基于JAK/STAT信号通路探讨六味地黄丸治疗绝经后骨质疏松症肾阴虚证的免疫机制

The study shows the mechanisms of Liuweidihuangwan treating Kidney Yin deficiency are related to improvement the immunity of human body. But the mechanisms of genomics have not been clarified. The research group found ASB1 was related to postmenopausal osteoporosis（POP） with Kidney Yin deficiency . ASB1 is one of SOCS subfamily member.SOCS is a negative feedback factor of Janus activated kinase- signal transducers and acti-vator of transcriptions(JAK/STAT) pathway.JAK/STAT pathway is closely related to the immune system .We speculated the immunity mechanisms of Liuweidihuangwan treating POP with Kidney Yin deficiency may be related to the regulation of JAK/STAT pathway . In this study, Liuweidihuangwan has been treated POP with Kidney Yin deficiency for 3 months.① Real time PCR array which contained 41 immune-related genes are applied to observe the gene expression of subjects before and after treatment.And differentially expressed genes are screened. Real time PCR array、 Western Blot technology are applied to validate immune-related gene and protein changes of JAK/STAT pathway .At the same time, clinical validation were dealt to detect the change of immune factors of POP with Kidney Yin deficiency .we hope to find out the related gene of Liuweidihuangwan treating POP with Kidney Yin deficiency ,to provide a theoretical basis for the immune mechanisms of Liuweidihuangwan treatment POP with Kidney Yin deficiency .

研究表明六味地黄丸治疗肾阴虚证机理与其改善人体免疫机能相关，但其基因组学机制尚未阐明。课题组前期发现ASB1可能是绝经后骨质疏松症（POP）肾阴虚证的关联基因；ASB1属于SOCS亚家族成员，SOCS是JAK/STAT信号通路的负反馈因子，而JAK/STAT通路与免疫密切联系。因此，课题组推测：六味地黄丸治疗POP肾阴虚证的免疫机制可能与其调控JAK/STAT通路相关。 本研究拟：选择POP肾阴虚证受试者，六味地黄丸治疗3个月，①用含有41个与免疫相关基因的JAK/STAT信号通路芯片，观察受试者治疗前、后免疫基因的表达变化，筛选差异表达基因；②用 RT-PCR、Western Blot技术验证差异表达基因及蛋白水平，结合受试者治疗前后血清中免疫因子变化佐以证明。旨在JAK/STAT通路中找出六味地黄丸治疗POP肾阴虚证的关联基因，为揭示六味地黄丸治疗POP肾阴虚证免疫机制提供理论依据。

临床用六味地黄丸治疗绝经后骨质疏松症（POP）肾阴虚证具有很好的疗效。其治疗机理被认为与改善人体免疫机能相关，但其基因组学机制尚未阐明。课题组提出假说：六味地黄丸治疗POP肾阴虚证的免疫机制可能与其调控JAK/STAT通路相关。本课题以POP肾阴虚证为研究对象，六味地黄丸治疗3个月后，用含有41个与免疫相关基因的JAK/STAT信号通路芯片观察受试者治疗前、后免疫基因的表达变化，筛选差异表达基因；用 RT-PCR、Western Blot技术验证筛选出来的差异表达基因及蛋白水平，结合受试者治疗前后血清中免疫因子变化佐以证明。结果：（1）六味地黄丸治疗POP肾阴虚证组3个月后，25个基因表达水平发生显著变化，其中7个免疫基因发生变化，4个基因上调，分别为：OSM、F2、IL4R、IRF1。3个基因下调，分别为：IFNG、PRLR、YY1。（2）经RT-PCR验证，六味地黄丸治疗POP肾阴虚证组3个月后，可显著上调IRFI mRNA 表达水平；OSM基因在治疗后表达虽有上调趋势，但统计学比较无明显差异； PRLR、IFNG和YY1基因表达水平下降,但统计学比较无显著性差异。（3）经Western Blot验证，六味地黄丸治疗3个月后，可显著上调OSM蛋白表达水平，IFNG、F2蛋白在药前、药后表达没有明显差异。（4）六味地黄丸治疗PMOP肾阴虚证3个月，降低血清IL-6、IL-8水平，TNF-α有下降趋势，但差异无统计学意义。结合文献分析，六味地黄丸治疗POP肾阴虚证的免疫机制可能与其调控JAK/STAT信号通路中的IRF1基因相关，IRF1基因可能是六味地黄丸治疗POP肾阴虚证的一个重要关键基因。这或许能为防治POP提供一个新的靶点基因，为推广六味地黄丸治疗提供强有力的科学依据，对促进祖国传统中药国际化具有重要的意义。