基于miRNA-146a介导TLR4信号通路探讨补肾益气方蠲痹胶囊治疗绝经后骨关节炎的分子机制

Postmenopausal osteoarthritis is one of the most common types in OA, but its pathogenesis is not yet clear. Recent studies have found that miRNA-146a plays an important role in the development and progression of OA by regulating TLR4 pathways via a negative feedback. Our previous researches have found that Juanbi capsule has a significant therapeutic effect on early to mid-stage osteoarthritis, especially on patients after menopause. After Juanbi capsules are given to the OA guinea pig by gavage, the TLR4, NF-κB and TNF-α proteins in osteoblasts of synovium and subchondral bone decrease dramatically, and the cartilage destruction is significantly inhibited.We propose that Juanbi capsule may function in the treatment of osteoarthritis after menopause by adjusting miRNA-146a-mediated TLR4 pathways. Therefore, our project intends to study through in vivo and in vitro experiments. The in vivo experiment is made through the OA guinea pig model with kidney deficiency caused by removal of ovary, and the in vitro experiment is conducted by means of co-culture of kidney-deficiency animals’ serum, osteoblasts and synovial cell. From the perspective of miRNA-146a-mediated TLR4 pathways, the molecular mechanism of Juanbi Capsule treatment of postmenopausal osteoarthritis is deeply explored in order to further clarify the mechanism of action of Juanbi Capsule for OA treatment, and to provide an experimental basis for Traditional Chinese Medicine treatment of OA.

绝经后骨关节炎（Osteoarthritis,OA）是OA中最常见的一种类型，目前发病机制尚不明晰。近期研究发现，miRNA-146a以负反馈的方式调控TLR4通路在OA中发挥重要作用。我们前期研究发现，蠲痹胶囊能够对早中期OA尤其是绝经后OA患者具有明显的治疗作用，且给OA豚鼠给予蠲痹胶囊后，其滑膜及软骨下成骨细胞TLR4、NF-κB和TNF-α蛋白明显下降，软骨的破坏受到明显抑制。我们推测，蠲痹胶囊可能通过调节miRNA-146a介导TLR4通路来达到治疗绝经后骨性关节炎的作用。基于此，本项目拟采用体内和体外实验进行研究。体内实验采用切除卵巢所致的肾虚型OA豚鼠模型，体外实验采用肾虚型OA的动物血清与成骨细胞和滑膜细胞共培养的方法，从miRNA-146a介导TLR4通路角度，深入探讨蠲痹胶囊治疗绝经后OA的分子机制，以期进一步阐明蠲痹胶囊治疗OA的作用机理，为中医治疗OA提供实验依据。

骨关节炎是一种以关节软骨的变性、破坏及骨质增生为特征的慢性关节病，发病部位多在负重关节，主要是关节软骨、细胞外基质和软骨下骨的合成与正常退变之间失去平衡的结果，最终导致患病关节结构畸形、功能障碍。其发病机制十分复杂，至今仍不十分清楚。“绝经后骨性关节炎”是OA中最常见的一种类型，中医认为绝经后骨关节炎患者主要证候为肾虚。.本次研究通过豚鼠KOA成纤维样滑膜细胞及软骨下成骨细胞的分离培养与鉴定，采用双侧卵巢切除的方法建立肾虚型豚鼠OA模型，并用改良的豚鼠灌胃方法给予实验动物不同剂量蠲痹胶囊灌胃，观察蠲痹胶囊对miRNA-146a及TLR4/MyD88通路中相关因子表达的影响，采用RNA干扰技术，通过腺病毒感染方法,分别抑制滑膜细胞和成骨细胞TRAF6上游因子MyD88和下游因子Iκβ的表达，然后用蠲痹胶囊含药血清（肾虚型OA动物血清）与细胞共培养方法，观察MyD88及其下游相关因子的表达，采用基因敲除技术，转染miR-146a mimic和miR-146a inhibitor，分别上调和下调miRNA-146a的表达，然后用蠲痹胶囊含药血清（肾虚型OA动物血清）与细胞共培养方法，观察TLR4通路相关因子的表达。应用免疫荧光双标观察滑膜细胞NF-κBp56蛋白的表达；RT-PCR法测定miRNA-146a、TLR4、MyD88、TRAF6、IκβmRNA表达；Western-blot法测定TRAF6、NF-κBp56蛋白表达；ELISA检测细胞上清液中雌二醇、IL-1β、TNF-α、MMP-13水平。.结果发现，补肾益气中药治疗OA的机理为：通过调节成纤维样滑膜细胞及成骨细胞miRNA-146a的表达来调控TLR4/NF-κB通路，从而抑制滑膜的炎症及软骨下骨的退变，达到治疗OA的作用。