“过咸伤骨”，基于相关离子通道研究高盐影响骨代谢的分子机制及补肾中药复方干预

“Salty entering the kidney，the kidney governing the bone，too salty to hurt the bone”, which is one of the important contents of the connection between five taste and viscera in Traditional Chinese Medicine (TCM) theory, and modern medical research has also found that high salt (sodium chloride) intake can lead to bone loss, but the specific mechanism is not clear.Ion channel plays an important role in the function of bone cells and renal tubular calcium reabsorption. According to the chemical composition of sodium chloride and the pre experimental results, we proposed the hypothesis that sodium chloride may affect the bone metabolism through ion channels associated with chloride and sodium ions.In this study, we will use immunohistochemistry, gene repression, laser scanning confocal microscopy, patch clamp and other techniques, firstly make a high turnover bone metabolism model in ovariectomized rats to observe the effects of high salt diet on expression and distribution of related ion channels in bone and renal tubular in ovariectomized rats; Secondly use several cell lines including bone cells and renal tubular epithelial cell lines in vitro to observe the expression and activity of the related ion channels during the differentiation of osteoblasts and osteoclasts, and the expression of the related ion channels in renal tubular epithelial cell lines in different concentrations of sodium chloride medium; And further to observe the intervention of a compound Chinese medicine for reinforcing kidney in the above conditions. To the end of the study，we will get a new scientific interpretation of the TCM theory of “too salty to hurt the bone”，which is important for the prevention and treatment of osteoporosis under the guidance of TCM theory and the understanding of pathophysiologic mechanism of high salt affecting bone metabolism; And we will get the overall role of a compound Chinese medicine for reinforcing kidney treating osteoporosis through regulating related ion channels.

“咸入肾，肾主骨，过咸伤骨”是中医理论中五味与脏腑相关的重要内容之一，现代研究也发现高盐（氯化钠）摄入可导致骨量丢失，但具体机制尚不清楚。离子通道在骨系细胞功能活性和肾小管钙重吸收中起着重要作用。我们根据化学结构和预实验结果提出了氯化钠可能通过氯和钠相关离子通道影响骨细胞功能活性和尿钙排泄从而影响骨代谢的科研假说，研究应用免疫组化、基因阻遏、LSCM、膜片钳等技术，①采用去卵巢大鼠高转换骨代谢模型，观察高盐饮食对骨组织和肾小管相关离子通道表达和分布的影响；②采用体外培养的多种细胞系，观察相关离子通道在氯化钠对成骨和破骨细胞分化及肾小管上皮细胞活性影响中的作用；③并进行补肾中药复方的干预研究，评价相关性。目的意义：①从离子通道角度阐释中医理论“过咸伤骨”的科学内涵，对于中医理论指导防治骨质疏松及探讨氯化钠影响骨代谢的病理生理学机制方面有重要意义，②进一步阐释补肾中药多靶点的网络药理学机制。

离子通道不仅参与骨系细胞的活性与功能，而且影响肾脏对钙的重吸收，前期研究显示ENaC、NCC、ClC-3、ClC-5、ClC-7、NCX1等离子通道不仅参与骨系细胞的活性与功能，并且可能是高盐影响骨代谢的最直接的效应分子。本研究以此为切入点，探究高盐摄入影响骨代谢的分子机制，以经典的去卵巢OP大鼠模型为平台，采用MicroCT、LSCM、免疫组化、Western blot、qPCR及RNA干扰等技术，从动物、细胞、分子多层次观察比较补肾中药在低盐或高盐状态下的干预研究，从一条新途径阐明补肾中药复方的多靶点网络药理学机制，并明确补肾中药服用时使用淡盐水送服的作用。研究发现，高盐诱导的骨丢失主要是提高了骨吸收程度而不是减少骨形成，高盐饮食可引起股骨组织的相关离子通道ENaCα、ClC-3、ClC-5的表达下调，NCC、NCX1和CLC7的表达上调，从而直接影响骨系细胞活性，引起骨形成的减少和骨吸收的增加，改变骨代谢的平衡，也影响着骨代谢相关指标的变化。而高盐饮食引起肾小管相关离子通道ENaCα、ClC-3、ClC-5的表达上调，NCC、NCX1和CLC7的表达下调，增加尿量和氯化钠的排泄，从而影响尿钙的排泄，维持体内钠和钙的平衡，动员骨骼钙的排出，引起骨量丢失，补肾中药复方的干预能够不同程度的调节相关离子通道的改变，对高盐引起的骨代谢失衡具有一定的改善和治疗作用。高NaCl在诱导细胞损伤、凋亡时，上调NCC、OPN并下调1α-羟化酶、BMP-7、CLC-5基因和蛋白的表达，而BKF能增强细胞活力，抑制由高NaCl上调的NCC、OPN基因和蛋白的表达，上调由高NaCl抑制的1α-羟化酶、BMP-7、CLC-5基因和蛋白的表达，高NaCl及BKF对细胞1α-羟化酶、BMP-7的抑制与促进作用可能通过NCC起调节作用，且NCC亦可能调控高NaCl环境下细胞OPN的过表达。