基于FAK-PI3K-AKT通路磷酸化修饰探讨针刀力学效应调控膝骨关节炎软骨细胞自噬的机制

Knee Osteoarthritis（KOA）is a senile disease that severely affects the quality of life. Biomechanical factors play an important role in the pathogenesis, abnormal cartilage stress leads to cartilage degeneration. The effectiveness of acupotomy on KOA is extremely precise. Our previous study found that acupotomy loose the muscle-tendon system of KOA can recuperate the mechanical imbalance of knee joint, benign stress mediated the phosphorylation of FAK-PI3K-AKT pathway in cartilage, and promoted the anabolism and alleviated the degradation of cartilage. The level of chondrocytes autophagy of KOA decreases is one of important factors which caused cartilage metabolic imbalance and degeneration. Does the mechanical and biological effects of acupotomy protect cartilage by regulating the autophagy level of chondrocytes. After the stimulation of acupotomy, where is the key effective target of FAK-PI3K-AKT pathway which was mediated by mechanical signal and modified on phosphorylation? To investigate this issue, acupotomy threapy was used as intervention in this experiment, estimating the level of chondrocytes autophagy by immunohistochemistry and electron microscopy,and seeking the key effective target of FAK-PI3K-AKT signal pathway which regulated chondrocytes autophagy through measure of phosphorylation modified proteomics and bioinformatics,to reveal the scientific connotation of loose the tendon to treat the bone by acupotomy theory.

膝骨关节炎（KOA）是严重影响生活质量的老年性疾病，生物力学为发病的关键因素之一，关节软骨应力异常导致软骨退变。针刀疗效确切，前期研究发现针刀松解KOA肌肉-肌腱，可恢复膝关节力学失衡，良性应力激活软骨细胞FAK-PI3K-AKT通路磷酸化，促进其合成代谢，缓解软骨降解。大量研究已证实KOA软骨细胞自噬水平下降是引起代谢失衡和软骨退变的重要因素。而针刀对KOA的效应，是否通过调节软骨细胞自噬水平影响合成代谢；其力学信号介导FAK-PI3K-AKT通路磷酸化调控软骨细胞的关键靶点何在，尚未清楚。为解决这一问题，本项目以针刀为干预KOA的手段，运用免疫组化法及电镜评估KOA兔软骨细胞自噬水平，并通过磷酸化修饰蛋白组学和生物信息学，寻找FAK-PI3K-AKT通路调控软骨细胞自噬的关键靶点，揭示针刀“调筋治骨”治疗KOA的科涵。

背景及目的：针刀治疗KOA疗效确切，但针刀对KOA软骨细胞自噬的影响尚不明确。本项目评估针刀力学效应对KOA兔软骨细胞自噬的影响并探寻其调控软骨细胞自噬的关键靶点。.方法:以改良Videman法制备KOA兔模型，分别行针刀、电针干预。光镜观察各组兔软骨病理变化，MRI扫描观察软骨变化; 表面张力测试器检测各组兔膝周软组织张力变化；肌骨超声检测各组兔膝周软组织超声弹性模量变化；透射电镜评估各组兔软骨细胞自噬水平；分子生物学、组织化学技术检测软骨中Akt、mTOR、Beclin-1、LC3、p62表达变化；运用磷酸化修饰蛋白组学和生物信息学筛选调控软骨细胞自噬的关键靶点。.结果:(1)成功制备KOA兔模型;(2)形态学：针刀干预后光镜下软骨细胞表浅层剥脱、排列紊乱得到改善，显著降低软骨Mankin病理评分; 针刀组软骨细胞胞质中自噬囊泡数目较电针组增加；(3)生物力学：针刀干预可不同程度提高膝关节内外侧副韧带超声弹性模量、降低膝周肌群表面张力；(4)影像学：针刀干预可改善KOA兔软骨损伤，减轻关节间隙变窄及膝关节积液，改善膝关节骨髓水肿；(5)分子生物学：针刀干预一定程度调控KOA软骨细胞中Akt、mTOR、Beclin1、LC3、p62的表达；运用蛋白组学及磷酸化修饰蛋白组学，针刀通过调控HSP90AB1、CAPNS1、STAT1、CTSV、SLC3A2、HMOX1蛋白表达以及调控磷酸化蛋白SPP1、HSP90AA1、PLCG1的表达激活KOA软骨细胞自噬；(6)针刀缓解KOA兔软骨退变的可能机理:1.针刀通过改善膝周软组织力学性能纠正关节力学失衡状态，恢复软骨细胞良性应力环境;2.针刀力学效应引起的良性应力刺激激活软骨细胞自噬；3.针刀通过调控HSP90AB1、CAPNS1、STAT1、CTSV、SLC3A2、HMOX1蛋白表达以及调控磷酸化蛋白SPP1、HSP90AA1、PLCG1的表达激活KOA软骨细胞自噬。.结论:相比于电针干预，针刀干预激活KOA软骨细胞自噬的关键在于其生物力学效应，针刀通过改善膝周软组织力学性能纠正关节力学失衡状态，恢复关节内良性应力刺激激活KOA软骨细胞自噬，主要通过调控软骨细胞中HSP90AB1、CAPNS1、STAT1、CTSV、SLC3A2、HMOX1蛋白表达及SPP1、HSP90AA1、PLCG1磷酸化蛋白表达。