从Akt-mTOR信号通路研究中西药合用治疗糖尿病肾病的机制

Diabetic nephropathy(DN) is a major public problems in the world and is now the main cause of end-stage renal disease. The mechanisms leading to the development and progression of renal injury are not well known.Therefore ,it is important to understand the pathogenesis of DN and to develop new therapies for its prevention and treatment.Recent investigations have revealed that injuries to podocytes play a critical role in the development of DN and glomerulosclerosis.These glomerular epithelial cells with their intercellular junctions (slit diaphragms) between foot processes contribute to the filtration barrier. The proteinuria is major associated with podocyte injury in glomerular diseases including DN. It was rencently reported that the activity of mTOR complex 1 (mTORC1),a rapamycin -sensitive protein kinase complex, is enhanced in the podocytes of animals and patients with diabetic nephropathy, and administration of rapamycin prevents the injuries of podocytes and the progression of DN in animal models of both type 1 and type 2 diabetes. However, rapamycin increases the risk of proteinuria in chronic allograft nephropathy.The mechanism of rapamycin causing poteinuria remains unclear. The mTORC1 function in podocytes is essential for the integrity of the filtration barrie. It was reported that podocyte-specific mTORC1 knockout mice develop significant protienuria, and the phosphorylation of Akt on residue Thr308 in podocytes is strongly increased in these mice. These data underline the importance of the balance between PI3K-Akt-mTORC1 axis for podocyte biology. Our previous studies revealed that Tujian Mixture ( a Chinese herb formulation) inhibit the activation of Akt on residue Thr308 in glomerular mesangial cells cultured in high concentrations of glucose. This program is to investigate the effect of Tujian Mixture on the Akt-mTORC1 axis in podocytes of diabetic nephropathy, and to elucidate if Tujian mixture could enhance the effect of rapamycin on prevention of diabetic nephropathy and elimilate the risk of rapamycin to the podocytes injuries, through inhibition of the phospahorylation of Akt on residue Thr308 in podocyte caused by rapamycin, to explore a new pathway of prevention and treatment of diabetic nephropathy through the combination of Chinese herb and rapamycin.

糖尿病肾病已经成为终末肾衰的首要原因，探索防治糖尿病肾病的有效措施是目前重要的课题。肾小球足细胞mTORC1活性的异常升高是糖尿病肾病发生蛋白尿的关键机制之一，雷帕霉素特异性抑制mTORC1可以防止糖尿病肾病发生发展，但过度抑制足细胞mTORC1也会造成足细胞的损伤，其机制可能与mTOC1上游蛋白Akt(Thr380)反馈性异常磷酸化有关。我们既往的研究发现中药菟箭合剂具有抑制肾小球系膜细胞Akt(Thr308)磷酸化的作用。本项目拟从PI3K-Akt-mTOR信号通路入手研究中西药合用治疗糖尿病肾病的机制，阐明菟箭合剂是否可抑制雷帕霉素导致的Akt(Thr308)反馈性异常磷酸化，达到增强雷帕霉素对糖尿病肾病的治疗效果、同时防止雷帕霉素潜在的造成肾小球足细胞损伤的作用，探索一种中西药各自发挥优势，具有减毒增效的联合用药治疗糖尿病肾病的新模式。

既往有研究发现雷帕霉素可通过抑制肾小球足细胞PI3K/Akt/mTOR信号通路的活性，从而防止足细胞裂孔隔膜相关蛋白的丢失达到减少糖尿病动物模型尿蛋白的作用，成为一种潜在的糖尿病肾病治疗药物，但很少有人关注其可能的毒副作用。本项目研究采用STZ诱导的1型糖尿病大鼠模型，在模型制作成功后4周开始分别给予小剂量雷帕霉素（0.5mg/kg隔日一次腹腔注射）、大剂量雷帕霉素（5mg/kg隔日一次腹腔内注射）、中药复方黄芪菟箭合剂、黄芪菟箭合剂合用小剂量雷帕霉素或大剂量雷帕霉素治疗12周。初步获得的结果提示小剂量雷帕霉素具有显著降低糖尿病大鼠尿白蛋白排泄率的作用，中药复方黄芪菟箭合剂治疗也具有显著降低糖尿病大鼠尿白蛋白排泄率作用，其降低尿蛋白排泄率的作用与小剂量雷帕霉素无显著差异。但雷帕霉素治疗对1型糖尿病大鼠有显著降低体重、增加大鼠死亡率的毒副作用，大剂量雷帕霉素（5mg/kg隔日腹腔内注射）治疗12周致死率达70%。黄芪菟箭合剂与小剂量雷帕霉素合用可降低糖尿病大鼠死亡率。黄芪菟箭合剂降低1型糖尿病大鼠尿白蛋白排泄率的作用机制可能与其通过提高PI3K/Akt/mTOR信号通路的天然抑制物PTEN的表达、抑制Akt（Thr308）活化有关，而雷帕霉素的毒副作用可能与其反馈性提高Akt（Thr308）活性有关。