"培土制水法"益肾降浊冲剂治疗慢性肾功能衰竭和尿毒症多脏器损伤的线粒体机制的研究

Our research group has brought forward “the hypothesis in cell biology of the essence of five Zang organs in traditional Chinese medicine(TCM)” since 2002. It deems that the essence of five Zang organs in TCM is the structures in cell and sub-cell. Five Zang organs in TCM are the five internal organs in cell. The five internal organs in cell are five function systems, which are taken by five structures in sub-cell，e.g. Mitochondria are the Micro Substance of “Spleen” in TCM.. By 20 years’ clinical practice, It has been proven that Soluble Granules Yishenjiangzhuo , which is constructed by the therapy principle of“Strengthening Earth to Restrain Water” in TCM, has great curative effects of chronic renal failure (CRF). And it has been proven by our previous animal experiment that the Soluble Granules Yishenjiangzhuo not only protect renal tubular epithelial cell in hypermetabolism (“Overstrain Impairing Spleen” in TCM), but also improve the ability to resisting uremic toxins ( “Disturbance of Spleen Due to Damp-pathogen” in TCM). It do this by protecting mitochondria ( “Strengthening Earth” in TCM）and reducing apoptosis..Basing on research above, Its pharmacological mechanism which we speculated is that restraining apoptosis from the way of mitochondria. Thus, this project intend to research the molecular mechanism and therapeutic target on damage of renal tubules in hypermetabolism , and that of organs in multi-system of body by the uremic toxins.

“培土制水法”益肾降浊冲剂，作为福建省人民医院院内制剂治疗慢肾衰(CRF)已20年。我们前期研究表明，CRF肾脏的持续损伤、以及尿毒症多脏器损害，均与氧自由基、线粒体、细胞凋亡相关；该冲剂具治疗上述两症的疗效，且能保护线粒体、减少氧自由基和凋亡。.基于以上研究，提出假说：①CRF时大量肾单位受损，剩余的健全肾小管处于超负荷的高代谢状态，线粒体过劳，导致氧化磷酸化效率降低、活性氧增多，可能激活了线粒体途径的细胞凋亡，其证应属“劳倦伤脾”。②尿毒症毒素可能通过影响线粒体，导致多脏器细胞凋亡、坏死，其证当属“湿气困脾”。③“培土制水法”的双重疗效，机制可能均是通过健“脾”，保护线粒体、抑制细胞凋亡。.本项目拟从整体动物和细胞培养两方面，研究活性氧、线粒体（膜流动性、膜电位、氧化损伤和呼吸功能）、细胞凋亡与上述双重疗效的关系，探讨“培土制水法”治疗CRF的分子机制和靶点，为治疗CRF提供新思路。

实验分为两个部分，一部分基于“肾脏高代谢学说”的以线粒体为中心的慢肾衰进展的机理研究及益肾降浊冲剂药理作用机制；另一个部分探讨尿毒症毒素引起的心脏损伤及益肾降浊干预的药理作用机制。.第一部分 在肾脏方面： .动物实验方面：用5/6肾切除术建立大鼠慢肾衰动物模型，用益肾降浊冲剂治疗，检测结果显示尿毒症大鼠血清中肌酐、尿素氮水平明显升高，肾脏组织中出现炎症及纤维化改变、氧自由基水平升高、肾小管上皮细胞凋亡数量增加，线粒体凋亡途径上促凋亡蛋白表达明显上升，而益肾降浊冲剂能够显著改善这些情况。.细胞实验部分：用H2O2模拟肾小管上皮细胞高代谢状态，用益肾降浊冲剂含药血清治疗，检测结果显示：高代谢肾小管上皮细胞内细胞活力明显下降，细胞毒性明显上升，细胞内氧自由基水平上升，细胞膜电位发生下降，细胞凋亡率上升，线粒体凋亡途径促凋亡蛋白及基因表达明显上升，经过益肾降浊冲剂干预之后，这些情况明显改变。.第二部分 在心脏方面： .动物实验方面：用益肾降浊冲剂治疗5/6肾切除术慢肾衰大鼠，实验结果显示尿毒症大鼠血清中肌酸激酶同工酶及乳酸脱氢酶水平明显升高，心脏组织中出现心肌细胞肥大等病理改变、ATP水平升高、心肌细胞凋亡数量增加，线粒体凋亡途径上促凋亡蛋白及基因表达明显上升，而益肾降浊冲剂能够显著改善这些情况。.细胞实验部分： 1、原代心肌细胞的培养和鉴定。2、用尿毒素硫酸吲哚酚（IS）干预心肌细胞进行细胞建模。3、用益肾降浊冲剂中活性成份中药单体大黄素及黄芪多糖进行干预治疗，检测细胞活力、细胞内氧自由基、细胞凋亡及线粒体凋亡途径相关基因及蛋白，摸索实验技巧。4、用益肾降浊冲剂含药血清重复第3步骤。实验结果显示，黄芪多糖、大黄素和益肾降浊冲剂含药血清，均能明显改善IS损伤心肌细胞所致的细胞活力，细胞内氧自由基，细胞凋亡率，以及线粒体凋亡途径促凋亡蛋白及基因表达。