温针灸调控膝骨关节炎模型兔关节软骨中PI3K/Akt信号通路的变化

Effect of warm acupuncture on PI3K/Akt signaling pathway in articular cartilage of a rabbit knee osteoarthritis model

背景:温针灸临床治疗膝骨关节炎疗效显著,但基础治疗机制尚未明确。目的:观察温针灸对兔膝骨关节炎模型关节软骨中PI3K/Akt信号通路及其下游因子Bcl-2、Bax表达的影响。方法:取6月龄新西兰兔40只,采用随机数字表法分为正常组、模型组、药物组、温针灸组,每组10只。模型组、药物组、温针灸组兔右后肢膝关节伸直位以石膏管型固定4周,制备膝骨关节炎模型。造模成功后,模型组每日置于兔架上固定15 min,药物组每天行硫酸氨基葡萄糖灌胃治疗(77 mg/kg,1次/d),温针灸组每天行温针灸治疗(15 min/次,1次/d)。连续治疗28 d后,利用苏木精-伊红染色观察软骨损伤程度,Tunel法检测软骨细胞凋亡,RT-PCR法检测软骨组织中PI3K、Akt、Bcl-2、Bax的mRNA表达,Western blot法检测软骨组织中PI3K、Akt、Bcl-2、Bax的蛋白表达。结果与结论:(1)苏木精-伊红染色显示,模型组软骨细胞数量减少,细胞排列无序,层次紊乱,软骨面被破坏,Mankin评分显著升高;药物组与温针灸组软骨细胞数量明显增多,基质着色深,Mankin评分降低;(2)与正常组相比,模型组软骨细胞凋亡率增高(P<0.05);与模型组相比,药物组、温针灸组软骨细胞凋亡率降低(P<0.05);(3)RT-PCR与Western blot检测显示,与正常组相比,模型组PI3K、Akt、Bcl-2的mRNA与蛋白表达降低(P<0.05),Bax的mRNA与蛋白表达升高(P<0.05);与模型组相比,温针灸组与药物组PI3K、Akt、Bcl-2的mRNA与蛋白表达升高(P<0.05),Bax的mRNA与蛋白降低(P<0.05);(4)结果表明温针灸可通过调控PI3K/Akt信号通路引起下游因子Bcl-2表达升高、Bax表达降低,进而抑制软骨细胞过度凋亡,达到保护关节软骨的作用。

BACKGROUND:Warm acupuncture is an effective treatment for knee osteoarthritis;however,the therapeutic mechanism is not clear OBJECTIVE:To observe the effects of warm acupuncture on PI3 K/Akt signaling pathway and expression of downstream factors Bc1-2 and Bax in the a rticular cartilage of a rabbit model of knee osteoarthritis.METHODS:Forty 6-month-old New Zealand rabbits were randomly divided into blank group,model group,glucosamine sulfate group and warm acupuncture group,with 10 rabbits in each group.The blank group was not modeled,and in the other three groups,the knee joint of the rabbit right hindlimb was immobilized in extension position using a plaster tube for 4 weeks to prepare the rabbit knee osteoarthritis model.After successful modeling,the blank group was not interfered.The rabbits in the model group were fixed in the rabbit frame for 15 minutes daily,those in the glucosamine sulfate group we re treated with glucosamine sulfate by gavage at 77 mg/kg daily,and those in the warm acupuncture group were treated with warm acupuncture for 15 minutes daily,for 28 continuous days in total.After the intervention,hematoxylin-eosin staining was performed to observe the seve rity of ca rtilage damage.Cell apoptosis in chondrocytes was observed using TUNEL assay.mRNA and protein expressions of PI3 K,Akt,Bcl-2 and Bax in ca rtilage tissue were detected by RT-PCR and western blot,respectively.RESULTS AND CONCLUSION:The model group had a rough surfa ce of a rticular cartilage,disordered arrangement and reduced number of chondrocytes,and significantly increased Mankin score,while the glucosamine sulfate and warm acupuncture groups showed increased number of chondrocytes,deeply stained matrix,and reduced Mankin score.Compared with the blank group,the apoptotic rate of chondrocytes was significantly increased in the model group(P<0.05).Compared with the model group,the apoptotic rate of chondrocytes was significantly decreased in the glucosamine sulfate and warm acupuncture groups(P<0.05).Compared with the blank group,the mRNA and protein expressions of PI3 K,Akt and Bcl-2 were significantly decreased and the mRNA and protein expressions of Bax were significantly increased in the model group(P<0.05).Compared with the model group,mRNA and protein expressions of PI3 K,Akt and Bcl-2 were significantly increased and the mRNA and protein expressions of Bax were significantly decreased in the glucosamine sulfate and warm acupuncture groups(P<0.05).To conclude,warm acupuncture can increase the expression of bcl-2 and decrease the expression of Bax by regulating PI3 K/Akt signaling pathway,and then inhibit the excessive apoptosis of chondrocytes to protect a rticular cartilage.