Objective: To compare the effects of manual acupuncture (MA), electroacupuncture (EA), and moxibustion on knee joint cartilage morphology, serum inflammatory factors interleukin (IL)-6 and IL-10, matrix metalloproteinase 13 (MMP13), and intestinal flora composition in knee osteoarthritis (KOA) model rats. Methods: Forty male specific-pathogen-free Sprague-Dawley rats aged seven weeks were randomly divided into a normal group (n=8) and a KOA modeling group (n=32). The KOA model was established using sodium iodoacetate induction. The KOA modeling rats were further randomly divided into a model group, an MA group, an EA group, and a moxibustion group, with 8 rats in each group. In the MA, EA, and moxibustion groups, interventions targeting the right Futu (ST32) and Zusanli (ST36) were performed for 15 min, once every other day, for 14 sessions. The normal and model groups were bundled on the self-made fixation frame for 15 min. The rat knee joint diameter was measured on the 8th day of adaptive feeding, after successful modeling, and after the 14th intervention. Lequesne behavioral scoring was performed after successful modeling and after the 14th intervention. After the 14th intervention, hematoxylin-eosin (HE) staining and Masson staining were performed with the cartilage sections of the right knee joint. The pathomorphological changes of the rat joint cartilage were observed and quantified by Mankin's score. Enzyme-linked immunosorbent assay was used to detect the rat serum levels of IL-6, IL-10, and MMP13. Additionally, 16S rDNA sequencing was used to detect the composition of rat fecal flora. Results: Compared to the normal group, the right knee joint diameter and the Lequesne score were significantly increased in the model group (P<0.01). Compared to the model group, the right knee joint diameter and the Lequesne score of rats in the MA, EA, and moxibustion groups were significantly reduced (P<0.01), with no significant differences among the three intervention groups (P>0.05). HE staining and Masson staining revealed disordered cartilage structure in the model group, which was improved following interventions in the MA group, EA group, and moxibustion group. Mankin's score was significantly higher in the model group versus the normal group (P<0.05) while significantly lower in the MA, EA, and moxibustion groups versus the model group (P<0.05). Serum analysis showed elevated IL-6 and MMP13 levels and reduced IL-10 level in the model group versus the normal group (P<0.05). Compared to the model group, the serum IL-6 level was significantly reduced (P<0.05), and the IL-10 level was significantly increased (P<0.05) in the MA, EA, and moxibustion groups, but without statistical differences among the three intervention groups (P>0.05); moreover, the MMP13 level in the moxibustion group was significantly lower than that in the model group (P<0.05). The alpha diversity analysis of intestinal flora showed no statistical difference in the number of operational taxonomic units (OTUs) and alpha diversity index among groups (P>0.05). Intestinal flora beta diversity analysis revealed significant differences among groups (P<0.05). Intestinal flora composition analysis showed significantly increased relative abundance of Lactobacillus (P<0.05) and significantly decreased relative abundance of Eubacterium_coprostanoligenes (P<0.05) in the model group compared to the normal group; compared to the model group, the relative abundances of Firmicutes, Lactobacillus, and Romboutsia in the MA, EA, and moxibustion groups were significantly reduced (P<0. 05); the relative abundance of Bacteroidetes and Lachnospiraceae_NK4A136 in the MA group increased significantly (P<0.05); Bacteroidetes and Ruminococcaceaae_UGC-005 increased significantly in the moxibustion group (P<0.05). Conclusion: MA, EA, and moxibustion effectively reduced knee joint swelling, improved cartilage tissue morphology, optimized intestinal flora composition, down-regulated expression levels of serum pro-inflammatory factors IL-6 and MMP13, and increased expression level of anti-inflammatory factor IL-10 in KOA rats. Among them, moxibustion exhibited the most obvious regulatory effect on inflammatory factors.
