| Background:Knee osteoarthritis(KOA)is a kind of chronic degenerative osteoarthritis with high morbidity and high disability.On the basis of previous studies,28 Clean New Zealand male rabbits were made by Videman’s method with left hind leg extension fixation for 8 weeks.By comparing the intervention effect of acupotomology(AP)and electro-acupuncture(EA)on different parts of KOA rabbits,the effects of acupotomology and electro-acupuncture on cartilage morphology,muscle and cartilage MRI imaging,autophagy level of chondrocytes,protein and gene expression of key factors in autophagy related pathways were observed,and the improvement effect of acupotomology and electro-acupuncture on joint symptoms and signs of KOA rabbits was clarified.The difference of therapeutic effect of acupuncture laid a theoretical basis and experimental basis for the follow-up study.Objective:To investigate the effect of acupotomology on the changes of cartilage morphology and MRI imaging,to study the effect of acupotomology on the autophagy level and histopathological regulation of chondrocytes,to discuss the molecular mechanism of PI3K/Akt-mTOR pathway and related factors expression in the treatment of KOA model rabbits by acupotomology.To demonstrate the scientific basis and connotation,lay a theoretical foundation for future experimental research,and provide experimental basis for clinical treatment of the disease.Methods:28 totally,6 months old Clean New Zealand rabbits,male,which were randomly allocated as 4 groups,normal,model,AP and EA group.Videman’s method was used to establish 21 rabbits’ model except the normal group.24 rabbits were used in the experiment because of the infection and death in the process of experiment,6 rabbits in each group.In the acupotomology group,6 rabbits in the normal group and 6 rabbits in the model group,which participated in the fixation without treatment.In the acupotomology group,the rabbits were given acupotomology treatment once a week.The biceps femoris,semitendinosus muscle,semitendinosus muscle tendon ending points and the nodular focus points of the peripheral tendons of the knee were released once a week and lasted for 3 weeks.In the electro-acupuncture group,the acupoints "Weiyang","Ququan","Nei Xiyan" and"Dubi" were selected for acupuncture treatment,every other day,20 min for each time,dense-sparse wave,frequency 2/100 Hz,strength 3 mA.At end of the week after treatment,3.0 T field Magnetic Resonance Imaging(MRI)was used to examine the cartilage of knee joint and the surrounding muscles of each group,and the image data were collected and analyzed.After the imaging examination,24 rabbits were sacrificed by injection of excessive anesthesia,and the articular cavity of the knee was dissected for gross morphological observation.24 rabbits were used for specimen examination and 6 rabbits for each group.The articular cartilage of the knee was cut and put into the fixative for Optical Microscopy(OM)and Transmission Electron Microscopy(TEM)observation.The pathological sections of cartilage stained with safranine O fast green(SAf-O/FG)were observed under optical microscope,and the Mankin’s score was used for statistical analysis.Transmission electron microscopy was used to observe the autophagy level of chondrocytes and the distribution and arrangement of collagen fibers in the extracellular matrix.The changes of autophagy level of chondrocytes in each group were observed.Western blot(WB)and Reverse Transcription Polymerase Chain Reaction(RT-PCR)were used to detect and observe the protein and gene expression level of key factors in the autophagy related pathway PI3K/Akt-mTOR of chondrocytes.The protein and gene expression level of relevant factors in the chondrocyte pathway of each group were analyzed,and the activation of autophagy related pathway and the expression regulation level of relevant factors by acupotomy and electro-acupuncture were compared.Results:The gross morphological observation demonstrated that normal group,the knee joint was complete and transparent with bright color,a small amount of transparent fluid can be observed in the joint cavity,and no obvious hyperemia and edema can be seen in the synovium.The morphology of cartilage on both sides of the tibiofemoral joint surface and patella medial joint surface was complete with white and transparent color,and there was a small amount of fluid in the cartilage joint cavity.The articular cartilage in the model group was gray white,with poor integrity,obvious soft