基于PI3K/Akt/mTOR自噬信号探讨虫草益肾颗粒对糖尿病肾病足细胞损伤的治疗机制

Diabetic kidney disease(DKD) is a major cause of end-stage renal disease in the world now, which is threatening peoples health. DKD have been rising significantly, the proportion which occupy in hemodialysis patients is continuously increasing. Therefore, it is very important to find new therapeutic targets. Recently, our studies have found that ChongCaoYiShsen Particle could inhibit oxidative stress reaction and reduce proteinuria. As we known, Oxidative stress can lead to autophagic response, and hence we infer that ChongCaoYiShsen Particle participates in the podocyte injury through PI3K/Akt/mTOR autophagy pathway , which delay DKD progress and protect renal function..Based on the above words, we put forward the scientific hypothesis: promoting autopshagy can lighten pathology changes in glomerulus and podocyte injury, which is the key mechanisms for ChongCaoYiShsen Particle treating DKD. This study aim to confirm the phenomenon of autophagy at the cellular level, clarify whether ChongCaoYiShsen Particle mediates the mTOR pathway and upstream and downstream key regulatory factors, and compare the therapeutic effects of autophagy regulator and ChongCaoYiShsen Particle. In this study, we will further intervene in the animal models by using ChongCaoYiShsen Particle combined with autophagy modulators, and further study the therapeutic mechanism of ChongCaoYiShsen Particle. The project team are expected to provide us a promising avenue for DKD therapy and prevention.

糖尿病肾病（DKD）是目前全球终末期肾病的主要原因，其所占透析患者的比例正在日益攀升，严重威胁着人们的健康。因此，寻找新的治疗靶点是突破DKD治疗瓶颈急待解决的关键科学问题之一。新近，我们研究发现虫草益肾颗粒可抑制氧化应激反应，降低蛋白尿，而氧化应激又是导致自噬的机制，因此，我们推断虫草益肾颗粒可能干预足细胞PI3K/Akt/mTOR自噬通路，以延缓DKD进展，保护肾脏功能。为此，我们提出科学假说：虫草益肾颗粒通过调节肾小球足细胞自噬，来减轻肾小球病理改变和足细胞损伤，是其防治DKD的关键作用机制之一。本项目拟在细胞水平上对自噬现象进行确证，明确虫草益肾颗粒介导mTOR通路和上游、下游关键的因子调控作用，并对比自噬调节剂和虫草益肾颗粒的治疗效果；在动物水平上，用虫草益肾颗粒对疾病模型鼠进行干预，深入研究虫草益肾颗粒的治疗机制。虫草益肾颗粒和自噬靶点药物的干预将为DKD治疗提供新的思路。

糖尿病肾病是糖尿病病人最重要的合并症之一，近来研究证实，该病实为一种足细胞病，其损伤程度是预测糖尿病肾病进展的重要指标。自噬失衡可导致足细胞损伤，从自噬角度探讨足细胞损伤的防治成为研究热点。我们前期的研究发现虫草益肾颗粒可抑制氧化应激反应，降低蛋白尿，而氧化应激又是导致自噬的机制，因此，我们提出科学假说：虫草益肾颗粒通过调节肾小球足细胞自噬，来减轻肾小球病理改变和足细胞损伤，是其防治糖尿病肾病的关键作用机制之一。本课题通过糖尿病肾病大鼠模型和体外高糖诱导足细胞损伤模型，电镜观察足细胞超微结构，激光共聚焦显微镜下观察各组足细胞LC3点状聚集物分布情况，WB检测PI3K/AKT/mTOR自噬通路的蛋白表达情况及自噬活性标记物LC3、P62的表达变化，以探讨虫草益肾颗粒对肾小球足细胞自噬的调控机制。体内实验结果：透视电镜下观察到正常组足细胞足突排列规则，大小均一有序，基底膜厚薄一致，模型组足细胞足突广泛融合，基底膜未见明显增厚，与模型组相比，虫草益肾颗粒组细胞的足突融合也明显减少。WB检测PI3K/AKT/mTOR自噬通路的蛋白表达情况，结果显示，与正常组比较，模型组中p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR表达下调，与模型组比较，虫草益肾颗粒能上调p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR的表达，提示虫草益肾颗粒可促进足细胞的自噬。体外实验结果：高糖处理后小鼠MPC5细胞中LC3II/I表达上调，p-Akt/ Akt、p-mTOR/ mTOR、p62表达下调，表明高糖会抑制肾脏细胞自噬，促进肾脏细胞凋亡，造成肾脏细胞损伤，经虫草益肾颗粒治疗后上述现象得到显著改善。以上实验证实：高糖会引起肾脏细胞损伤，虫草益肾可逆转由高糖引起的肾脏细胞损伤；高糖会抑制肾脏细胞的自噬功能，经虫草益肾颗粒治疗后上述现象得到显著改善，表明虫草益肾可逆转由高糖引起的肾脏细胞自噬功能的抑制，促进肾脏细胞自噬功能的恢复。综上所述：虫草益肾颗粒可通过促进肾小球足细胞自噬，来减轻肾小球的足细胞损伤。