肾苏Ⅳ干预lncRNA H19/DIRAS3调控足细胞自噬阻抑肾小球足细胞损伤的机制研究

Podocyte injury is now considered to be the centerpiece in the pathological course of glomerular injury. The umbalanced autophagy was found to exacerbate podocyte injury, which has become a hot spot in the research of podocyte injury. In recent researches it was elaborated that lncRNA/DIRAS3 participate in the process of autophagy regulation, but the mechanism remained unclear.It brought up to us that whether Shensu-Ⅳ was able to regulating mTOR pathway through lncRNA/DIRAS3 regulation, as our former research proved that Shensu-Ⅳwas able to alleviate podocyte injury by regulating its mTOR pathway. Intend to provide theoretical basis of TCM in the treatment of chronic kidney podocyte injury, we are going to investigate the effect of Shensu-Ⅳ on the podocyte autophagy regulation by the means of H19 gene silencing through siRNA interference, lncRNA H19/DIRAS3 assessment through RT-PCR, electron microscopy of podocytes and autophagosome, and multiple assessment of mTOR, p-mTOR, LC3-Ⅰ, LC3-Ⅱ, and p62 using Western blot in the following project.

足细胞损伤是肾小球疾病的关键病理环节，自噬失衡可导致足细胞损伤及肾小球硬化加重，从自噬角度探讨足细胞损伤的防治成为研究热点。最新研究表明，lncRNA H19/DIRAS3参与调控细胞自噬，但其对足细胞自噬的调控机制尚不明确。前期研究已证实中药复方肾苏Ⅳ可通过调控足细胞自噬mTOR信号通路，促进足细胞损伤修复。我们假说肾苏Ⅳ可能通过干预lncRNA H19/DIRAS3调控mTOR信号通路，促进足细胞自噬以阻抑足细胞损伤。本项目通过复制PAN肾病大鼠模型及体外诱导足细胞损伤模型，采用siRNA干扰技术沉默H19，RT-PCR检测lncRNA H19、DIRAS3，电镜观察足细胞超微结构及自噬体，Western blot检测mTOR、p-mTOR及自噬活性标记物LC3-Ⅰ、LC3-Ⅱ、p62表达变化，以探讨肾苏Ⅳ对肾小球足细胞自噬的调控机制，旨为中医药防治慢性肾脏病足细胞损伤提供理论依据。

足细胞损伤是肾小球疾病的关键病理环节，自噬失衡可导致足细胞损伤及肾小球硬化加重，从自噬角度探讨足细胞损伤的防治成为研究热点。研究表明，lncRNA H19/DIRAS3参与调控细胞自噬，但其对足细胞自噬的调控机制尚不明确。前期研究已证实中药复方肾苏Ⅳ可通过调控足细胞自噬mTOR信号通路，促进足细胞损伤修复。我们假说肾苏Ⅳ可能通过干预lncRNA H19/DIRAS3调控mTOR信号通路，促进足细胞自噬以阻抑足细胞损伤。本项目通过复制PAN肾病大鼠模型及体外诱导足细胞损伤模型，采用siRNA干扰技术沉默H19，RT-PCR检测lncRNA H19、DIRAS3，电镜观察足细胞超微结构及自噬体，Western blot检测mTOR、p-mTOR及自噬活性标记物LC3-Ⅰ、LC3-Ⅱ、p62表达变化，以探讨肾苏Ⅳ对肾小球足细胞自噬的调控机制。实验证实：3周和6周模型组大鼠肾小球肥大，囊腔狭窄，部分节段出现球囊黏连，系膜基质增多，系膜区增宽，基底膜增厚，肾小管上皮细胞肿胀，肾间质出现部分纤维化。各干预组光镜观察可见不同程度的减轻，中剂量和高剂量最为明显。第6周末，模型组大鼠足细胞自噬体数量明显少于正常组；肾苏Ⅳ各组足细胞自噬体数量较模型组明显增多。3周和6周时，模型组p62表达水平明显升高，LC3-II/LC3-I明显下降，肾苏IV干预组p62表达水平明显下降，LC3-II/LC3-I明显升高；其中以中剂量与高剂量最为明显。分离SD大鼠足细胞成功，shLncRNA H19-3干扰效果约70%以上，后续选择该进行后续的实验。与对照组相比，shLncRNA H19组和shLncRNA H19+嘌呤霉素+肾苏IV组LncRNA H19、mTOR、p-mTOR和p62表达明显下降，DIRAS3、LC3-II/I表达明显升高，嘌呤霉素组LncRNA H19、mTOR、p-mTOR和p62表达明显升高，DIRAS3、LC3-II/I表达明显下降；与shLncRNA H19+嘌呤霉素组相比，shLncRNA H19+嘌呤霉素+肾苏 IV组的LncRNA H19、mTOR、p-mTOR和p62表达明显下降，DIRAS3、LC3-II/I的表达明显升高。证实肾苏IV通过干预lncRNAH19/DIRAS3调控mTOR信号通路，促进肾小球足细胞自噬从而阻抑足细胞损伤。