从调控PI3K/Akt和AMPK表达失衡-激活自噬-恢复软骨细胞稳态探讨补肾壮骨防治OA的分子机制

The prescription Niuxizhuanggu granule has been used clinically for many years.From preliminary study it has been proved to restrain inflammatory cytokines and apoptosis.Its effective constituent astragaloside and panax notoginside can increase the activity of autophagy.The activigy of chondrocyte autophagy decrease with age,but the profilin mTOR over-express.Based on above analysis the hypothesis is put forward.Autophagy is probably the target of Niuxizhuanggu granule regulating chondrocyte homeostasis. PI3K/Akt/mTOR and AMPK/mTOR are the important signal pathway of regulating mTOR.By keeping them balance Niuxizhuanggu granule decrease the expression of mTOR ,increase the activity of autophagy,recove the chondrocyte homeostasis to cure OA.Our study intend to verify the objectivity of the hypothesis on the lay of function and structure .We will focus on Hartley guinea pig using the RT-PCR WB Transmission electron microscopy TUNEL and so on.We intend to explain the therapeutic molecular mechanism of Tonifying Kidney to Strenthen Bone Method on Osteoarthritis and the meaning of the theory of kidney dominating bone. Thus we can provide a new theoretical basis for the treatment of OA by Tonifying Kidney to Strenthen Bone Method.

补肾壮骨方(牛膝健步颗粒)临床治疗骨性关节炎（OA）有多年工作积累，前期实验证实该方能抑制OA软骨细胞炎性细胞因子表达和凋亡，其有效成份黄芪甲苷、三七总皂苷能提高细胞自噬活性。软骨细胞自噬活性随年龄降低，而其抑制蛋白mTOR高表达。据此提出假说：自噬可能是补肾壮骨方整体调节软骨细胞细胞稳态的靶点，PI3K/Akt/mTOR和AMPK/mTOR是调控mTOR的重要通路，补肾壮骨方可能通过调控两者的表达失衡，抑制激活的mTOR，提高自噬活性，恢复软骨细胞稳态，起到防治OA的作用。本课题拟以Hartley豚鼠自发性骨关节炎模型为研究对象，采用RTPCR、WB、透视电镜、TUNEL等技术，从结构与功能层面验证假说的客观性，研究结果将从调控PI3K/Akt/mTOR和AMPK/mTOR双通路表达、自噬和软骨细胞稳态的角度说明补肾壮骨法治疗OA的分子机制，阐释肾虚骨衰的理论内涵，为防治OA提供新思路。

骨性关节炎(Osteoarthritis，OA)是一种常见的退行性骨关节病，是导致老龄人群疼痛、残疾的主要原因之一。镇痛药和非甾体类消炎药是现代医学治疗OA的主要手段，尚缺乏更加有效的预防和治疗方法，在前期国家自然科学基金(81072805)的研究证实了中医补肾壮骨法能够抑制OA软骨细胞IL-1β/MAPK、NF-κB信号通路相关分子的表达，抑制基质金属蛋白酶表达，从而抑制细胞凋亡保护细胞外基质。. 本研究在前期基础上进一步探究补肾壮骨法通过自噬调控软骨细胞稳态的具体机制。研究表明补肾壮骨方干预后能够上调骨关节炎豚鼠软骨中AMPK、ULK1、Beclin-1、LC3蛋白表达，能上调骨关节炎豚鼠软骨AMPK/mTOR通路，并下调PI3K/Akt-mTOR通路，补肾壮骨方可能是通过调节这两条通路从而激活自噬治疗骨关节炎。同时补肾壮骨方干预后能上调II 型胶原的mRNA的表达，从而减少骨关节炎软骨细胞的肥大分化，并下调VEGF mRNA的表达而保护软骨细胞。. 本研究验证了自噬可能是补肾壮骨方整体调节软骨细胞细胞稳态的靶点，补肾壮骨方可能通过调控PI3K/Akt/mTOR和AMPK/mTOR两条通路的表达失衡，抑制激活的mTOR，提高自噬活性，恢复软骨细胞稳态，起到防治OA的作用。本研究从结构与功能层面验证了假说的客观性，研究结果从调控PI3K/Akt/mTOR和AMPK/mTOR双通路表达、自噬和软骨细胞稳态的角度说明了补肾壮骨法治疗OA的分子机制，阐释了肾虚骨衰的理论内涵，为防治OA提供了新思路。