[Effect of needle knife on autophagy and matrix degradation of chondrocytes in rabbit knee osteoarthritis].

Abstract

Objective: To explore effects of needle knife on autophagy and matrix degradation-related proteins of chondrocytes in rabbits with knee osteoarthritis (KOA).

Methods: Eighteen healthy male New Zealand rabbits aged 6 months, with a body weight of (2.0±0.5) kg, were selected and randomly divided into blank group, model group and needle knife group, with 6 rabbits in each group. KOA rabbit animal model was established by fixing and immobilizing left hind limb of rabbits in an extended position with a plaster cast for 6 weeks, following the modified Videman method. The control group was not immobilized. Both of control group and model group were not intervened. The needle knife group received needle knife intervention once a week for 4 weeks. One week later, Kellgren-Lawrence(K-L) grading and Magnetic Resonance Knee Osteoarthritis Score (MOAKS) were used to evaluate bone spurs, joint space, cartilage smoothness and effusion on the left knee joint of rabbits; Lequesne MG score was used to evaluate effects of acupuncture knife on symptoms, signs and functional activities of rabbit's knee joint. After killing rabbits, cartilage morphology of left knee joint was observed;pathological changes of cartilage on the left knee joint of rabbits were examined by hematoxylin O-eosin staining under a light microscope;TdT-mediated dUTP nick end labeling (TUNEL) staining technique was used to detect apoptosis rate of chondrocytes on the left knee joint of rabbits. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect autophagy of chondrocytes on the left knee joint and expressions of regulatory molecules for cartilage matrix degradation, including myosin-like bcl2 interacting protein (Beclin-1), light chain 3 (LC3), miR-107, cysteine aspartate-specific protease 1 (Caspase-1), interleukin-1β (IL-1β), collagen typeⅡ(CollagenⅡ), matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-13 (MMP-13), at mRNA and protein levels.

Results: Compared with blank group, the surface of left knee joint cartilage in model group showed defects, with unclear structure, reduced matrix staining, disordered cell arrangement, mostly in clustered distribution, significantly reduced in quantity, and chaotic and unclear marginal lines;the apoptosis rate of chondrocytes increased, while K-L grading level, MOAKS score, Lequesne MG score rose, and visual assessment score also increased. The expressions of Caspase-1, IL-1β, MMP-3, and MMP-13 mRNA and proteins in cartilage tissue were upregulated, and all the differences were statistically significant (P<0.01). The expressions of Beclin-1, LC3, miR-107, CollagenⅡmRNA, as well as Beclin-1, LC3Ⅱ/Ⅰ, and CollagenⅡproteins in cartilage tissue were all decreased, and the differences were statistically significant (P<0.01). Compared with model group, the surface of left knee joint cartilage in needle knife group was smoother, with fewer defects, clearer structure, more uniform and normal matrix staining, more orderly cell arrangement and an increase in cell number, and the ridges were clearer and more complete;K-L grade, MOAKS, and Lequesne MG scores decreased, the visual assessment score decreased(P<0.05), apoptosis rate of chondrocytes decreased, and mRNA and protein expressions of Caspase-1, IL-1β, MMP-3, and MMP-13 in cartilage tissue were all downregulated;all the differences were statistically significant (P<0.01);expressions of Beclin-1, LC3, miR-107, CollagenⅡmRNA, and Beclin-1, CollagenⅡproteins in cartilage tissue were significantly increased (P<0.01), and expression of LC3Ⅱ/Ⅰprotein in cartilage tissue was also increased, with a statistically significant difference (P<0.05).

Conclusion: Based on the theory of meridians, acupuncture and knife therapy could promote chondrocyte autophagy by regulating the expression of proteins such as Beclin-1, LC3, miR-107, and Caspase-1, reduce apoptosis, inhibit the degradation of cartilage matrix, lower the level of inflammation, delay degeneration of articular cartilage, and exert functions of protecting cartilage and improving knee joint pain and functional disorders.