基于肠上皮细胞肌球蛋白轻链激酶途径探讨肾衰II号方改善CKD微炎症状态的作用机理

Progress and complication of chronic kidney disease (CKD) are closely related to microinflammatory state. Meliorated Renal Failure Decoction for renal failure and effective prescription for the treatment of CKD, based on clinical studies confirmed that renal failure II can reduce the micro-inflammatory status of patients with CKD and improve the lrregular defecation and other TCM syndromes.Recent studies have shown that the intestinal barrier dysfunction caused by intestinal endotoxin into the blood increased, can cause or aggravate the immune inflammatory response to accelerate the process of CKD. we put forward the concept of "renal failure II to protect the intestinal barrier, thereby improving the microinflammatory state, delay the progress of CKD" , The rats were treated with 5/6 nephrectomized chronic renal failure model rats. Probiotics were used as controls. Biochemical, cellular and molecular levels, immunohistochemistry, immunofluorescence, pathology and electron microscopy were used，A new mechanism of the renal failure group II improving the intestinal permeability by the myosin light chain kinase pathway,In addition, a study in vitro used indole-induced intestinal epithelial monolayer cell permeability injury cell model.To reveal the clinical efficacy of CKD in the prevention and treatment of CKD, and to provide scientific basis for clinical research and clinical guidance.

慢性肾脏病（CKD）进展及并发症同微炎症状态密切相关。前期临床和实验研究证实扶正降浊，活血化瘀的肾衰II号方治疗CKD疗效显著，同时可降低CKD患者微炎症状态并改善大便不实中医证候，延缓疾病进展。而新近研究报道，CKD中肠道屏障功能下降引起的肠源性内毒素入血增多，可引起或加重免疫炎症反应从而加速CKD进程。鉴此，我们提出“肾衰II号方通过肌球蛋白轻链激酶途径保护肠道屏障，从而降低微炎症水平，延缓CKD进展”假说。拟在体内采用5/6肾切除慢性肾衰模型大鼠为研究对象，以益生菌为对照，以肠道屏障破坏关键环节肠上皮细胞间紧密连接破坏为切入点，从血内毒素、肠通透性、小肠紧密连接蛋白、肌球蛋白轻链激酶途径等探讨肾衰II号方改善肠通透性作用的新机制。另以吲哚诱导肠上皮单层细胞通透性损伤的体外细胞模型研究，进一步明确肾衰II号方作用机制环节。为肾衰II号方防治CKD的新药研发和临床应用推广提供科学依据。

我院肾病科长期运用中药“肾衰II号方”治疗CKD临床疗效显著。近年来系例基础研究证实该方能通过改善肾脏血氧供应环境，减少肾脏细胞凋亡及自噬，降低炎症因子，抗氧化应激等多途径、多靶点的综合药理途径发挥抗肾损伤作用。本研究旨在从调节肠道紧密连接蛋白保护肠道屏障功能角度探讨肾衰II号方改善5/6肾切除大鼠微炎症状态的作用机制。研究结果：1）与模型组比较，肾衰II号方可显著降低升高的肌酐、尿素氮、血浆内毒素水平（P<0.05）；2）治疗组炎症因子：C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）、白介素-2（IL-2）、白介素-6（IL-6）、单核细胞趋化蛋白-1（MCP-1）下降（P<0.05）；3）病理结果显示治疗组肾脏、肠道病理变化减轻；4）肾衰II号方组紧密连接ZO-1、Occudin、Claudin-1蛋白表达优于模型组（P<0.05），mRNA表达无差异。5）肾衰II号方组MLCK、p-MLC蛋白表低于模型组（P<0.05），MLCK mRNA表达低于模型组（P<0.05）。研究结果提示养血活血、降浊化瘀为功效的肾衰II号方可保护5/6肾衰大鼠肾功能，改善微炎症状态，其机制同该方介导MLCK-MLC通路修复肠道紧密蛋白，改善肾衰大鼠的肠道屏障功能相关。这为肾衰II号方临床运用与开发提供依据的同时，也为从“肾－肠”相关方向展开中医药诊治慢性肾脏病提供了可参考的方法同思路。