Abstract: BACKGROUND: Previous studies have demonstrated that moxibustion can alleviate osteoarthritis-induced cartilage lesions and delay the progression of knee osteoarthritis. In a previous study conducted by our research group, it was found that regulating the circPan3/miR-667-5p/Ghrelin signaling pathway can enhance the level of autophagy in chondrocytes, thereby exerting a cartilage protective role in knee osteoarthritis. 
OBJECTIVE: To explore the effect of moxibustion on knee osteoarthritis cartilage in rats based on the circPan3/miR-667-5p/Ghrelin signaling pathway. 
METHODS: Sprague-Dawley rats were randomly divided into three groups (n=10 per group): sham operation, model, and moxibustion groups. A rat model of knee osteoarthritis was established in the latter two groups by anterior cruciate ligament transection and medial meniscus resection. Rats in the sham operation group underwent arthrotomy only. At 6 weeks after modeling, moxibustion intervention at Zusanli and Shenyu acupoints was applied in the moxibustion group, 20 minutes daily for 4 weeks, while the other two groups did not receive any special treatment. After 4 weeks, histopathological staining was performed to observe the degree of synovial lesions and cartilage damage in the knee joint. Additionally, changes in the serum levels of inflammatory cytokines, interleukin-6 and tumor necrosis factor-α, were detected by enzyme-linked immunosorbent assay. Furthermore, chondrocytes isolated from the above three groups of rats were divided into three groups: a normal control group, a model group, and a moxibustion group. The expression levels of cartilage matrix anabolic molecules (type II collagen and Sox9), matrix degradation molecules (matrix metalloproteinase 13 and ADAMTS5), autophagy markers (ATG5, Beclin-1, microtubule-associated protein 1 light chain 3), Ghrelin, CircPan3, and miR-667-5P were determined by western blot and qRT-PCR in each group.
RESULTS AND CONCLUSION: After 4 weeks of treatment, the model group exhibited significant thickening of the synovium, infiltration of inflammatory cells, narrowing of the joint space, rough and uneven cartilage surfaces, thinning, reduction in the number of chondrocytes, a significant increase in synovitis and Osteoarthritis Research Society International scores, and a significant up-regulation of interleukin-6 and tumor necrosis factor-α levels (P < 0.000 1). In comparison, the sham operation group showed no significant changes. Compared with the model group, the moxibustion group exhibited a reduction in synovial thickening, a decrease in inflammatory cell infiltration, an increase in joint space, a reduction in cartilage roughness and loss of integrity, an increase in the number of chondrocytes, a reduction in synovitis and Osteoarthritis Research Society International scores, and a significant reduction in the levels of interleukin-6 and tumor necrosis factor-α (P < 0.000 1). The results of in vitro experiments demonstrated that the expression of matrix metalloproteinase 13, ADAMTS5, and miR-667-5P was elevated, while the expression of type II collagen, Sox9, autophagy markers, Ghrelin, and Circpan3 was reduced in the model group compared with the normal control group (P < 0.01). Moxibustion was found to reverse these changes in the moxibustion group compared with the model group (P < 0.01). To conclude, moxibustion may enhance chondrocyte autophagy by modulating the circPan3/miR-667-5p/Ghrelin signaling pathway, which may inhibit knee osteoarthritis inflammation and cartilage destruction, exerting a protective effect on cartilage.

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, moxibustion, ghrelin, cartilage, autophagy, rat, signaling pathway