Zhen Ci Yan Jiu. 2025 Oct 25. pii: 1000-0607(2025)10-1114-10. [Epub ahead of print]50(10): 1114-1123
OBJECTIVES: To observe the effect of electroacupuncture (EA) on knee function and symptoms, intestinal flora and TLR4/MyD88/NF-κB signaling pathway of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its potential mechanism underlying improvement of knee function by regulating the gut-joint axis response of KOA.
METHODS: Thirty-two Japanese white rabbits were randomly divided into blank, model, glucosamine and EA groups, with 8 rabbits in each group. The KOA model was reproduced by using modified Hulth method. The rabbits in the EA group were given EA (2 Hz/100 Hz, 0.5-1.5 mA) at bilateral "Xuehai"(SP10), "Yinlingquan"(SP9) and "Dubi"(ST35) for 15 min, once daily, 5 times a week for 4 weeks. The rabbits of the glucosamine group received glucosamine solution once daily, 5 times a week for 4 weeks. The knee function was evaluated by Lequesne MG index of severity for osteoarthritis (total scores of pain, swelling, range of motion, and gait) after modeling and treatment. H.E. staining was used to observe the histopathological changes of the knee cartilage tissue. Immunofluorescence histochemical method was used to detect the expressions of MyD88 and NF-κB p65, and Wes automated protein blot quantitative analysis technique was employed to detect the protein expressions of TLR4, MyD88 and NF-κB p65 in the knee cartilage tissue. Analysis of the species structure changes of intestinal flora was detected by 16S rDNA high-throughput sequencing.
RESULTS: After modeling, compared with the blank group, the Lequesne MG score, immunofluorescence intensity of MyD88 and NF-κB p65, and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly increased in the model group (P<0.05). H.E. staining showed thin and rough surface of the knee cartilage, clusters and disordered arrangement of chondrocytes, a few cellular nuclei being dark blue-purple in staining, with some nuclei being shrank, ruptured, and disappeared in the model group. In comparison with the model group, the Lequesne MG score, fluorescence intensity of MyD88 and NF-κB p65, and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly decreased in both EA and glucosamine groups (P<0.05). The effects of EA were significantly superior to glucosamine in down-regulating the protein expression levels of TLR4, MyD88 and NF-κB (P<0.05). H.E. staining showed relatively complete surface of the knee cartilage, slightly hypertrophic chondrocytes with relatively clear nucleus and relatively complete tide line in both EA and glucosamine groups. Results of 16S rDNA sequencing showed that compared with the blank group, the Shannon index was obviously increased (P<0.05), the relative abundance of Proteobacteria, Actinobacteria, Escherichia coli-Shigella and Clostridium UCG-014 was elevated, and the relative abundance of Firmicutes, Verrucomicrobia, Comamonas and Acmea bacteria was decreased in the model group. In comparison with the model group, the Chao1 index and Shannon index were increased (P<0.05), the relative abundance of Proteobacteria, Actinobacteria and Escherichia coli-Shigella was decreased, while the relative abundance of Firmicutes, Comamonas and Nobilus was significantly increased in both EA and glucosamine groups.
CONCLUSIONS: EA can significantly improve the symptoms and function of knee joint in KOA rabbits, which may be associated with its functions in inhibiting cartilage injury and inflammation reaction by regulating TLR4/MyD88/NF-κB signaling pathway and intestinal flora diversity through the "gut-joint" axis.
Keywords: Electroacupuncture; Inflammation; Intestinal flora; Knee osteoarthritis; TLR4/MyD88/NF- κB signaling pathway