To observe the protective effect of electroacupuncture (EA) on the knee joint cartilage tissue of rats with knee osteoarthritis (KOA), so as to explore the potential mechanism of miR-335-5p involved in the treatment of EA for KOA.

A total of 42 adult male Sprague-Dawley (SD) rats were randomly divided into blank, model and EA groups, with 14 rats in each group. The model was established by injecting a sodium glutamate iodoacetate solution (80 μg/μL) into the right joint cavity. On the 15th day after the model establishment, rats in the EA group received EA intervention at “Yanglingquan” (GB34) and “Xuehai” (SP10) on the right side, and the needles were retained for 15 minutes. The intervention was carried out every other day for a total of two weeks. After the twoweek intervention, the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of the rats were detected; a gait analyzer was used to detect the gait adaptability changes induced by pain; the pathological changes of the knee joint cartilage tissue were observed after hematoxylin-eosin (HE) staining; qPCR and Western blot were used to detect the expression levels of miR-335-5p, Dickkopf related protein 1 (DKK-1), β-catenin, and matrix metalloproteinase 13 (MMP-13) mRNAs and proteins in the cartilage tissue respectively; immunofluorescence staining was used to observe the positive fluorescence expression of β-catenin in the right knee joint cartilage tissue.

In the model group, HE staining showed a large area of chondrocyte necrosis, with the dissolution and disappearance of cell nuclei, accompanied by a macrophages-predominant inflammatory cell infiltration; cartilage surface defects in local areas, necrosis of trabecular bone and proliferation of fibrous tissue in local areas, and new capillary formation in the proliferative areas were also observed. In the EA group, pathological manifestations such as inflammatory cell infiltration and fibrous tissue proliferation were significantly alleviated. Compared with the blank group, the PWMT, PWTL, the support duration, footprint area, and average ground pressure of the right hind paw, and the mRNA and protein expressions of DKK-1 in the right knee joint cartilage tissue were decreased (P<0.001) in the model group, while the expressions of miR-335-5p, mRNA and protein expressions of β-catenin and MMP-13, and the fluorescence intensity of β-catenin in the right knee joint cartilage tissue were increased (P<0.001, P<0.01). All the above indicators in the EA group were all reversed (P<0.001, P<0.05, P<0.01) in comparison with the model group.

EA can significantly improve the pain symptoms of KOA rats, promote the recovery of motor function, and improve the pathological morphology of the knee joint cartilage tissue. This may be related to regulating the expression of miR-355-5p and Wnt/β-catenin pathway, as well as inhibiting the release of cartilage extracellular matrixrelated degrading enzymes.

To observe the effect of Guasha on inflammation factors, apoptosis and autophagy in the cartilage tissue of knee joint in rats with knee osteoarthritis(KOA), so as to explore its mechanisms underlying improvement of KOA.

A total of 51 male SD rats were randomized into three groups: blank control, KOA model and Guasha(n= 17 in each group). The rats in the blank control group received intra-articular injection of 0.9%NaCl solution in the right knee joint. The KOA model was established by intraarticular injection of glutamate sodium iodoacetic acid in the right knee joint. For rats of the Guasha group, Guasha(at a frequency of 1 time/s, and an applied pressure of 0.3—0.5 kgf) was applied to “Yanglingquan”(GB34) and “Xuehai”(SP10) areas of the right leg, once every other day, for 7 consecutive sessions. The circumference of the right knee was measured, The histopathological changes of right knee cartilage were observed after H. E. staining. The contents of inflammatory factors interleukin(IL)-1β and tumor necrosis factor(TNF)-α in the right knee articular cartilage tissue were assayed using ELISA. The expression levels of autophagy-related key molecule Beclin-1(homologous series of yeast Atg6), light chain protease complication 3 type II/I(LC3II/LC3 I), ubiquitin binding factor 62(P62) and cysteine aspartate protease-3(Caspase-3) mRNAs and proteins of the right knee articular cartilage tissue were measured using real-time fluorescent quantitative PCR and Western blot, separately. The apoptosis of chondrocytes was assayed using TUNEL staining, and the immunoactivity of LC3 determined using immunofluorescence staining.

After modeling, the right knee circumference of the model and Guasha groups was significantly increased compared with the blank control group(P＜0.01), and after the intervention, the knee circumference of the Guasha group was markedly decreased in comparison with that of the model group(P＜0.05). Results of H.E. staining showed obvious degeneration and defects in the cartilage tissue, necrosis of a large number of chondrocytes, fibrous hyperplasia, accompanied by inflammatory cell infiltration, osteoclast increase, fibroplasia and bone trabecular destruction in the model group, which was relatively milder in the Guasha group. Compared with the blank control group, the expression of Beclin-1 and LC3 mRNAs and proteins, and LC immunofluorescence intensity in the right knee articular cartilage tissue were significantly down-regulated(P＜0.01,P＜0.001), whereas the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, contents of IL-1β and TNF-α in the right knee articular cartilage tissue considerably increased(P＜0.01,P＜0.001) in the model group. In contrast to the model group, the Guasha group had an apparent increase in the expression levels of Beclin-1 and LC3mRNAs and proteins and LC immunofluorescence intensity in the right knee articular cartilage tissue(P＜0.05), and a pronounced decrease in the expression of P62 and Caspase-3 mRNAs and proteins, the apoptosis rate, and contents of IL-1β and TNF-α in the right knee articular cartilage tissue(P＜0.05,P＜0.01).

Guasha stimulation of GB34 and SP10 can improve joint cartilage damage in KOA rats, which may be associated with its functions in inhibiting the excessive release of inflammatory factors and apoptosis, possibly by down-regulating the expression of P62 and Caspase-3 mRNAs and proteins and up-regulating the expression of Beclin-1 and LC3 mRNAs and proteins, and by promoting autophagy of chondrocytes.