针刀对膝骨关节炎兔软骨细胞磷酸化黏着斑激酶、磷酯酰肌醇-3激酶、聚集蛋白聚糖基因及蛋白表达的影响

Phosphorylated Focal Adhesion Kinase,Phosphinositides 3 Kinase and Aggrecan Genes and Proteins in Cartilage Cells Are Probably Involved in Needle Knife Intervention Induced Improvement of Knee Osteoarthritis in Rabbits

目的:观察针刀对膝骨关节炎(KOA)兔关节软骨磷酸化黏着斑激酶(p-FAK)、磷酸化磷酯酰肌醇-3激酶(p-PI 3K)、聚集蛋白聚糖(Aggrecan)基因和蛋白表达的影响,探讨针刀干预促进软骨细胞修复的可能力学转导通路。方法:新西兰大白兔随机分为正常组、模型组、模型抑制剂组、针刀组、针刀抑制剂组、电针组、电针抑制剂组,每组7只。采用改良Videman左后肢伸直位固定制动法复制KOA模型。电针组、电针抑制剂组电针左侧"梁门""血海""内膝眼""外膝眼",每周3次,治疗4周。针刀组、针刀抑制剂组分别采用针刀治疗,每周2次,治疗4周。采用Real-time PCR法和Western blot法检测各组家兔膝关节软骨p-FAK、p-PI 3K、Aggrecan基因和蛋白表达水平。结果:与正常组相比,模型组p-FAK、p-PI 3K mRNA和蛋白表达水平明显升高(P<0.05,P<0.01),Aggrecan mRNA和蛋白表达水平明显下降(P<0.01)。与模型组比较,电针组p-FAK、p-PI 3K、Aggrecan蛋白表达水平明显升高(P<0.05,P<0.01),针刀组p-FAK、p-PI 3K、Aggrecan mRNA和蛋白表达水平均明显升高(P<0.05,P<0.01)。与电针组相比,针刀组p-FAK蛋白及p-PI 3K、Aggrecan mRNA和蛋白表达水平均明显升高(P<0.05,P<0.01)。与相同干预方法组相比,模型抑制剂组(除p-PI 3KmRNA外)、电针抑制剂组、针刀抑制剂组p-FAK、p-PI 3K、Aggrecan mRNA和蛋白表达水平均明显下降(P<0.05,P<0.01)。结论:FAK-PI 3K信号通路在针刀干预后关节软骨的修复过程中起到了重要的介导作用。

Objective We have demonstrated that needle knife（acupotomy）treatment can improve knee osteoarthritis（KOA）in rabbits.The present study was designed to examine its effect on expression of phosphorylated focal adhesion kinase（p-FAK）,phosphinositides 3 kinase（p-PI 3 K）and Aggrecan genes and proteins in the knee-joint cartilage tissues of KOA rabbits,so as to explore its partial molecular mechanism underlying improvement of KOA.Methods Forty-nine New Zealand male rabbits were randomly divided into normal control,model,model＋inhibitor,needle knife,needle knife＋inhibitor,electroacupuncture（EA）,EA ＋inhibitor groups（n=7 in each）.The KOA model was established by modified Videman method（left hindlimb extension immobilization and ankle dorsal flexion 60 O）.Acupotomy relaxing manipulation was applied to the lateral collateral ligament and patellar ligament of the left knee-joint,two times a week for 4 weeks,and EA（2 Hz/100 Hz,3 mA）was applied to the left＂Liangmen＂（ST 21）,＂Xuehai＂（SP 10）,＂Neixiyan＂（EX-LE 4）and＂Dubi＂（ST 35）for 20 min,three times a week,for4 weeks.About 2 hbefore every needle-knife or EA treatment or at the corresponding time-point,intra-articular cavity injection of PF-562271（a specific antagonist of FAK,200 !mol/L,0.5 mL）was performed in the three inhibitor groups.The expression levels of p-FAK,p-PI 3 K,Aggrecan genes and proteins in the cartilage tissues were measured with quantitative Real-time PCR and Western blot,separately.Results After modeling,the expression levels of p-FAK and p-PI 3 Kgenes and proteins were significantly up-regulated（P〈0.05,P〈0.01）,while those of Aggrecan protein and mRNA considerably down-regulated in the model group in comparison with the normal group（P〈0.01）.Following 4 weeks＇ needle-knife or EA treatment,the expression levels of p-FAK and p-PI 3 Kand Aggrecan proteins in both EA and needle-knife groups,and Aggrecan mRNA in the needle knife group were significantly up-regulated（P〈0.05,P〈0.01）.After administration of p-FAK antagonist,modeling-induced up-regulation of expression of p-FAK mRNA and protein and p-PI 3 Kprotein,as well as modeling-induced down-regulation of Aggrecan mRNA and protein were significantly suppressed in the model＋inhibitor group（P〈0.05,P〈0.01）,and needle knife-induced and EA-induced up-regulation of expression of p-FAK,p-PI 3 Kand Aggrecan mRNAs and proteins was notably suppressed respectively in comparison with the needle knife and EA groups（P〈0.05,P〈0.01）.The effect of needle knife was significantly superior to that of EA in up-regulating p-PI 3 Kand Aggrecan mRNAs as well as p-FAK,p-PI 3 Kand Aggrecan proteins（P〈0.05,P〈0.01）.Conclusion Needle knife intervention can up-regulate the expression levels of p-FAK,p-PI 3 Kand Aggrecan proteins and mRNAs in the cartilage tissue of the knee-joint in KOA rabbits,suggesting an involvement of FAK-PI 3 Ksignaling in the needle knifeinduced improvement of KOA.