| Objective:To observe the effect of acupotomy on the rectus femoris muscle injury in KOA rabbits by the expression of Akt,mTOR,4EBP1 and p70s6k and to explore the underling mechanism of Akt/mTOR signaling pathway in KOA treatment by acupotomy,and also attempts to explore the effect and mechanism of needle knife for KOA based on a National Natural Science Foundation Porject.Methods:36 six-months old,health and sanitary level,New Zealand rabbits were randomly divided into four groups(n=9 in each group),normal group,model group,acupotoy group,electro-acupuncture group.8 rabbits of each group were measured while the remaining one was used for validation of morphological modeling after 4 weeks.The left knee joint was fixed with plaster bandage in extenson position to establish the rabbit model of knee joint injury with the Modified Videman method.Acupotomy and electro-acupuncture were respectively applied in acupotomy,and electro-acupuncure group.EA group:left"Yanglingquan(GB34)with Yinlingquan(SP9)","Neixiyan(Ex-LE40)with Waixiyan(ST35)",using rarefaction-dense and continuous wave of 2/100Hz,3mA,for 10 min each time 3 times/week,4 weeks in total.Acupotomy group:the front edge of the midpoint,the starting and the ending point of the medial collateral ligament,lateral collateral ligament and patellar ligament.The ligament edge line and loosening direction should keep parallel,twice a week respectively,twice a week,4 weeks in total.In the fist part of the study,bilateral knee passive range(PROM)and the Lequesne MG total index was evaluated before excuted.MRI were used to assess the radiological changes of KOA rabbit knee jonints.HE staining was used to observe the morphology of rectus femoris muscle,myocardial fiber rupture and abnormal cells thought the ordinary optical microscope.Ultra structural changes of the rectus femoris were observed using transmission electron microscope.In the second part,the Akt,mTOR,4EBP1 and p70s6k were analyzed by real-time fluorescence quantitative RT-PCR assay.The protein and mRNA expression of Akt,mTOR,4EBP1 and p70s6k of rectus femoris were examined by western blotting analysis.Result:(1)Acupotomy and Electro-acupuncture therapy can effectively articular pain and movement disorder in KOA rabbits,by reducing Lequesne MG knee arthritis index(pain,joint swelling,and deformity of knee joint)(p<0.01),passive motion(PROM)could be improved by Acupotomy therapy significantly.(2)Compared with the normal group,the wet weight of rectus femoris was decreased significantly in model group(p<0.01).Compared with model group,the wet weight was increased in Electro-acupuncture group(p<0.05).Compared with normal group,the Max.girth of thigh was decreased significantly in model group(p<0.01).Compared with model group,the Max.girth of thigh was increased in Acupotomy group significantly.It was also found to have a certain improving effect on knee joint stride length and frequency especially in Electro-acupuncture group(p<0.01,p<0.05).(3)From morphology observation,repairing effects of Acupotomy and electro-acupuncture therapy on skeletal muscle was definte.Under the microscope,HE staining of the rectus femoris showed that both the Acupotomy and elctro-acupuncture therapies could effectively promoting the recovery of skeletal muscle fiber structure.The cross-sectional area of muscle fibers were extremely reducted(p<0.01)in Model group and also enlarged in Electronical-acupuncture group and Acupotomy group(p<0.01).(4)Under transmission electron microscope:skeletal muscle fibers arrangement in Electro-acupuncture appeared appeared more orderly than those in the model group as the Z line.Also in the Model group,the myofibrils were disordered,disappearance of local myofibrils,widened gaps,abnormal Z-line structures,large numbers of mitochondria,In the needle knife group,the myofibrils were arranged in order,the thickness was uniform,the sarcomere was clear,and the bright and dark bands were obvious.In the electroacupuncture group,the myofibrils were arranged disorderly,the thickness was more uniform,the sarcomeres were clearer,and the bright and dark bands were distinguishable.A small amount of Z lines were abnormally arranged,and the number of mitochondrial swellings slightly increased.(5)One week after model establishment,the left knee joints of KOArabbits in each group showed MRI findings:normal rabbits had normal soft tissues around the knee joints,normal articular surface clearances were normal,meniscal shadows were clear,and no vegetation was observed;the model group KOA rabbit knee joints.Peripheral soft tissue such as rectus femoris muscles and other obvious atrophy,high-density shadow in the left knee joint cavity,synovial hyperplasia changes,femoral articular surface proliferation and deformation,joint clearance was significantly narrower than the normal group joint space was significantly reduced There was even cartilage damage;atrophy of the soft tissue such as the rectus femoris muscle around the knee joint of the KOA rabbit in the needle-knife group was restored,which was more plump than the model group's rectus femoris,and no obvious growth was seen in the joint cavity.The knee gap is wider than the model group and is relatively even,but the high-density shadow,synovial hyperplasia,and the articular cartilage articular surface of the tibial plateau are less smooth in the left knee joint cavity;the soft tissue around the knee joint of the electroacupuncture group KOA is straight.The atrophy of the muscles was restored and was more full than the rectus femoris muscle in the model group.The high-density shadow(synovial hyperplasia)changes appeared in the left knee joint cavity and the degree was higher than that of the model group.Alleviation of defects in the articular surface of the femur resulted in lipoid hyperplasia of the articular surface of the tibia,which was significantly narrower than the normal joint space.