Zhen Wang,1,* Chi Zhao,1,2,* Mengmeng Li,1,2,* Lili Zhang,1,* Jieyao Diao,1 Yiming Wu,2 Tao Yang,2 Mingwei Shi,2 Yang Lei,2 Yu Wang,3 Miaoxiu Li,4 Yanqin Bian,5 Yunfeng Zhou,1 Hui Xu1,2 

1College of Acupuncture and Massage, Henan University of Chinese Medicine, Zhengzhou, People’s Republic of China; 2Acupuncture and Massage 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; 4College of Acupuncture and Massage, Shanghai University of Chinese Medicine, Shanghai, People’s Republic of China; 5Orthopaedic Research Laboratory, University of California, Davis, CA, USA

*These authors contributed equally to this work

Correspondence: Hui Xu, Email 15036065036@163.com

Background: Tuina therapy has demonstrated its potential in modulating autophagy-related factors in knee osteoarthritis (KOA); however, its core therapeutic targets and specific mechanisms require systematic elucidation through interdisciplinary research.
Objective: This study investigated the mechanism by which Tuina alleviates KOA progression using multidimensional approaches, including Mendelian randomization (MR), in vivo experiments, and machine learning.
Methods: Genetic data from genome-wide association studies of 60 cytokines and KOA were analyzed using MR analysis to identify autophagy-related factors significantly associated with KOA. A KOA rat model was established via intra-articular injections of L-cysteine–activated papain solution into the right knee. The key autophagy-related cytokines identified by MR were validated using enzyme-linked immunosorbent assay (ELISA). Cartilage degeneration and autophagic activity were assessed through histological evaluation via safranin O/fast green (SO/FG) staining and ultrastructural analysis via transmission electron microscopy (TEM). A Support Vector Machine (SVM) algorithm was used to conduct a secondary analysis of the experimental dataset to predict comprehensive therapeutic effects and synergistic correlations among the indicators.
Results: MR analysis revealed causal relationships between eight cytokines and KOA at genetically determined levels, with seven exhibiting autophagy-related regulatory properties. Tuina significantly alleviated pain and improved motor function. SO/FG staining and TEM revealed reduced cartilage destruction, increased cartilage thickness, and decreased chondrocyte autophagy. ELISA revealed that Tuina downregulated interferon gamma, parathyroid hormone, interleukin (IL)– 1β, matrix metalloproteinase-3, and IL-17 levels in the cartilage while upregulating osteocalcin and transforming growth factor-β 1 levels. Furthermore, Tuina demonstrated superior comprehensive and classified curative effects compared with celecoxib. IFN-γ and PTH were strongly correlated with both relative autophagy level and cartilage area ratio. Autophagy-related cytokines had the strongest correlation with the degree of cartilage degeneration.
Conclusion: Tuina therapy alleviates pain and functional impairment in KOA by suppressing chondrocyte autophagy and delaying cartilage degeneration. This mechanism may involve the regulation of autophagy-related cytokines.

Keywords: knee osteoarthritis, autophagy-related cytokines, Tuina, Mendelian randomization, in vivo experiment, machine learning