针刀对膝关节骨关节炎兔软骨细胞早衰标志物p16^(Ink4a)和p21^(Waf1/Cip1)表达的影响

Effect of acupotomy on expressions of cellular senescence markers p16^(Ink4a) and p21^(Waf1/Cip1) in cartilage tissue of rabbits with knee osteoarthritis

目的:观察针刀对膝关节骨关节炎(KOA)兔软骨细胞早衰标志物p16^(Ink4a)、p21^(Waf1/Cip1)表达的影响,分析针刀是否可通过抑制软骨细胞早衰治疗KOA。方法:新西兰兔随机分为正常组、模型组、针刀组和电针组,每组6只。采用改良Videman法左后肢伸直位制动制备KOA模型。针刀组选取兔左后肢膝关节股内外侧肌肌腱止点、股直肌肌腱止点、股二头肌肌腱止点及鹅足滑囊等高应力点行针刀松解治疗,1次/周,治疗3周。电针组选取兔左后肢“血海”“梁丘”“内膝眼”和“外膝眼”进行电针治疗,20 min/次,隔日1次,治疗3周。治疗前后,各组兔进行患侧膝关节Lequesne MG评分和膝关节被动活动度(PROM)检测。治疗结束后采用免疫组织化学法和Western blot法分别检测患侧膝关节软骨组织p16^(Ink4a)、p21^(Waf1/Cip1)的阳性表达和蛋白表达水平。结果:治疗前后,与正常组相比,模型组兔患侧膝关节Lequesne MG评分显著升高(P<0.01),PROM显著减小(P<0.01)。治疗后,与正常组相比,模型组兔患侧膝关节软骨组织p16^(Ink4a)、p21^(Waf1/Cip1)阳性表达和蛋白表达水平显著升高(P<0.01);与模型组相比,针刀组和电针组兔的患侧膝关节Lequesne MG评分显著降低(P<0.01),PROM显著增加(P<0.01),膝关节软骨组织p16^(Ink4a)、p21^(Waf1/Cip1)阳性表达和蛋白表达水平均显著降低(P<0.01,P<0.05);与电针组相比,针刀组患侧膝关节Lequesne MG评分降低(P<0.05),PROM增加(P<0.05),膝关节软骨组织p16^(Ink4a)、p21^(Waf1/Cip1)阳性表达和蛋白表达水平均显著降低(P<0.05,P<0.01)。结论:针刀干预能够下调软骨细胞中早衰标志物p16^(Ink4a)、p21^(Waf1/Cip1)的表达,提示针刀力学效应可能通过抑制软骨细胞早衰缓解软骨退变以治疗KOA。

Objective To observe the effect of acupotomy on the expressions of p16^(Ink4a)and p21^(Waf1/Cip1)in knee osteoarthritis(KOA)rabbits,so as to analyze whether acupotomy can treat KOA by inhibiting the cellular senescence of chondrocytes.Methods Twenty-four New Zealand male rabbits were randomly divided into normal,model,acupotomy and electroacupuncture(EA)groups,with 6 rabbits in each group.The KOA model was established by left hindlimb straightening fixation.After modeling,rabbits in the acupotomy group were treated with acupotomy loosening therapy on high stress points around the affected knee joints such as tendons attachment points of vastus medialis,vastus lateralis,rectus femoris,biceps femoris and pes anserine bursa,once a week for 3 weeks.In the EA group,“Xuehai”(SP10),“Liangqiu”(ST34),“Neixiyan”(EX-LE4)and“Waixiyan”(ST35)on the affected hindlimb were selected for EA treatment(3 mA,2 Hz/100 Hz),20 min each time,once every other day for 3 weeks.Before and after treatments,the knee Lequesne MG score and passive range of motion(PROM)of the affected knee joint were evaluated.After the treatments,the expressions of p16^(Ink4a)and p21^(Waf1/Cip1)in the cartilage tissue of the affected knee joint were detected by immunohistochemistry and Western blot respectively.Results Before and after treatment,compared with the normal group,the Lequesne MG score was significantly increased(P<0.01),the PROM was significantly decreased(P<0.01)in the model group.After treatment,compared with the normal group,the positive expression and protein expression levels of p16^(Ink4a)and p21^(Waf1/Cip1)were significantly increased(P<0.01)in the model group;compared with the model group,the Lequesne MG score was significantly decreased(P<0.01),the PROM was significantly increased(P<0.01),the positive expression and protein expression levels of p16^(Ink4a)and p21^(Waf1/Cip1)were significantly decreased(P<0.01,P<0.05)in the acupo-tomy and EA groups;compared with the EA group,the Lequesne MG score was decreased(P<0.05),the PROM was increased(P<0.05),the positive expression and protein expression levels of p16^(Ink4a)and p21^(Waf1/Cip1)were decreased(P<0.05,P<0.01)in the acupotomy group.Conclusion Acupotomy intervention can down-regulate the expressions of cellular senescence markers p16^(Ink4a)and p21^(Waf1/Cip1)in chondrocytes,indicating that acupotomy therapy may alleviate cartilage degeneration by inhibiting chondrocyte premature cellular senescence to treat KOA.