To Explore The Mechanism Of Acupotomy Intervention On Autophagy Of Rabbit Chondrocytes In Knee Osteoarthritis Based On AMPK-mTOR-ULK1 Signaling Pathway

Objective The purpose of this study was to investigate the effect of acupotomy on chondrocyte autophagy in rabbits with knee osteoarthritis and its possible mechanism,so as to gain a more profound comprehension of the action of acupotomy in treating knee osteoarthritis.MethodsThe Videman fixation technique was employed in this research to construct a KOA rabbit model,and then,all rabbits were randomized into one of four groups: Control;KOA(Model);KOA + acupotomy(Acupotomy),and KOA + sham acupotomy(Sham acupotomy),and treatment was carried out for 3 weeks.Improved knee joint function was observed by Lequesne MG knee joint behavior score in KOA rabbits.Hematoxylin-eosin staining(HE)revealed morphological changes in chondrocytes,while electron microscopy revealed autophagy.Quantitative fluorescence in real-time(q RT-PCR)was utilized to identify m RNA expressions of adenylate activated protein kinase(AMPK),mammalian target of rapamycin(m TOR),UNC-51-like kinase 1(ULK1),and mammalian homology of yeast ATG6(Beclin-1).To further probe the expression of essential proteins of AMPK-m TOR-ULK1 signaling pathway and microtubule-associated protein 1A/1b-light chain 3(LC3)in cartilage,Western blotting(WB)was utilized.Results(1)Improved Lequesne MG knee behavioral score showed that acupotomy could improve local signs and functions in KOA rabbits.(2)HE examination showed that the model group of rabbits had thin cartilage layer,scattered cells and unclear layers,while the number of immature chondrocytes near the cartilage layer was small,and the nuclei were flattened,crumpled,heterochromatin increased,and a few cells were swollen.The acupotomy group saw a return to normal cartilage surface,a slight increase in juvenile chondrocytes,even chromatin distribution,and normal staining of nuclei and nuclei,as well as a clear and orderly structure of cartilage tissue.Conversely,the pseudoacupotomy group’s cartilage layer remained thin and rough,with fewer juvenile chondrocytes and an unclear structure.(3)Transmission electron microscopy showed that there were fewer autophagosomes in chondrocytes in the model group,the nuclei were wrinkled and crescent-shaped,the chromatin edges were concentrated in the inner layer of the nuclear membrane,which had been ruptured,and the cells were significantly enlarged,and the cytoplasm appeared vacuoles of different sizes.Some small vacuoles fused with each other to form large vacuoles,forming balloon-like transformation,and most mitochondria were swollen and changed,the crists were shortened or even disappeared,and vacuum-like structures appeared.Rough endoplasmic reticulum dilatation;In the acupotomy group,autophagosomes were increased,the double-layer membrane structure was clear and complete,nuclear shrinkage was restored,the nuclear membrane boundary was obvious,chromatin aggregation was reduced,the number of organelles in the cells was increased,and there was no vacuole-like structure.The number of autophagosomes in chondrocytes in the pseudoacupotomy group was still less,the nuclei shrank,the cytoplasm had vacuoles of different sizes,the organelles were less,and the mitochondria were swollen.(4)Electron microscopy scanning revealed the cartilage of the model group to be rough and uneven,with deep cracks and a great many collagen fibers exposed.In contrast,the acupotomy group’s cartilage was smooth and collagen fibers were less conspicuous.The sham needle knife group’s cartilage surface was similar to that of the model group,yet still had a considerable amount of collagen fibers and deep cracks.(5)The q RT-PCR and WB detection revealed that the m RNA expressions of AMPK,ULK1,Beclin-1 and p-AMPK,as well as the protein expressions of p-AMPK,p-ULK1,Beclin-1 and LC3Ⅱ/LC3Ⅰ,were lower in the model group than in the normal group.Additionally,m TOR m RNA and p-m TOR protein expression were higher in the model group than the normal group(P <0.01).Moreover,the expression of these indexes in acupotomies were higher than those in both the model and sham acupotomies(P<0.05).ConclusionsThe KOA model rabbits’ knee cartilage injury can be significantly improved by acupotomy therapy,potentially due to its AMPK-m TOR-ULK1 signaling pathway mediating autophagy and thus diminishing cartilage deterioration.