基于ER/RANK/NFATc1通路探讨壮骨止痛方调控破骨细胞分化的miRNA机理

Zhuanggu Zhitong decoction has showed satisfied effect on postmenopausal osteoporosis. Inhibiting ER / RANK signaling pathway in osteoclast is an important mechanism of Zhuanggu Zhitong decoction on postmenopausal osteoporosis, but it is not entirely clear on the osteoclast ER / RANK pathway details and regulation mechanisms. This project is based on the osteoclast ER / RANK / NFATc1 signaling pathway and puts forward research hypothesis that miRNAs maybe the targets regulated by Zhuanggu Zhitong decoction which regulate the ER/RANK/NFATc1 signaling pathway in osteoclasts. At first, animal experiments and cell experiments will be designed to verify the hypothesis,then antagomir and agomir technology will be further applied to explore the mechanisms of Zhuanggu Zhitong prescription regulation of osteoclast ER / RANK / NFATc1 signaling pathway in cell and animal level, meanwhile, the mechanismsthat Zhuanggu Zhitong prescription regulatesthe miRNA will be unveiled through detecting those gene expression regulating miRNA maturation. This project is the first time to investigate from the miRNA level Zhuanggu Zhitong prescription regulation of ER / RANK / NFATc1 signaling mechanism in osteoclasts from gene post-transcription and further clarify Zhuanggu Zhitong prescription regulation of osteoclast signal transduction mechanism, which will provide guidance for the clinical application of Zhuanggu Zhitong prescription better.

壮骨止痛方对绝经后骨质疏松症具有良好防治作用，抑制破骨细胞ER／RANK信号通路是壮骨止痛方抗绝经后骨质疏松症的重要作用机理，但尚不完全清楚其对破骨细胞ER／RANK信号通路的具体作用细节及其调控机理。本项目以破骨细胞ER／RANK／NFATc1信号通路为中心，提出壮骨止痛方可能从miRNA水平调节破骨细胞ER／RANK／NFATc1信号通路的研究假说。先从动物实验和细胞实验初步验证研究假说，并进一步应用antagomir和agomir技术从细胞和动物水平探讨壮骨止痛方调控破骨细胞ER／RANK／NFATc1信号通路的机理，同时通过检测对miRNA成熟过程相关基因的影响探讨壮骨止痛方调控miRNA的机理。本项目首次从miRNA探讨壮骨止痛方调控破骨细胞ER／RANK／NFATc1信号通路机理，不仅丰富和深化中医药抗绝经后骨质疏松症作用机理，而且为临床更好地应用壮骨止痛方提供指导。

项目根据实验的实际情况将研究分为壮骨止痛方全方和复方单味药（包括有效成分）两部分。复方研究部分先行制备壮骨止痛方药物给予复制去卵巢骨质疏松大鼠模型，分别收集短期给药（6周）和长期给药（12周）大鼠血清，利用RANKL诱导BMMs分化成破骨细胞模型研究大鼠血清对破骨细胞分化的抑制作用，复方长期含药血清体现抑制破骨细胞形成的作用，但是模型长期血清却没有体现促进破骨细胞形成的作用，正常长期血清也没有体现统计学意义的差异，经过多次实验均显示该结果，导致复方含药血清细胞实验受阻。复方单味药（包括有效成分）选择齐墩果酸、补骨脂乙素、牛膝提取物、西芹总黄酮先期进行破骨细胞诱导实验，筛选其对破骨细胞ER/RANK/NFATc1通路的抑制靶点和miRNA，然后再从动物实验验证复方对ER/RANK/NFATc1通路的抑制靶点和miRNA。结果表明，齐墩果酸直接作用雌激素受体，并通过促进miR-503表达而调控ERα/RANK/NFATc1调控破骨细胞形成与溶骨功能；西芹总黄酮（TFC）通过抑制p38/c-Fos/NFATc1通路和NF-κB通路显著抑制破骨细胞分化和骨吸收功能，有促进miR-124、miR-9718表达的趋势；川牛膝提取物对ERα/RANK/NFATc1信号通路多个节点具有抑制作用，体现川牛膝提取物抗骨质疏松作用的“多成分、多途径、多靶点”作用特点；补骨脂乙素显著抑制破骨细胞分化和骨吸收功能，并通过抑制c-Fos/NFATc1通路和NF-κB通路显著抑制破骨细胞分化和骨吸收功能，通过抑制miR-193-3p表达而抑制NFATc1。复方动物实验由于新冠肺炎疫情影响和前述复方含药血清细胞实验消耗较长时间，导致相关检查还没有完全结束。项目已经初步阐明ERα/RANK/NFATc1信号通路是壮骨止痛方抑制破骨细胞分化途径之一，并通过miR-503、miR-193-3p、miR-124、miR-9718等对ERα/RANK/NFATc1信号通路进行调控。发表2篇SCI收录论文和2篇CSCD收录论文，已培养毕业4位研究生。