tissue proliferation around,thin cartilage thickness,obvious fascial adhesion and cracks on the surface.The cartilage in the acupotomy group was light white,part of the cartilage surface defection was not found,there was edema at the tendon attachment,fascial adhesion,synovium hyperplasia and osteophyte formation.In the electro-acupuncture group,the cartilage was gray,some cartilage surface defects,a small amount of osteophyte hyperplasia,synovium hyperplasia,hyperemia,joint fluid increase,fascial adhesion osteophyte hyperplasia was not obvious.MRI:In normal group,the articular cartilage surface was smooth,the cartilage space on both sides of the knee joint was uniform and equal width,and there was a small amount of joint cavity effusion with long T1 and T2 signals,while the swelling and effusion of fascia and soft tissue were not obvious.The contour of soft tissue such as muscles around the knee joint was clear,the shape of cartilage and subchondral bone was clear,the density of marrow cavity was well-distributed and intensive,meniscus was clear,and there was no growth.In the model group,the thickness of cartilage became thinner,the space of articular cavity was smaller than that in the normal group,showing the equal T1 long T2 signal shadow,the joint capsule was enlarged,the effusion was increased,the subchondral bone edema was obvious,showing the long T1,T2 signal shadow,the soft tissue swelling and the fascial effusion were obvious,showing the slightly long T1 and T2 signals,and the suprapatellar tendon edema was also visible,showing the long T1 and T2 signals,the soft tissue around the knee joint was obviously atrophied,and the left lower part was obvious.There was obvious synovial hyperplasia in the articular cavity of the limb,serious defects of the articular surface of the femur and vague meniscus outline.Compared with the model group,there was a significance for the cartilage thickness of AP group than model group.The articular space was equal width with a small amount of effusion,showing long T1 and T2 signal shadows.The articular soft tissue was atrophied,and the articular cartilage surface of the tibia was defective.The articular surface of the rabbits in the acupotomy group was smooth,and the articular cavity and fascia were seen.The effusion showed as long or slightly long T1 and T2 signals.The atrophy of rectus femoris had obvious synovium hyperplasia and tibial cartilage defection.There was no obvious accretion in the joint cavity,but the bilateral joint space was narrower than normal group,the articular cartilage surface of femur was smooth,slightly rough than normal group.The articular cartilage surface of tibial plateau was damaged and unclear,and a small amount of vegetations were seen.The articular cartilage surface of rabbits in the electro-acupuncture group was smooth,and part of the articular cavity effusion was seen,showed as long T1 and T2 signals,and some joints of rabbits presented as slightly longer T1 and T2 signals soft tissue injury and effusion.There was no bone edema,the shape of suprapatellar tendon was normal.The cartilage space of osteoarthritis side of knee joint became narrow,the rectus femoris atrophy could be seen,and some fatty like high-density changes appeared in the knee joint cavity of both lower limbs,demonstrated the synovium hyperplasia had been changed.The femoral joint surface was damaged,the tibial joint surface was not smooth,and there was a defection,the surface covered with growth,the joint space was not clear,which was significantly reduced compared with the normal group.Walther grade score result showed that there was a significant difference between model and normal group(P=0.018,P<0.05),model and AP group(P=0.015,P<0.05).The results of MRI semi quantitative score showed that compared with the normal group,the semi quantitative MRI scores of the knee joint in the model group were significantly different(P=0.000,P<0.05,P<0.01),the scores of the electro-acupuncture group were significantly different(P=0.000,P<0.05,P<0.01),the scores of the acupotomology group were significantly different(P=0.001,P<0.05,P<0.01).Compared with the model group,the scores of the electro-acupuncture group were significantly different(P=0.009,P<0.05,P<0.01)<0.01),there was significant difference in the score of AP group(P=0.000,P<0.05,P<0.01).Compared with the electro-acupuncture group,there was significant difference in the score of AP group(P=0.003,P<0.05,P<0.01).The results of Mankin’s score showed that