Objective: To observe the effect of acupotomy on the expression of Beclin-1, Bcl-2 and Caspase-3 in the cartilage tissue in rabbits with knee osteoarthritis (KOA), so as to explore its mechanism underling improvement of KOA.
Methods: Twenty-four healthy male New Zealand rabbits were randomly and equally divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman's methods, the KOA model was established by left hind limb immobilization with a plaster cast for 6 weeks. The severity of KOA (knee pain, swelling and motor function) was assessed using Lequesne score, and the rabbits with a score below 4 were excluded. The acupotomy was applied to "Hedingci" (the attachment of the quadriceps tendon to the patella at the upper edge), "Binneixia" (the medial patellar supporting band attachment of medial inferior patellar margin), "Binwaixia" (the lateral patellar supporting band attachment of the lower lateral patellar margin), "Chengfeijian" (the lateral collateral ligament of the knee passes over the lateral joint space), "Weiyangci" (the medial margin of biceps femoris at the lateral end of popliteus), "Yinlingci" (the medial tibial attachment of anserinus tendon) on the left hind limb once a week for 4 weeks. One week after the last intervention, the left knee joint dysfunction severity(pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne score. Histopathological changes of the cartilage were observed under light microscope after H.E. staining. The apoptosis of chondrocytes was observed after terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) staining. The autophagolysosomes of chondrocytes were observed using transmission electron microscopy. The expression levels of Beclin-1, Bcl-2 and Caspase-3 (related factors of autophagy and apoptosis) were detected using Real-time PCR and Western blot separately.
Results: In comparison with the blank control group, the Lequesne score, apoptosis rate, expression levels of Caspase-3 mRNA and protein were significantly increased (P<0.001), and the number of autophagolysosomes, expression levels of Beclin-1 and Bcl-2 mRNAs and proteins considerably decreased (P<0.001) in the model group. Relevant to the model group, the acupotomy group had an obvious decrease in Lequesne score, rate of apoptosis, and expression levels of Caspase-3 mRNA and protein (P<0.001) and an apparent increase in the number of autophagolysosomes and expression levels of Beclin-1 and Bcl-2 mRNAs and proteins (P<0.001). Findings of H.E. staining showed severe damaged cartilage surface, with a large number of exfoliation defects, few chondrocytes on the surface and disordered arrangement of transitional cells in the model group, which was relatively milder in the acupotomy group.
Conclusion: Acupotomy can mitigate knee-joint pain and improve functional activity in KOA rabbits, which may be associated with its functions in promoting autophagy and suppressing apoptosis by up-regulating expressions of Beclin-1 and Bcl-2 mRNAs and proteins and down-regulation of Caspase-3 mRNA and protein.
目的：基于Beclin-1、Bcl-2、Caspase-3对自噬和凋亡的调控，观察针刀对膝关节骨关节炎(KOA)兔膝关节软骨的保护作用，探讨针刀治疗KOA的可能机制。方法：雄性新西兰兔随机分为空白组、模型组和针刀组，每组8只。采用改良Videman法将兔左后肢以伸直位石膏固定6周建立KOA模型。针刀组选取左侧“鹤顶次”“髌内下”“髌外下”“成腓间”“委阳次”“阴陵次”行针刀治疗，每7 d干预1次，共4次。干预结束1周后，采用Lequesne膝关节级别评分法评估兔左膝关节疼痛、肿胀程度和功能；HE染色法观察各组兔膝关节软骨病理形态，TUNEL染色法检测膝关节软骨细胞凋亡率；透射电镜法观察膝关节软骨细胞自噬溶酶体；Real-time PCR法和Western blot法检测膝关节软骨组织中自噬和凋亡调控分子Beclin-1、Bcl-2、Caspase-3 mRNA和蛋白表达水平。结果：与空白组比较，模型组兔左膝关节Lequesne评分、软骨细胞凋亡率、膝关节软骨组织中Caspase-3 mRNA及蛋白表达水平均升高(P<0.001)，软骨细胞胞质内自噬溶酶体减少(P<0.001)，软骨组织中Beclin-1、Bcl-2 mRNA和蛋白表达水平均降低(P<0.001)。与模型组比较，针刀组兔左膝关节Lequesne评分、软骨细胞凋亡率、膝关节软骨组织中Caspase-3 mRNA及蛋白表达水平均降低(P<0.001)，软骨细胞胞质内自噬溶酶体增多(P<0.001)，软骨组织中Beclin-1、Bcl-2 mRNA和蛋白表达水平均升高(P<0.001)。空白组膝关节软骨表面光滑，软骨细胞均匀呈柱状排列；模型组膝关节软骨表面破损严重，出现大量剥脱缺损，表层软骨细胞稀少，细胞排列紊乱；针刀组膝关节软骨表面较光滑，表层细胞稍减少，软骨细胞排列趋于空白组。结论：针刀治疗能通过调控Beclin-1、Bcl-2、Caspase-3等蛋白表达而上调KOA兔膝关节软骨细胞自噬水平，减少其软骨细胞凋亡，进而发挥保护软骨和改善关节疼痛及功能障碍的作用，这可能是针刀治疗KOA的作用机制之一。.
Keywords: Acupotomy; Apoptosis; Autophagy; Knee osteoarthritis.