Effects of acupotomy therapy on chondrocyte ferroptosis based on the Piezo1/GPX4/ACSL4 signaling axis

To observe the effect of acupotomy on the piezoelectric mechanical gating ion channel component 1 (Piezo1)/glutathione peroxidase 4 (GPX4)/long-chain acyl coenzyme A synthetase 4 (ACSL4) signaling axis of knee chondrocytes in knee osteoarthritis (KOA) rabbits, so as to explore its mechanisms underly improvement of KOA.

Twenty-four male New Zealand rabbits were randomly divided into blank control, KOA model and acupotomy groups, with 8 rabbits in each group. The KOA model was established by immobilizing the left hind limb (with a plaster cast) in an extended position for 6 weeks according to the modified Videman method. For rabbits of the acupotomy group, the needle-knife release technique was applied to the musculotendon lesion points (“Hedingci” “Binwaishang” “Binneishang” “Chengfeijian” “Weiyangci” and “Yinlingci”) of the left knee, once a week for 4 weeks. The degree of the left knee joint dysfunction was assessed by referring to Lequesne MG’s functional disability scale (pain reaction [0—3 points], gait [0—3 points], joint motion [0—3 points] and swelling [0—2 points]) before and after the intervention. Histopathological changes of the left knee joint cartilage tissue (chondrocytes) were observed after H.E. staining. The mitochondrial structure of the cartilage chondrocytes was observed using transmission electron microscopy (TEM). The mRNA expression levels of Piezo1, GPX4 and ACSL4 in the cartilage tissue were detected by using real-time qPCR. The protein expression levels of Piezo1, GPX4 and ACSL4 in the cartilage tissue were detected by using Western blot.

Compared with the blank control group, the model group showed a significant increase in the Lequesne MG score (P<0.01), expression levels of Piezo1 and ACSL4 mRNAs and proteins (P<0.01), and a notable decrease in the expression levels of GPX4 mRNA and protein (P<0.01, P<0.05). Compared with the model group, the acupotomy group had an evident decrease in the Lequesne MG score (P<0.05), expression levels of Piezo1 and ACSL4 mRNAs and proteins (P<0.01, P<0.05), and an evident increase in the expression levels of GPX4 mRNA and protein (P<0.01, P<0.05). The results of H.E. staining showed a rough surface of the cartilage of the left knee joint, wrinkled and broken nucleus, being sparse in number, scattered arrangement and with uneven level in the model group. Whereas in the acupotomy group, the cartilage surface of the knee joint was relatively flat, the nucleus was clear and increased in the number and the arrangement was in order, and the level was relatively distinct. The results of TEM showed shrunk and irregular chondrocytes, ruptured cell membrane, multiple dissolved cytoplasm, formation of some vacuoles, round or oval mitochondria with reduction in the number, shortening in the ridge, even disappearance, and dissolved matrix in a large area in the model group. In the acupotomy group, the chondrocytes were relatively regular in shape, with complete membrane and uniform cytoplasm, and the mitochondria were more in the number, with more uniform matrix and more ridges.

Acupotomy can improve the motor function in KOA rabbits, which may be associated with its functions in inhibiting chondrocyte ferroptosis by regulating Piezo1/GPX4/ACSL4 signaling axis, and relieving cartilage injury.