compared with the normal group,the Mankin’s score of the rabbits in the model group,the acupotomy group and the electro-acupuncture group showed a significant forward regulation.The model group had a significant difference compared with the normal group(P=0.000,P<0.05,P<0.01).There was a significant difference between the electro-acupuncture group and the normal group(P=0.000,P<0.05,P<0.01),the acupotomy group had a significant difference compared with the normal group(P=0.000,P=0.001,P<0.05,P<0.01).The EA group had a significant difference compared with model group(P=0.000,P<0.05,P<0.01),AP group had a significant difference compared with the model group(P=0.000,P<0.05,P<0.01).It had no significant difference between comparison results of Mankin’s score of acupotomy group and electro-acupuncture group(P=0.368,P>0.05),but the difference between each group had significant difference(P=0.000,P<0.05,P<0.01).The outcome for transmission electron microscopy showed that in normal group,collagen fibers were intensive,glycogen granules,endoplasmic reticulum and lipid granules were visible in the cytoplasm,shape of cells were round,nuclear membrane was complete and clear,chromatin was well-distributed,autophagy was observed in some of the cytoplasm,microfilaments were arranged in bundles,density was uniform,nucleus was complete,cytoplasm was distributed well,collagen fibers were regularly arranged.In the model group,cartilage was fine most of the cells are irregular in shape,unclear in outline,membrane was incomplete,a small amount of autophagy in cytoplasm,pyknosis of nucleus but obvious in nuclear membrane,obvious chromatin clusters in nucleus,pool like expansion of endoplasmic reticulum,disintegration,sparseness or disappearance of mitochondrial cristae,degradation and disorder of collagen fibers in matrix.The arrangement of chondrocytes in acupotomy group was irregular,the distribution of collagen fibers in matrix was intensive and degraded,the cell membrane was complete and clear,autophagic vesicles appeared in cytoplasm,the atrophy of chondrocytes was not obvious,the shape and shape of chondrocytes were regular,there was clustering phenomenon of chromatin in cells,rough endoplasmic reticulum,Golgi body and mitochondria were obvious,and the arrangement of microfilaments in cytoplasm was disorder.The arrangement of collagen fibers under cartilage is disordered.The autophagy level of chondrocytes in the electroacupuncture group was slightly better than that in the model group,and the autophagy level of chondrocytes had better level for AP group than model and electro-acupuncture group.Western blot results showed that compared with the normal group,the expression level of beclin-1 protein in the model group was significantly lower,there was no significant difference between groups(P>0.05).The expression level of beclin-1 protein in the electro-acupuncture group had upregulated,it had no significant difference(P>0.05).The expression level of beclin-1 protein in the acupotomy group was significantly higher,it had no significant difference compared with normal group(P>0.05).It appeared that comparison between model and EA group,the expression for beclin-1 protein in the electro-acupuncture group raised,but it had no significance between model and EA groups(P>0.05).The expression level of beclin-1 protein in the acupotomology group raised significantly,it had significance between electro-acupuncture and acupotomy groups(P<0.05,P=0.029),but there was no extremely signifi cant difference(P<0.01).There was no significant difference in the expression level of beclin-1 between the AP group and the electro-acupuncture group(P>0.05).In comparison for normal group,the mTOR protein expression of the model group increased slightly,but if had no significance between model and normal groups(P>0.05).In the comparison for model group,the content of protein expression(P>0.05),the expression level of mTOR protein in the electro-acupuncture group decreased significantly,but it had no significance between EA and model group(P>0.05).The expression level of mTOR protein in the acupotomy group decreased significantly,but it had no significance between acupotomy and model group(P>0.05).Compared with the model group,the expression level of mTOR protein in the electro-acupuncture group was significantly lower,and there was no significant difference between groups(P>0.05).The expression level of mTOR protein in the acupotomy group was significantly lower,and there was no significant