基于CircSERPINE2-miR-1271-5P-ERG轴探讨针刀对膝关节骨关节炎兔膝关节软骨细胞凋亡的影响

Effect of needle-knife on chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis based on CircSERPINE2-miR-1271-5P-ERG axis

目的:基于CircSERPINE2-miR-1271-5P-E26特异性转化相关基因(ERG)轴,观察针刀对膝关节骨关节炎(KOA)兔膝关节软骨细胞凋亡的影响,探讨针刀治疗KOA的作用机制.方法:将36只新西兰大白兔随机分成正常组、模型组、针刀组、假针刀组,每组9只.除正常组外,其余3组采用改良Videman法制备KOA模型.造模成功后,针刀组于患侧膝关节股四头肌肌腱、内外侧副韧带附近条索状粘连、结节行针刀干预,假针刀组于针刀组进针点旁侧仅刺入针刀,不做任何操作.均每周1次,共3次.采用Lequesne MG行为学评分于干预前后评估各组兔膝关节损害程度.干预后,采用HE染色法和透射电镜观察各组兔膝关节软骨组织形态和软骨细胞超微结构;TUNEL法检测各组兔膝关节软骨细胞凋亡水平;实时荧光定量PCR法检测各组兔膝关节软骨组织CircSERPINE2、miR-1271-5P、ERG mRNA表达.结果:干预后,与正常组比较,模型组Lequesne MG行为学评分升高(P<0.01);与模型组和假针刀组比较,针刀组Lequesne MG行为学评分降低(P<0.01).模型组、假针刀组软骨细胞数量及细胞器数量减少,细胞核固缩,线粒体肿胀或消失;针刀组软骨细胞数量及细胞器数量增加,未见细胞核明显固缩,线粒体无明显肿胀.与正常组比较,模型组软骨细胞凋亡数量增多(P<0.01);与模型组和假针刀组比较,针刀组软骨细胞凋亡数量减少(P<0.01,P<0.05).与正常组比较,模型组CircSERPINE2、ERG mRNA表达降低(P<0.01),miR-1271-5P mRNA表达增加(P<0.01);与模型组和假针刀组比较,针刀组CircSERPINE2、ERG mRNA表达增加(P<0.01),miR-1271-5P mRNA表达降低(P<0.01).结论:针刀可以减轻KOA兔膝关节损害,减少软骨细胞凋亡,可能与上调CircSERPINE2和ERG mRNA表达、抑制miR-1271-5P mRNA表达有关.

Objective To observe the effect of needle-knife on the chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis(KOA)based on the CircSERPINE2-miR-1271-5P-E26 specific transformation-related gene(ERG)axis,and to explore the mechanism of needle-knife for KOA.Methods Thirty-six New Zealand white rabbits were randomly divided into a normal group,a model group,a needle-knife group and a sham needle-knife group,9 rabbits in each group.The rabbits in the model group,the needle-knife group and the sham needle-knife group were treated with modified Videman method to prepare KOA model.After successful modeling,the rabbits in the needle-knife group were treated with needle-knife at cord adhesion and nodules near quadriceps femoris tendon and internal and external collateral ligament on the affected knee joint;the rabbits in the sham needle-knife group were treated with sham needle-knife baside the needle insertion point of the needle-knife group(needle-knife was only inserted,without any operation).The treatment was given once a week,3 times in total.The Lequesne MG behavioral score was used to evaluate the knee joint damage in each group before and after intervention.After intervention,HE staining and transmission electron microscopy were used to observe the cartilage tissue morphology and ultrastructure of chondrocytes in the knee joint in each group;TUNEL method was used to detect the level of chondrocyte apoptosis in the knee joint;real-time fluorescence quantitative PCR was used to detect the expression of CircSERPINE2,miR-1271-5P and ERG mRNA in knee cartilage tissue in each group.Results After intervention,compared with the normal group,the Lequesne MG behavioral score in the model group was increased(P<0.01).Compared with the model group and the sham needle-knife group,the Lequesne MG behavioral score in the needle-knife group was decreased(P<0.01).In the model group and the sham needle-knife group,the number of chondrocytes and organelles was decreased,the cell nucleus was shrunk,mitochondria was swelling or disappeared;in the needle-knife group,the number of chondrocytes and organelles was increased,the cell nucleus was not obviously shrunk and the mitochondria was not obviously swelling.Compared with the normal group,the level of chondrocyte apoptosis in the model group was increased(P<0.01);compared with the model group and the sham needle-knife group,the level of chondrocyte apoptosis in the needle-knife group was decreased(P<0.01,P<0.05).Compared with the normal group,the expression of CircSERPINE2 and ERG mRNA in the model group was decreased(P<0.01),and the expression of miR-1271-5P mRNA was increased(P<0.01);compared with the model group and the sham needle-knife group,the expression of CircSERPINE2 and ERG mRNA in the needle-knife group was increased(P<0.01),and the expression of miR-1271-5P mRNA was decreased(P<0.01).Conclusion Needle-knife could reduce the knee joint damage and chondrocyte apoptosis in KOA rabbits,which may be related to up-regulating the expression of CircSERPINE2 and ERG mRNA,and inhibiting the expression of miR-1271-5P mRNA.