Zhen Wang,1,* Hui Xu,1,2,* Zheng Wang,1,* Yu Wang,3,* Jieyao Diao,1 Juntao Chen,1 Yuchen Xie,4 Lijuan Zhang,5 Miaoxiu Li,6 Yanqin Bian,7 Yunfeng Zhou1 

1College of Acupuncture and Massage, Henan University of Chinese Medicine, Zhengzhou, People’s Republic of China; 2Tuina Department, The Third Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, People’s Republic of China; 3College of Computer Science, Xidian University, Xian, People’s Republic of China; 4Tuina Department, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou, People’s Republic of China; 5Rehabilitation Department, Jiaozuo Coal Industry (Group) Co. Ltd. Central Hospital, Jiaozuo, People’s Republic of China; 6College of Acupuncture and Massage, Shanghai University of Chinese Medicine, Shanghai, People’s Republic of China; 7Orthopaedic Research Laboratory, University of California, Davis, CA, USA

*These authors contributed equally to this work

Correspondence: Hui Xu; Yunfeng Zhou, Email 15036065036@163.com; zyf5680198@126.com

Background: Knee osteoarthritis (KOA) is on the rise due to lifestyle changes, obesity, and aging, yet effective treatments are lacking. Traditional Chinese manual therapy (Tuina) is promising for KOA. However, its mechanism remains unclear.
Objective: This study aims to determine the effects of Tuina on a rat KOA model, focusing on the role of chondrocyte apoptosis and autophagy mechanisms.
Methods: KOA was induced in rats by intra-articular injection of L-cysteine-activated papain into the right knee. Thirty-six male Sprague Dawley (SD) rats were randomly divided into blank, model control, Tuina, and positive drug groups. Paw withdrawal threshold tests, knee joint swelling, and passive range of motion assessed knee behavior. Cartilage tissue cytology, cytokine contents, and the mRNA and protein expression of PI3K/AKT/mTOR signaling pathway components were analyzed using HE and TUNEL staining, ELISA, RT-qPCR, and Western blotting, respectively. In addition, we used machine learning methods to conduct a secondary analysis of the dataset from the in vivo experiments in rats to verify the findings.
Results: Tuina significantly relieved pain and joint swelling, and improved range of motion. Staining showed reduced articular cartilage destruction and apoptosis. Tuina reduced the serum levels of IL-1β, IL-17, MMP-3, and MMP-13. Tuina downregulated Bax, ULK1, Beclin-1, LC3-II/I and upregulated PI3K, AKT, mTOR, and BCL-2 in cartilage tissue. The machine learning results indicated an 83.33% accuracy for the prediction model, remaining stable through both uni- and multivariate analyses. Tuina yielded the best comprehensive efficacy on KOA as well as better rat behavior and PI3K/AKT/mTOR signaling pathway improvement effect than positive drugs, while its cytokine-reducing ability was comparable to that of positive drugs.
Conclusion: Tuina can alleviate cartilage tissue injury in KOA, relieve inflammation, and reduce chondrocyte apoptosis and autophagy, the underlying mechanisms of which may be associated with activation of the PI3K/AKT/mTOR signaling pathway.

Keywords: knee osteoarthritis, apoptosis, autophagy, tuina, PI3K/AKT/mTOR signaling pathway, in vivo experiment, machine learning