difference between groups(P>0.05),had increased than AP group,and it had no significance between electro-acupuncture group and acupotomy group(P>0.05).Compared with the normal group,Akt protein expression level of the model group was significantly lower,it had no significance between normal and model groups(P>0.05),Akt protein expression level of the electro-acupuncture group had been raised,it had no significance between EA and model groups(P>0.05).Akt protein expression level of the acupotomology group was significantly higher,it had no significance between AP and model group(P>0.05).Compared with the model group,the Akt protein expression level of the electro-acupuncture group decreased,there was no significant difference between groups(P>0.05).The Akt protein expression level of the acupotomology group increased,there was no significant difference between groups(P>0.05),the Akt protein expression of the acupotomology group and the electro-acupuncture group increased significantly,but it had no significance between acupotomy and electro-acupuncture group(P>0.05).RT-PCR results showed that,compared with the normal group,the expression level of mRNA of mTOR in the model group slightly increased,meanwhile it had no significance and extremely significance between normal and model groups(P>0.05).In comparison of normal and model group,the content level for mRNA of mTOR in the electro-acupuncture group significantly increased,and it had no significance and extremely significance between EA group and normal group(P=0.000,P<0.05,P<0.01).It had significance and extremely significance between the electro-acupuncture group and the model group(P=0.000,P<0.05,P<0.01),the mRNA expression level of the AP group had been increased than normal and model group,it had no significance between acupotomy group and the normal group(P=0.287,P>0.05),and it had no significance between acupotomy group and the model group(P>0.05).It had no significance between acupotomy and electro-acupuncture groups(P=0.170,P>0.05).The expression level of mTOR mRNA in acupotomology group had been increased in normal and model group,it had no significance between AP and normal groups(P=0.287,P>0.05),and it had no significance and extremely significance between acupotomology group and electro-acupuncture group(P=0.170,P>0.05).In comparison of normal group,content expression of beclin-1 mRNA had been reduced for model group,it had no any significances between normal and model groups(P>0.05).The expression level of beclin-1 mRNA in the electro-acupuncture group increased significantly compared with the normal group and model group,it had no any significances between EA and normal group,EA and model group(P=0.000,P=0.001,P<0.05,P<0.01).The expression level of beclin-1 mRNA in acupotomology group was higher than that in normal group and model group,and lower than that in electro-acupuncture group,had significance between model group(P=0.000,P<0.05,P<0.01).The expression level of beclin-1 mRNA in acupotomology group was not significantly different from that in electro-acupuncture group(P>0.05).In comparison of normal group,Akt mRNA content expression in the model group slightly increased,and there was no significant difference between groups(P>0.05).Akt mRNA expression level in the electro-acupuncture group significantly increased,and it had significance and extremely significance between the electro-acupuncture group and the normal group(P=0.005,P<0.05,P<0.01).Akt mRNA expression level for AP group had significantly more than normal group(P=0.002,P<0.05,P<0.01).In comparison of model group,Akt mRNA content expression was more than model group(P=0.014,P<0.05),Akt mRNA for AP group had significantly more than model group(P=0.014,P<0.05).And there was extremely significant difference between AP and model groups(P=0.004,P<0.05,P<0.01),but it had no significance between acupotomy and electro-acupuncture groups(P>0.05).Conclusions:Acupotomology can significantly improve the symptoms and signs of the joint of KOA model rabbits under long-term immobilization,decrease the degree of cartilage pathological damage,initiate autophagy of chondrocytes to inhibit apoptosis and thus alleviate the degeneration of chondrocytes and matrix.Significantly regulated the expression of Akt and mTOR genes,enhance the expression of Beclin-1 protein and gene,inhibit PI3K/Akt-mTOR pathway and forward regulate autophagy level,effectively protect chondrocyte damage. |
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