鼠肠道屏障延缓慢性肾脏病进展的作用机制  

Study on the Mechanism of Bupi Yishen Formula on Improving Intestinal Barrier and Delaying CKD Progression Based on Intestinal Flora in 5/6 Nephrectomy Rats

作　　者：莫业南 李茵[2,3] 揭西娜 王立新[2,3] 吉春兰 卢钊宇[2,3] 刘旭生 Mo Yenan;Li Yin;Jie Xina;Wang Lixin;Ji Chunlan;Lu Zhaoyu;Liu Xusheng(Nephrology Department of Integrated Traditional Chinese and Western Medicine Hospital of Southern Medical University,Guangzhou 510515,China;Department of Nephrology,Guangdong Hospital of Traditional Chinese Medicine,Guangzhou 510120,China;The Second Clinical Medical College of Guangzhou University of Traditional Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]南方医科大学中西医结合医院,广州510515 [2]广东省中医院,广州510120 [3]广州中医药大学第二临床医学院,广州510006

出　　处：《世界科学技术-中医药现代化》2022年第4期1539-1548,共10页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会面上项目(81873261):补脾益肾颗粒通过调整肠道微生态影响芳烃受体AHR通路治疗慢性肾脏病的机制研究,负责人:刘旭生。

摘　　要：目的探讨补脾益肾方延缓5/6肾切除大鼠慢性肾脏病进展的作用机制。方法雄性SD大鼠随机分为4组:假手术组、5/6肾切除模型组、补脾益肾方低剂量组、补脾益肾方高剂量组。假手术组和5/6肾切除模型组予蒸馏水灌胃,补脾益肾方低剂量组和高剂量组分别给予低剂量和高剂量补脾益肾方治疗,灌胃为期4周。检测各组大鼠血肌酐、尿素氮、24 h尿蛋白定量、硫酸吲哚酚、二胺氧化酶;肾脏组织采用PAS和Masson染色;结肠组织采用HE染色;Western-blot检测ZO-1、Occludin、Claudin-1三种紧密连接蛋白及IL-22在结肠组织的表达水平;采用16S rDNA高通量测序并进行生物信息学分析。结果与假手术组相比,5/6肾切除组大鼠的血肌酐、尿素氮、24 h尿蛋白定量、血清硫酸吲哚酚和二胺氧化酶水平明显升高;与5/6肾切除组相比,补脾益肾方高剂量组大鼠的血肌酐、尿素氮、24 h尿蛋白定量、血清硫酸吲哚酚和二胺氧化酶水平明显下降,肾脏病理损伤缓解;补脾益肾方亦可调整肠道菌群,提高益生菌Prevotella(普氏菌属)、Phascolarctobacterium(考拉杆菌属)和Megamonas(巨单胞菌属)的相对丰度,降低Clostridiaceae(梭菌科)、Haloplasmataceae(盐扁菌科)、Micrococcaceae(微球菌科)、Pseudomonadaceae(假单胞菌科)和Ruminococcus(瘤胃球菌)的相对丰度;促进紧密连接蛋白(ZO-1、Occludin和Claudin-1)及IL-22的表达,减轻结肠固有层和粘膜层水肿及炎症细胞浸润;减少毒素入血,血清中的硫酸吲哚酚水平下降,进而延缓CKD进程。结论补脾益肾方延缓5/6肾切除大鼠的CKD进展可能是通过调节肠道菌群,修复肠道屏障功能,降低血清尿毒症毒素水平,以实现肾保护作用。Objective To explore the underlying mechanism of the protective effect of Bupi Yishen Formula(BYF) on 5/6 nephrectomy(5/6Nx) rats. Methods We divided 40 male SD rats randomly into four groups: Sham Group, 5/6Nephrectomized Model Group(5/6Nx Group), Low-dose BYF Group and High-dose BYF Group. Pure water was given to Sham Group and 5/6Nx Group, while treatment of low-or high-dose BYF was given to L-BYF Group and H-BYF Group respectively for 4 weeks. At the end of the study, Serum creatinine(Scr), blood urea nitrogen(BUN), 24-hour urinary protein(24 h-Upro), indoxyl-sulfate(IS) and diamine oxidase(DAO) were measured in each group. PAS and Masson staining were used in renal tissue and HE staining was used in colon tissue. Western-blot was performed to detect the expression levels of tight junction proteins(ZO-1, Occludin, claudin-1) and IL-22 in colon tissues. 16S rDNA high-throughput sequencing was used for bioinformatics analysis. Results Compared with Sham Group, SCR,BUN, 24h-Upro, IS and DAO of rats in 5/6Nx Group were significantly increased. BYF administration could reduce the levels of SCR, BUN, 24h-Upro, IS and DAO, and alleviate the pathological damage of kidney in 5/6 nephrectomy rats.BYF could also regulate intestinal flora, manifesting as increased Prevotella, Phascolarctobacterium and Megamonas and decresed Clostridiaceae, Haloplasmataceae, Micrococcaceae, Pseudomonadaceae and Ruminococcus in relative abundance, promoted the expression of tight junction proteins(ZO-1, Occludin and Claudin-1) and IL-22, and reduced the edema and inflammatory cell infiltration in the lamina propria and mucous layer of the colon. With the decrease of uremic toxin in the blood, thus delayed the CKD process.Conclusion BYF might regulate intestinal flora, repair the intestinal barrier function, reduce serum uremic toxin level and finally delay the progression of CKD in 5/6 nephrectomy rats.

关 键 词：补脾益肾方 慢性肾脏病 肠道菌群 肠道屏障 

分 类 号：R285.5[医药卫生—中药学]