[Effect of needle knife on mTOR/Atg/ULK1/Beclin-1 axis and chondrocyte autophagy in rats with knee osteoarthritis].

To observe the effect of needle knife on chondrocyte autophagy and expressions of autophagy-related protein and mammalian target of rapamycin (mTOR) in rats with knee osteoarthritis (KOA), and to explore the possible mechanism of needle knife for KOA.A total of 42 SD rats were randomly divided into a normal group, a model group and a needle knife group, 14 rats in each group. Except for the normal group, the other two groups were injected with the mixture of papain and L-cysteine into the left hind knee joint to establish the KOA model. After modeling, the rats in the needle knife group were treated with needle knife at strip or nodule around the quadriceps femoris and medial and lateral collateral ligament on the affected side, once a week for 3 times (3 weeks). The changes of left knee circumference in each group were observed; the chondrocytes and ultrastructure of left knee joint were observed by HE staining and electron microscope; the mRNA and protein expressions of autophagy-related genes (Atg5, Atg12, Atg4a), Unc-51 like autophagy activated kinase 1 (ULK1), autophagy gene Beclin-1 and mTOR in left knee cartilage were detected by real-time fluorescence quantitative PCR and Western blot.After modeling, the left knee circumferences in the model group and the needle knife group were increased compared with those before modeling and in the normal group (P<0.05); after intervention, the left knee circumference in the needle knife group was smaller than that in the model group and after modeling (P<0.05). Compared with the normal group, the number of chondrocytes was decreased, and a few cells swelled, nuclei shrank, mitochondria swelled and autophagosomes decreased in the model group; compared with the model group, the number of chondrocytes was increased , and most cell structures returned to normal, and autophagosomes was increased. Compared with the normal group, the mRNA and protein expressions of Atg5, Atg12, Atg4a, Beclin-1 and ULK1 in the knee cartilage in the model group were decreased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were increased (P<0.05). Compared with the normal group, the mRNA and protein expressions of mTOR in the knee cartilage in the model group were increased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were decreased (P<0.05).The needle knife intervention could improve knee cartilage injury in rats with KOA, and its mechanism may be related to reducing the expression of mTOR and up-regulating the expressions of Atg5, Atg12, Atg4a, ULK1 and Beclin-1, so as to promote chondrocyte autophagy and delay the aging and degeneration of chondrocytes.目的：观察针刀对膝关节骨关节炎（KOA）大鼠软骨细胞自噬及自噬相关蛋白、哺乳动物雷帕霉素靶蛋白（mTOR）表达的影响，探究针刀干预KOA的可能机制。方法：将42只SD大鼠随机分为正常组、模型组和针刀组，每组14只。除空白组外，其余2组采用左侧后肢膝关节腔注射木瓜蛋白酶和L-半胱氨酸混合液制备大鼠KOA模型。造模后，针刀组以大鼠患侧股四头肌腱和内、外侧副韧带附近的条索或结节作为进针点行针刀干预，每周1次，共干预3次（3周）。观察各组大鼠左侧膝围变化；HE染色法和透射电镜观察各组大鼠左侧膝关节软骨细胞及超微结构形态；实时荧光定量PCR和Western blot法检测大鼠左侧膝关节软骨组织自噬相关基因（Atg5、Atg12、Atg4a）、Unc-51样自噬激活激酶1（ULK1）、自噬基因Beclin-1及mTOR的mRNA和蛋白表达。结果：造模后，模型组、针刀组左侧膝围均较本组造模前及正常组增加（P<0.05）；干预后，针刀组左侧膝围小于模型组及本组造模后（P<0.05）。与正常组比较，模型组大鼠软骨细胞数量减少，少数细胞肿胀，细胞核皱缩，线粒体肿胀，自噬体减少；与模型组比较，针刀组大鼠软骨细胞数量增多，多数细胞结构恢复正常，自噬体增多。与正常组比较，模型组大鼠膝关节软骨组织Atg5、Atg12、Atg4a、Beclin-1、ULK1 mRNA和蛋白表达降低（P<0.05）；与模型组比较，针刀组大鼠以上指标表达升高（P<0.05）。与正常组比较，模型组大鼠膝关节软骨组织mTOR mRNA和蛋白表达升高（P<0.05）；与模型组比较，针刀组大鼠以上指标表达降低（P<0.05）。结论：针刀干预能改善KOA模型大鼠膝关节软骨损伤，其机制可能与降低mTOR的表达，上调Atg5、Atg12、Atg4a、ULK1、Beclin-1的表达，从而促进KOA大鼠软骨细胞自噬，延缓软骨细胞的衰老退变有关。.