(6)Immunohistochemical staining:No significant difference of 4EBP1 protein were found among groups(p<0.05).Compared with normal group,the expression of Akt,mTOR,4EBP1 and p70s6k protein was decreased significant in model group(p<0.01).Compared with model group,the expression of Akt,mTOR and p70s6k protein were increased significantly in both acupotomy and Electro-acupuncture group(p<0.01),and while the expression of p70s6k protein were increased(p<0.05)slightly in the Electro-acupuncuure group and increased significantly in the acupotomy group(p<0.01).(7)Western blot results:No significant difference of 4EBP1 protein were found among groups(p<0.05).Compared with normal group,the expression of Akt,mTOR and p70s6k protein was decreased significant in model group(p<0.01).Compared with model group,the expression of Akt,mTORprotein were increased significantly in acupotomy group(p<0.01),and while the expression of p70s6k protein were increased(p<0.05)slightly.Compared with model group,the expression of Akt was increased significantly in acupotomy group(p<0.01),and while the expression of p70s6k protein and mTORprotein were increased(p<0.05)slightly.(8)Rel time-PCR test results:Low level expressions of Akt mRNA,mTOR mRNA,and p70s6k mRNA were observed in rectus femoris damage in model group compared with normal group(p<0.01).Compared with model group,the expression of Akt mRNA was increased(p<0.05),and the expression of mTOR and p70s6k mRNA were increased significantly in acupotomy group.Compared with model group the expression of mTOR mRNA and p70s6k mRNA were both increased significantly in Electro-acupuncture group(p<0.01).However,there was no significant difference between groups(p>0.05)in the expression of 4EBP1 mRNA.Conclusion:(1)Knee joint behavior scores,measurement of passive joint motion,observation of femoral rectus by HE staining,ultrastructural changes of muscle fibers by transmission electron microscopy,measurement of thigh circumference,wet weight and wet weight ratio of quadriceps,and MRI It is proved that using the Videman left hind brake method to copy the KOA model will not only cause the muscles of the rectus femoris,the quadriceps around the knee,and even the entire thigh muscles to have damage and atrophy,but also cause the damage of the cartilage.Successful disuse KOA model.Through transmission electron microscopy,ultrastructural changes suggested that the KOA model caused damage to the rectus femoris muscle in rabbits.Both the needle and electroacupuncture groups promoted the regeneration of the rectus femoris.(2)The treatment methods of acupuncture and electro-acupuncture all have repair effects on the experimental rectus femoris muscle injury in experimental KOA rabbits.The cross-sectional area of muscle fibers is significantly increased after intervention of the needle-knife group,the passive motion of the joints,and the thigh circumference increase.Increased body weight,consistent with these indicators,showed an increase in muscle cells and an increase in the cross-sectional area of muscle fibers.Electro-acupuncture intervention showed an increase in wet weight of the quadriceps muscle,a significant increase in step size,and a significant increase in cadence.These measures combined together may indicate that muscle strength after electro-acupuncture intervention was improved,motor function and pain status Get restored.The difference in these indicators reflects that the mechanism of action of needle-knife and electro-acupuncture intervention is not the same.Although electro-acupuncture does not make muscle fibers thicker,but it makes the coordination of muscle movement to get the maximum recovery,but the needle knife can greatly restore the joint activity and increase the skeletal muscle cross-sectional area.(3)Knee-knife intervention can significantly increase the behavioral score of KOA model rabbits,increase the cross-sectional area of the rectus femoris fibers,increase the thigh circumference,and significantly increase body weight,while Akt,mTOR,and p70s6k The high expression of mRNA and protein both showed that the needle-knife intervention led to the activation of the Akt/mTOR pathway,thereby exerting a protective effect on the damaged atrophic skeletal muscle,thereby exerting a protective effect on the skeletal muscle.(4)Acupotomy intervention can effectively improve the clinical symptoms of KOA.The mechanism may be through the activation of Akt,mTOR and p70s6k expression,thereby making the Akt/mTOR signaling pathway play a role in promoting the increase and repair of skeletal muscles,activation of Akt/mTOR pathway It may be one of the mechanisms by which needle knives play a role in repairing skeletal muscle injury. |
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