Chondroprotective Effects of Combination Therapy of Acupotomy and Human Adipose Mesenchymal Stem Cells in Knee Osteoarthritis Rabbits via the GSK3β-Cyclin D1-CDK4/CDK6 Signaling Pathway

Abstract

Adipose-derived stem cells (ASCs) are highly chondrogenic and can be used to treat knee osteoarthritis (KOA) by alleviating cartilage defects. Acupotomy, a biomechanical therapy guided by traditional Chinese medicine theory, alleviates cartilage degradation and is widely used in the clinic to treat KOA by correcting abnormal mechanics. However, whether combining acupotomy with ASCs will reverse cartilage degeneration by promoting chondrocyte proliferation in KOA rabbits is unknown. The present study aimed to investigate the effects of combination therapy of acupotomy and ASCs on chondrocyte proliferation and to determine the underlying mechanism in rabbits with KOA induced by knee joint immobilization for 6 weeks. After KOA modeling, five groups of rabbits (acupotomy, ASCs, acupotomy + ASCs, model and control groups) received the indicated intervention for 4 weeks. The combination therapy significantly restored the KOA-induced decrease in passive range of motion (PROM) in the knee joint and reduced the elevated serum level of cartilage oligomeric matrix protein (COMP), a marker for cartilage degeneration. Furthermore, magnetic resonance imaging (MRI) and scanning electron microscopy (SEM) images showed that the combination therapy inhibited cartilage injury. The combination therapy also significantly blocked increases in the mRNA and protein expression of glycogen synthase kinase-3β (GSK3β) and decreases in the mRNA and protein expression of cyclin D1/CDK4 and cyclin D1/CDK6 in cartilage. These findings indicated that the combination therapy mitigated knee joint immobility, promoted chondrocyte proliferation and alleviated cartilage degeneration in KOA rabbits, and these effects may be mediated by specifically regulating the GSK3β-cyclin D1-CDK4/CDK6 pathway.

Keywords: ASCs; KOA; acupotomy; chondrocytes; proliferation.

Figure 1.

The intervention points of the acupotome.

Figure 2.

The combination therapy of acupotomy and ASCs blocked the KOA-induced decrease in the PROM score. The score analysis of PROM. Values are means ± SEMs. n = 6 per group. * p < 0.05, ** p < 0.01.

Figure 3.

The combination therapy of acupotomy and ASCs relieved cartilage degeneration and inhibited the increased levels of COMP in the serum of KOA rabbits. (A) MRI images in the coronal position. a: control group, b: model group, c: acupotomy group, d: ASCs group, e: acupotomy + ASCs group. (B) SEM images. (Magnification: a1, b1, c1, d1, e1 are at 5000×; a2, b2, c2, d2, e2 are at 10000×). (C) ELISA of serous COMP. Values are means ± SEMs. n = 6 per group. * p < 0.05, ** p < 0.01.

Figure 4.

The combination therapy of acupotomy and ASCs upregulated the decreased expression of cyclin D1, CDK4 and CDK6 in the cartilage of KOA rabbits. (A) Real-time PCR analysis of cyclin D1. (B) Real-time PCR analysis of CDK4. (C) Real-time PCR analysis of CDK6. (D) Western blot assay of cyclin D1. (E) Western blot assay of CDK4. (F) Immunohistochemical staining and optical density values of cyclin D1, CDK4 and CDK6. The values are expressed as the mean ± SEMs. n = 6 per group. * p < 0.05, ** p < 0.01. Scale bars = 250 μm.

Figure 5.

The combination therapy of acupotomy and ASCs affected chondrocyte proliferation by inhibiting the activation of GSK3β in the cartilage of KOA rabbits. (A) Real-time PCR analysis of GSK3β. (B) Western blot assay of GSK3β. (C) Immunohistochemical staining and optical density value of GSK3β. The values are expressed as the mean ± SEMs. n = 6 per group. * p < 0.05, ** p < 0.01. Scale bars = 250 μm.