补肾活血方通过调节钙磷代谢调控血管平滑肌细胞转分化的实验研究

Vascular calcification is one of the most severe complications in chronic kidney disease, especially in late end stage, being a major cause of cardiovascular disease related death. Vascular calcification is closely related with abnormal metabolism of calcium and phosphorus, characterized by the transformation of vascular smooth muscle cells(VSMCs) into osteoblast-like celles. Previous clinical experience and experimental data from our group indicated that the decoction of nourishing Qi and promoting blood (Bushenhuoxue Decoction) could improve phosphate retention in ESRD patients, bi-directionally regulate calcium metabolism and activate osteoblast activities. This study is planned to investigate whether the method mentioned above can inhibit differentiation of VSMCs to an osteoblastic phenotype by regulating the metabolism disorder of calcium and phosphate with in vitro and in vivo experiments. A mouse model with 5/6 kidney removed, and calcium and phosphate metabolism disorder induced by high phosphate food were developed. This model are then used to analyze the regulation of calcium and phosphate metabolism, interference of other calcification promoting f and inhibitory factors and by thereby antagonist the transformation of osteoblast like cells by Bushenhuoxue therepy. In the in vitro experiment, the VSMCs were generated by osteoblast culture stimulated with high concentration of phosphate, calcium and PTH, and then, the inhibition roles of Bushenhuoxue decoction on transformation of VSMCs into osteoblast cells were evaluated, and the underlying mechanisms are explained.

血管钙化是慢性肾脏病特别是终末期肾病患者严重并发症之一，是导致患者发生心血管疾病而致死的主要原因。血管钙化的关键在于血管平滑肌细胞（Vscular smooth mucle cell, VSMC）向成骨细胞样表型转化，与钙磷代谢异常密切相关。本课题组在以往临床及实验研究中上发现补肾活血方能够改善ESRD患者磷潴留，双向调节血钙，促进成骨细胞活动。本研究包括在体实验和体外实验两部分内容。在体实验拟以5/6肾切除+高磷饮食致慢性肾衰钙磷代谢紊乱的大鼠模型为对象，研究补肾活血方通过调节钙磷代谢、干预钙化促进因子及抑制因子，拮抗VSMC向成骨样细胞表型转化的作用来评价其防治血管钙化的作用。体外实验部分是以体外培养的高磷/高钙/高PTH刺激下的VSMCs为对象，给予补肾活血方含药血清进行干预，检测血管平滑肌细胞标记物等多项钙化调节因子，观察本方拮抗VSMCs向成骨样细胞转分化的作用，并探讨其机制。

慢性肾脏病（CKD）患者众多死亡原因中，因心血管疾病（CVD）而致死的约占总死亡人数的50%。血管钙化（VC）作为动脉粥样硬化的标志事件及心血管事件病死率的预测指标，增加了透析患者发生CVD及相关死亡的风险。VC发病机制复杂，其中OPG/RANK/RANKL系统及BMP-2/Runx2/Osterix信号通路及MGP、BMP-7因子近年来被认为在VC中发挥重要作用。本项研究从体内实验和体外实验两部分观察补肾活血方对CKD VC大鼠及VSMCs（即A7r5细胞）细胞表型转化的干预作用及相关机制。.体内实验：75只SD大鼠随机分为正常组、模型组及中药组。模型与中药组用腺嘌呤混悬液灌胃及高磷饲料喂养建立CKD VC模型，中药组同时按给予补肾活血方灌胃治疗，每日1次。实验结束时，观察肾组织变化及主动脉钙化结节情况，检测主动脉钙含量及碱性磷酸酶（ALP）活性，腹主动脉取血检测肾功能及全段甲状旁腺激素（iPTH）水平，检测肾脏指数，Western blot法检测主动脉α-平滑肌蛋白（α-SMA）、ALP、BMP-2、Runx2、Osterix蛋白的表达，Real-time PCR法检测主动脉OPG、RANKL 、MGP、BMP-7mRNA表达。补肾活血方能够改善CKD VC大鼠肾脏组织学、肾功能、改善钙磷代谢及继发性甲状旁腺功能亢进，减轻主动脉钙化程度，上调OPG/RANK/RANKL系统中OPG mRNA表达及MGP、BMP-7mRNA表达，下调BMP-2/Runx2/Osterix信号通路表达，这可能是其抑制CKD VC的作用机制。体外实验：通过体外高磷/高钙刺激VSMCs建立血管钙化模型，用补肾活血含药血清低、中、高三个剂量组进行体外干预。实验结束后观察各组细胞形态，细胞层钙含量及ALP活性，各组α-SMA、ALP、BMP-2、Runx2、Osterix蛋白表达及OPG 、RANKL 、MGP、BMP-7mRNA表达。结果发现补肾活血含药血清可抑制体外高磷/高钙刺激下VSMCs向成骨样细胞表型转化，其可能通过上调OPG/RANK/RANKL系统中OPG mRNA表达及MGP、BMP-7mRNA表达、下调RANKL mRNA表达实现，并与下调BMP-2/Run2/Osterix信号通路表达有关。本项研究更好的阐述了补肾活血方的作用机制，为该方临床应用及推广提供基础