Abstract: BACKGROUND: Developed by the esteemed Chinese medicine master Wei Guikang, Tongan Decoction has proven highly effective in treating knee osteoarthritis. However, the mechanism of action is yet unclear. 
OBJECTIVE: To elucidate how Tongan Decoction modulates synovial macrophage polarization as a therapeutic strategy for knee osteoarthritis in a rat model.
METHODS: (1) We employed high-throughput microRNA sequencing and polymerase chain reaction to analyze the differentially expressed miRNA in synovial macrophages of normal and knee osteoarthritis rats. Predicted target genes of miR-27a were identified using bioinformatics databases, with subsequent validation through luciferase assays. A total of 68 Sprague-Dawley rats were randomly divided into normal control group (n=16), model group (n=16), miR-27a overexpression group (n=12), Tongan Decoction group (n=12), and Tongan Decoction+miR-27a inhibition group (n=12). The miR-27a overexpression group and the Tongan Decoction+miR-27a inhibition group were injected with miR-27a mimic and miR-27a inhibitor in the right knee joint cavity, respectively, once daily for 5 continuous days. On the 15th day after modeling, the Tongan Decoction group and the Tongan Decoction+miR-27a inhibition group were given Tongan Decoction by gastric lavage, and the other three groups were given saline by gastric lavage, once daily for 14 continuous days. After administration, behavioral tests, X-rays, hematoxylin-eosin staining of synovial and cartilage tissues of the knee joint and immunofluorescence staining of synovial tissues of the knee joint, RT-PCR, and Western blot were performed.
RESULTS AND CONCLUSION: High-throughput sequencing of miRNA showed low expression of miR-27a in synovial tissues of rats with knee osteoarthritis. The target gene of miR-27a was nuclear factor κB, and luciferase assay showed that the two could bind to each other. Behavioral assays showed that miR-27a overexpression or Tongan Decoction alleviated joint dysfunction in rats with knee osteoarthritis, and miR-27a inhibition antagonized the effect of Tongan Decoction. X-ray films and hematoxylin-eosin staining showed that miR-27a overexpression or Tongan Decoction reduced the degree of knee osteoarthritis and miR-27a inhibition weakened the therapeutic effect of Tongan Decoction. The results of RT-PCR and western blot assay showed that compared with the normal control group, the expression of interleukin 10 was reduced in the model group (P < 0.05), and the expression of matrix metalloproteinase 13, interleukin 1β, and nuclear factor κB was elevated in the model group (P < 0.05). miR-27a overexpression or Tongan Decoction could differently reverse the changes in the above-mentioned indexes, while miR-27a inhibition weakened the effect of Tongan Decoction. Immunofluorescence staining results showed that CD86 protein expression in the model group was higher than that in the normal control group (P < 0.05), and CD206 protein expression was lower than that in the normal control group (P < 0.05); miR-27a overexpression group and Tongan Decoction had lower CD86 protein expression than that in the model group (P < 0.05), and higher CD206 protein expression than that in the model group (P < 0.05); in the Tongan Decoction+miR-27a inhibition group, CD86 protein expression was higher (P < 0.05) and CD206 protein expression was lower than that in the Tongan Decoction group (P < 0.05). Tongan Decoction mitigates knee osteoarthritis by upregulating miR-27a expression and suppressing nuclear factor κB activity, which improves knee joint function and further treats knee osteoarthritis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: knee osteoarthritis, macrophage polarization, synovitis, microRNAs, Tongan Decoction, iodoacetic acid, nuclear factor κB, high-throughput sequencing, engineered tissue construction