基于“肠-肾轴”从肠道微生态研究三七注射液对慢性肾衰竭大鼠的保护作用机制

It is considered in the theory of the gut-kidney axis that, the disorder of micro-ecology in intestines is the important mechanism for the product of toxin and progress in the chronic renal failure. The regulation of the disorder of micro-ecological in intestines maybe is new way to delay the progress in the chronic renal failure. Our previous study discovered that, SanQi injection plays a role of anti-kidney fibrosis through blocking both the TGF-β1/P38MAPK and TGF-β/Smads pathway, suppressing the expression of ILK and MMP-7, interposing the net of inflammation. In this study, the disorder of micro-ecological in intestines is regarded as the entry point, we observe the pathology of Ileum, indoxyl-sulfate, endotoxin, D-lactic acid, TNF-αand IL-6, Bacterial flora species in intestines, bacterial translocation , the change of The genome sequence in the intestinal contents and the expression of TLR-4 both in the kidney and intestines through the way of pathology, ELISA, GC/TOF-MC analysis, Macro genome sequencing and Western blot. It is researched on both the disorder of micro-ecological in intestines for the chronic renal failure and the new mechanism and target of SanQi injection on the delay the progress in the chronic renal failure through the intestinal barrier, flora, metabolites, the intestinal immune system and the inflammation signaling, demonstrating the material basis on the theory of kidney controlling the urine and stool. It will provide theory basis on the clinical apply of Traditional Chinese medicine (TCM) for activating blood circulation to treat of micro-ecological in intestines.

“肠-肾轴”理论认为，肠道微生态失调是慢性肾衰竭（CRF）毒素产生及进展的重要机制，调控微生态失调可能成为延缓CRF进展新方法。我们前期研究发现，三七注射液通过阻断TGF-β1/P38MAPK和TGF-β/Smads信号通路、抑制ILK和MMP-7、干预炎症信号网络发挥抗肾纤维化作用。本课题以CRF相关肠道微生态失调为切入点，通过病理学、ELISA、GC/TOF-MC分析、宏基因组测序、Western blot等，观察各组5/6肾切除大鼠回肠病理、血清硫酸吲哚酚、血清内毒素、血清D乳酸、TNF-α和IL-6、细菌菌群种类及细菌移位、肠道内容物基因组序列、肾和肠组织TLR-4的改变，从肠道屏障、菌群、代谢物、肠道免疫系统以及炎症信号探讨CRF肠道微生态失调和三七注射液延缓肾衰竭进展的新机制和靶点，从而论证中医学“肾司二便”的物质基础，为活血化瘀中药临床应用于肠道微生态提供理论依据。

“肠-肾轴”理论认为，肠道微生态失调是慢性肾衰竭（CRF）毒素产生及进展的重要机制，调控微生态失调可能成为延缓CRF进展新方法。本课题以CRF相关肠道微生态失调为切入点，从肠道屏障、菌群、代谢物、肠道免疫系统以及炎症信号探讨CRF肠道微生态失调和三七注射液延缓肾衰竭进展的新机制和靶点。方法：采用5/6肾切除大鼠动物模型，观察各组5/6肾切除大鼠回肠病理、血清硫酸吲哚酚（IS）、血清内毒素、血清D乳酸（D-LA）、TNF-α和IL-6、细菌菌群种类及细菌移位、肠道内容物基因组序列、肾和肠组织TLR-4的改变。结果：三七注射液可降低CRF大鼠尿素氮、肌酐、IS、内毒素、D-LA、TNF-α、IL-6水平，降低CRF大鼠肾组织和肠组织TLR-4蛋白表达；减少肠道肠球菌和肠杆菌水平，同时升高双歧杆菌、乳酸杆菌水平，减少菌群在淋巴结、肾、肝、脾重要脏器的移位率；升高Shannon指数，Ace指数，Chao指数，降低Simpson指数；通过宏基因组分析了各组样本菌群在门、属水平的差异，采用线性判别分析各组样本菌群在门、纲、目、科、属、种的差异；通过UPLC-TOF/MS代谢组学方法，PLS-DA图提示正常组和模型组完全分离，三七高剂量组和三七中剂量组在正常组和模型组之间，更靠近正常组，而与模型组大体分离；对差异代谢物进行鉴别，最终从模型组与正常组肠道内容物中鉴别出67个差异代谢物，经三七高剂量治疗后对49个差异代谢物有调节作用；采用KEGG通路分析工具，对三七高剂量组与模型组鉴别出的潜在生物标志物进行通路富集分析和通路拓扑分析，匹配相关代谢通路；光镜和电镜下均可见肠道结构病理改变，经三七注射液治疗后上述病理改变均有减轻；PAS染色和甲苯胺蓝染色结果显示，三七注射液可调节杯状细胞和肥大细胞在CRF大鼠肠道组织的表达。结论：三七注射液具有良好的肾脏保护作用，其作用机制可能与保护肠道粘膜屏障、调整肠道菌群结构和维持内环境稳定、改善肠道菌群多样性、调节引起CRF差异代谢物的代谢过程及相关代谢通路、调节肠道免疫细胞的表达、干预肠道TLR4信号通路活化有关。