基于肾络瘀痹理论干预高血糖诱导足细胞ERS分子作用机制研究

By the Traditional Chinese medicine compound and syndrome of disease association study methods, In order to study molecular mechanisms of action of the Traditional Chinese medicine compound of TongLuoBaoShen (TLBF) and TongLuoBaoShen of prescription dismantlement intervene with hyperglycemia induced activation of podocyte Endoplasmic Reticulum Stress (ERS) to bring about podocytes injury to express gene expression and protein expression of GRP78、IBX-1 in early phases of Streptozotocin-induced diabetic nephropathy. podocytes injury in Streptozotocin-induced diabetic nephropathy based on podocyte cell volume reduction and density decreased and membrane molecule expression reduction. apoptotic signals CHOP、JNK and caspase12 pathway activation, NF-κB/P65 pathway activation Increased gene expression and protein expression of downstream inflammatory cytokines in podocyte cell injury in 6 weeks and 16 weeks of early phases of Streptozotocin-induced diabetic nephropathy. In order to study different target function molecular mechanism of prevention and treatment of Streptozotocin-induced diabetic kidney damage of the Traditional Chinese medicine compound of TongLuoBaoShen and TongLuoBaoShen of Different fractions intervene on to concern between activation of Endoplasmic Reticulum Stress (ERS) with podocyte cell volume reduction and density decreased and membrane molecule expression reduction, apoptotic signals JNK and caspase12 pathway activation, NF-κB/P65 pathway activation and increased gene expression and protein expression of downstream inflammatory cytokines relevant in early phases of Streptozotocin-induced diabetic nephropathy. We carry on a further research on Traditional Chinese medicine pathogenesis characteristics of Kidney collateral stasis stagnation the virtual ie deficiency of the kidney and Standard ie each node of stasis stagnation with water and poisonous on pathology of microcosmic syndrome differentiation characteristics. We carry on a further research on Traditional Chinese medicine mcrocosmic dialectic points of Specimens were divided and Tongzhi. We carry on a further research to be connected molecular mechanisms of action of activation of Endoplasmic Reticulum Stress with podocyte cell injury and progression of kidney fibrosis in renal pathological physiology in Streptozotocin-induced diabetic nephropathy. As we put forward Innovation theory of Kidney collateral stasis stagnation theory to lay a new foundation application on Clinical application of basic research in diabetic nephropathy.

采用方药与病证相关性研究方法，通过研究通络保肾复方及其拆方干预STZ诱导大鼠糖尿病高血糖造成肾脏足细胞内质网应激（ERS）引起的足细胞损伤（密度下降、数目减少、膜分子表达减少）与应激蛋白GRP78、IBX-1的表达、凋亡信号CHOP、JNK及caspase12通路、NF-κB/P65信号通路及其下游炎症细胞因子作用机制，深入探讨肾络瘀痹病机学特点本虚（肾虚）、标实（瘀、水、毒）的病理学微观辨证特征、标本分治和同治微观辨证要点。深入分析通络保肾复方及其不同组分、不同实验终点防治糖尿病高血糖造成肾脏损伤足细胞内质网应激与足细胞凋亡信号CHOP、JNK、caspase12通路、NF-κB/P65信号通路及下游炎症因子作用靶点，探讨足细胞ERS与足细胞损伤、肾脏病理学进展关联在糖尿病肾脏损伤"肾脏纤维化"发生发展中的作用机制和中药药理学作用优势靶点，为我们的肾络瘀痹理论在临床应用奠定新的应用基础。

在我国糖尿病肾病已经成为肾脏替代治疗的第二位病因，因此如何预防糖尿病肾病的发生、延缓疾病的进展已经成为当代医学迫切需解决的难题。由高血糖诱导的肾脏内质网应激方应（ERS）在糖尿病肾损伤过程中发挥着重要作用。而通络保肾复方是基于“肾络瘀痹”理论，直接针对糖尿病肾病“虚、瘀、水、热、毒”的病机学要点组方而成。关于其防治糖尿病肾病方面的作用，已在前期实验研究及临床疗效观察中取得满意结果。本实验通过腹腔注射STZ诱导高血糖肾损伤大鼠模型，分拆复方研究，将通络保肾复方分拆为通络保肾补虚组分、通络保肾解毒组，借助中医方药与病症相关分析，以方测证，结合最新糖尿病肾损伤机制—内质网应激学说（ERS），观察6周、16周两个实验终点时通络保肾复方及其拆方对高血糖肾损伤大鼠模型生化、普通光镜及电镜下肾组织改变，借助免疫组化法、westerbot、QT-PCR方法检测肾脏内质网应激启动因子IRE1α/GRP78，及肾脏ERS相关凋亡信号通路、NF-κBP65炎症信号通路的改变。实验结果证实：1.单纯中药治疗在改善糖尿病大鼠GLU、HbA1c等方面无明显效果。2.普通光镜及电镜下发现模型组肾小球体积增大，基底膜增厚及系膜基质增多，导致部分毛细血管腔受压变窄，肾小管上皮细胞胞浆疏松，水肿。与模型组大鼠相比，通络保肾复方治疗组及其拆方治疗组无论在6周，16时均能降低肾小球硬化指数、肾小管损伤指数、肾小球基质指数及肾小球基底膜厚度。3.免疫组化、westerbot及QT-PCR实验结果证实：与模型组大鼠相比，6周、16周时通络保肾复方及其拆方均可以抑制ERS启因子IRE1α/分子伴侣GRP78的表达，减少ERS相关凋亡通路JNK/CHOP/Caspase-12/Caspase-3的表达，同时可以抑制高血糖诱导肾脏NF-κBP65炎症号通路中P-65及Ikk的表达。结合中医学方药与病症相关性研究，以方测证，通过观察复方及拆方研究，可以推断出STZ诱导糖尿病高血糖肾脏损伤模型6周时以标实瘀、水、毒为主，兼有肾虚；16周时以肾虚为本，标实瘀、水、毒为辅。此为糖尿病肾病微观辩证提供了可靠依据，为临床防治糖尿病肾病提供了重要指导意义。