基于“补虚泻实”理论探讨黄芪甲苷、大黄酸调控PGC-1α/TGF-β/ROS通路抗慢性肾衰竭的协同机制

Chronic renal failure (CRF) is a serious threat to human health. The “BuXuXieShi” theory of Traditional Chinese Medicine treating CRF provides us a research idea for Traditional Chinese Medicine kidney protection. Astragaloside IV and Rhein, which is the effective component of the typical combination of Traditional Chinese Medicine "Astragalus and Rhubarb" for CRF treatment, has been widely confirmed to have renal protective effect. However, the combined application of them has been rarely reported. Our previous research found the degree of renal tissue injury in combined group was obviously better than the single group through 5/6 nephrectomy model. In vitro results showed that the combined group can reduce the level of ROS, decrease the expression of TGF-β of the key protein in renal interstitial fibrosis and increase the expression of the mitochondria-function regulator PGC-1α. It’s still need to be further verified the synergistic renal protection of the combined group whether based on the “BuXuXieShi” theory of Traditional Chinese Medicine through antioxidant action and regulate the expression of TGF-β and PGC-1α protein to co-degeneration the Epithelial-mesenchymal transition of renal tubular epithelial cells. In this project, Flow cytometry, Laser scanning confocal microscope, RT-PCR, Immunohistochemistry and other related technology were used to explore whether the synergistic effect of Astragaloside IV and Rhein combined with PGC-1α/TGF-β/ROS pathway to protect CRF based on the “BuXuXieShi” theory of Traditional Chinese Medicine on the animal, cellular and molecular levels. It will provide a theoretical basis for the clinical treatment of CRF in the future.

慢性肾衰竭（CRF）严重危害人类健康。中医“补虚泻实”理论治疗CRF为我们进行传统中药开发提供研究思路。黄芪甲苷及大黄酸作为抗CRF经典组合“黄芪、大黄”的有效单体，已广泛证实具有明显肾保护作用，但二者联用的研究却鲜有报道。本课题组预实验动物结果发现联用组肾组织损伤程度明显优于单用组；细胞实验显示联用能减少ROS生成，降低肾间质纤维化关键蛋白TGF-β及增加线粒体功能调节因子PGC-1α的表达。然而联用能否基于中医“补虚泻实”理论，通过抗氧化、调控TGF-β及PGC-1α蛋白表达共同降低肾小管上皮间质细胞转化的发生，产生抗CRF协同作用，有待验证。本项目拟采用流式细胞仪、激光共聚焦显微镜、RT-PCR及免疫组化等相关技术手段，在整体、细胞、分子水平研究黄芪甲苷、大黄酸联用是否基于“补虚泻实”理论调控PGC-1α/TGF-β/ROS通路产生抗CRF协同作用，为二者应用于临床提供理论依据。

目前临床上治疗慢性肾衰竭（CRF）的方法包括药物治疗和手术治疗，但效果均不理想。中医基于CRF的主要病机，认为“补虚泻实”理论是中医药治疗CRF的重要指导思想，且被广泛应用。黄芪、大黄“一补一泻”的配伍应用，与中医治疗CRF“补虚泻实”基本原则相契合。而黄芪甲苷、大黄酸作为黄芪、大黄的主要有效单体已被证实具有明显肾保护作用，我们前期证实了二者联用能减少ROS生成，降低肾间质纤维化关键蛋白TGF-β及增加线粒体功能调节因子PGC-1α的表达。本课题基于前期研究，通过5/6肾切除模型和TGF-β诱导的HK2细胞模型，对黄芪大黄是否能调控PGC-1α/TGF-β/ROS通路发挥抗CRF的作用进行验证。结果表明：黄芪甲苷和大黄酸单用组和联用均能改善5/6肾切除模型肾组织损伤的情况，且联用组的效果更好；MTT实验数据结果显示，黄芪甲苷和大黄酸合用能显著提高TGF-β诱导的HK2细胞活力，减少细胞凋亡，同时降低TGF-β蛋白水平，提高PGC-1α表达，提示，二者联用能够调控PGC-1α/TGF-β/ROS通路发挥抗CRF作用。此外，我们对大黄酸肾保护作用进行了系统研究，结果显示：大黄酸可以使LPS诱导的HK2细胞活性上升，抑制细胞凋亡，降低IκBα蛋白的磷酸化水平，抑制p50和p65的表达，提示大黄酸可能是通过调控NF-κB信号通路发挥的肾保护作用；通过5/6肾切除的大鼠和H2O2刺激的HK2细胞模型，发现大黄酸能够抑制氧化应激，使肾纤维化和肾细胞凋亡程度降低，证实了SIRT3/Foxo3α信号通路在大黄酸抑制肾的氧化损伤和纤维化的过程中扮演着至关重要的角色。综上结果看出，本课题的顺利完成，不仅系统研究了大黄酸、黄芪甲苷对CRF的药效作用及相关机制，同时为临床应用黄芪、大黄防治CRF提供重要依据。