基于miR-21调控NF-κB信号通路介导肾缺血再灌注诱导的凋亡和炎症探讨桂枝去桂加茯苓白术汤的肾保护作用机制的研究

There is no effective medicine for acute kidney injury (AKI). Ischemia-reperfusion (IR) is one of the primary pathogenesis of AKI. AKI belongs to spleen deficiency and water stopping and tri-jiao hysteresis in the traditional Chinese Medicine. The treatment should be dredged tri-jiao and dieresis. In previous clinical research, Guizhi qugui plus Fuling Baizhu decoction has been proved to sharply reduce serum creatinine, BUN and NGAL in the patients with acute kidney injury(AKI),which is in an unknown mechanism. MicroRNA-21 (miR-21) could regulate multiple target genes.Some researches have shown that miR-21 could mediate inflammation and apoptosis through the NF- kappa B signaling pathway to participate into the development of AKI.We found in the pre-experiment that the prescription Guizhi qugui plus Fuling Baizhu decoction may up-regulate the expression of microRNA-21 (miR-21) in renal tissues after ischemia-reperfusion. This project will investigate the rat model of renal IRI and renal tubular epithelial cell line and podocyte strain of miR-21 silented to probe the effects of Guizhi qugui plus Fuling Baizhu decoction on the expression of miR-21, NF- kappa B, inflammatory factors and apoptotic proteins in rat kidney tissue and renal tubular epithelial cell line, podocyte by means of immunohistochemistry, real-time fluorescent PCR and Western blotting . The project is helpful to provide the theoretical basis for the clinical treatment of Guizhi qugui plus Fuling Baizhu decoction for AKI.

急性肾损伤（AKI）目前尚无有效治疗药物，缺血再灌注（IR）是其主要发病机制之一。中医学认为，AKI属脾虚水停，湿滞三焦，治疗需畅三焦、利小便。前期临床研究发现“桂枝去桂加茯苓白术汤”治疗AKI患者，能显著降低血肌酐、尿素氮、NGAL，作用机制不明。miR-21能调控多个靶基因，已有研究表明，miR-21可通过NF-κB信号通路介导炎症和凋亡参与AKI的发生发展。我们预实验发现“桂枝去桂加茯苓白术汤”能上调IR肾组织miR-21的表达。本研究以肾IRI大鼠模型和miR-21沉默的肾小管上皮细胞株、足细胞株为研究对象，通过免疫组织化学法、实时荧光PCR和Western等方法探讨桂枝去桂加茯苓白术汤对大鼠肾组织和肾小管上皮细胞株、足细胞株miR-21、NF-κB、炎症因子和凋亡蛋白表达的影响。项目有助于为桂枝去桂加茯苓白术汤临床治疗AKI提供理论依据。

背景：缺血再灌注是急性肾损伤的主要发病机制，目前还没有发现有效治疗药物。AKI的临床表现根据辨证论治的中医学理论，应当归属于脾虚水停，湿滞三焦，治疗上需畅三焦、利小便。前期临床研究发现“桂枝去桂加茯苓白术汤”治疗AKI患者，能显著降低血肌酐、尿素氮、NGAL。为探究其机制，我们关注miR-21，已有研究表明其能调控多个靶基因，通过调控NF-κB信号通路介导炎症和凋亡参与AKI的发生发展。我们的预实验证实“桂枝去桂加茯苓白术汤”能上调IR肾组织miR-21的表达。. 方法：以肾IRI大鼠模型和miR-21沉默的肾小管上皮细胞株、足细胞株为研究对象，通过免疫组织化学法、实时荧光PCR和Western等方法探讨该方剂对大鼠肾组织和肾小管上皮细胞株、足细胞株miR-21、NF-κB、炎症因子和凋亡蛋白表达的影响。.结果：该方剂能明显降低缺血再灌注损伤大鼠模型的血清肌酐、尿素氮与NGAL，减轻其肾组织细胞损伤与细胞凋亡，降低肾组织TNF-α、NF-κB、IL-6、caspase-3与caspase-8蛋白质与mRNA表达水平，促进miR-21表达成倍数增长；进一步培养肾小管上皮细胞，验证了缺氧复氧也能刺激小管上皮细胞TNF-α、NF-κB、caspase-3表达增加和p38MAPK、ERK1/2和JNK1/2/3蛋白的磷酸化，桂枝去桂加茯苓白术汤干预能够下调缺氧复氧肾小管上皮细胞TNF-α、NF-κB、caspase-3表达，抑制MAPKs、ERS相关因子磷酸化，提高miR-21表达；抑制miR-21后，桂枝去桂加茯苓白术汤对缺氧复氧肾小管细胞TNF-α、NF-κB等因子的下调作用被逆转。低、中剂量的桂枝去桂加茯苓白术汤能够促进足细胞增殖，其增殖效果具有剂量依赖性，且能降低足细胞早期凋亡率及晚期凋亡率。.意义：研究结果证实桂枝去桂加茯苓白术汤对缺血再灌注诱导的AKI具有确切的防治效果，该方剂通过提高miR-21表达抑制p38MAPK、ERK1/2和JNK1/2/3蛋白磷酸化，减少TNF-α、NF-κB、IL-6等炎症因子及caspase-3、caspase-8等凋亡蛋白表达，减轻肾小管细胞结构和功能损伤。同时也提示miR-21是AKI发生和发展的重要作用因子，可能作为AKI的潜在治疗靶点。