| The mechanism of Tui-na on skeletal muscle disease is a hot spot in the field of Tui-na.Myofascial trigger points are one of the main causes of muscle tension pain.Activated myofascial trigger points can cause spontaneous and severe muscle pain,which is the main reason for patients to see a doctor.Modern medical research has found that local sarcomere contracture caused by ATP energy crisis is the key factor of its pathological mechanism.Mitochondrial dysfunction further leads to insufficient ATP production and aggravates the energy crisis.AMPK pathway inhibition is an important factor leading to pain point energy crisis and mitochondrial dysfunction.For the treatment of such diseases,Su Wen Tiao Jing Lun believes that Tui-na has the effect of “After pressing,the Qi is sufficient to warm it,so it will be refreshing without pain” pressing the method can warm the Qi,so it is fast but not painful".Based on this,this project takes the rat model of myofascial trigger points as the research object,and adopts the method of Pressing Manipulation to stimulate,mainly discussing:(1)the deactivation effect of Pressing Manipulation on myofascial trigger points;(2)The effect of pressing the method on the energy metabolism of the p myofascial trigger points;(3)The effect of pressing method on AMPK pathway related proteins in myofascial trigger points.Part OneObjective: By observying the influence of Pressing Manipulation on the local temperature,mechanical pain threshold,soft tissue tension,electromyography and muscle fiber morphology of myofascial trigger points,the deactivation effect of Pressing Manipulation is clarified.Methods: The rat model of MTrPs was established by blunt shock combined with centrifugal motion.36 SD rats were randomly divided into blank group,model group and Pressing group,with 12 rats in each group.The Pressing group was treated with Pressing Manipulation,7.5min each time,once every other day,for a total of 7 times.The model group and the blank group did not receive treatment.The general condition of rats was observed,threshold of pain was measured by von-frey,local soft tissue tension was measured by soft tissue tensiometer,electromyography was collected,and the changes of muscle fiber morphology and structure were observed by HE staining light microscope.Results:1.The rats in model group showed symptoms of mental depression,decreased activity,decreased diet and drinking water,yellow hair color and poor luster,and left hind limb weakness and claudication.In model group,the threshold of pain decreased,soft tissue tension increased,and the differences were statistically significant(P<0.05).Electromyogram of model group showed peak potential with increased amplitude,muscle fiber thickening,enlargement and nuclear inward migration under light microscope,and muscle fiber cross section area increased,the difference was statistically significant(P<0.05).2.The spirit of rats in Pressing group was improved,their food intake and water intake were improved,their hair gloss was improved,their left hind limb could walk Mechanical pain area increased,soft tissue tension decreased,the differences were statistically significant(P<0.05).The peak potential of electromyography disappeared,the morphology and structure of muscle fiber returned to normal under the light microscope,and the cross sectional area of muscle fiber decreased,the difference was statistically significant(P<0.05).Conclusion: The Pressing Manipulation can increase the mechanical pain threshold,improve the soft tissue tension,eliminate the spontaneous potential of the MTrPs,and promote muscle fiber repair.Part TwoObjective: To observe the effect of Pressing Manipulation on the energy metabolites,mitochondria and sensitizing substances of the myofascial trigger points,and explore the effect mechanism of Pressing Manipulation on myofascial trigger points.Methods: 48 SD rats were randomly divided into blank group,model group,Pressing group and lidocaine group,with 12 rats in each group.The rat model of MTrPs was established by blunt shock combined with centrifugal motion.The Pressing group was treated with Pressing Manipulaton,7.5min each time,once every other day,for a total of 7 times.The lidocaine group was treated with local injection of lidocaine at the MTrPs,once every 6 days,for 3 times in total.The model group and the blank group did not receive treatment.After treatment,ATP,glycogen,lactic acid and ATPase content in muscle were detected by high performance liquid method.The changes of local sensitization substances(CGRP,PGE2,SP and BK)were detected by ELISA.The ultrastructure of muscle fibers and the number,morphology and structure of mitochondria in MTrPs were observed by electron microscope.Results:1.The contents of ATP,muscle glycogen and ATPase in MTrPs of rats in model group decreased(P<0.05),while the contents of lactic acid,PGE2 and BK increased,and the differences were statistically significant(P<0.05);SP and CGRP contents only increased numerically,and the difference was not statistically significant(P>0.05).Under electron microscopy,the myofibrils were disordered,the light and dark bands were blurred,the Z-line was wavy,the hoof-knot tissue and endoplasmic reticulum were hyperplasia,and the distribution of mitochondria was irregular.The number of mitochondria was reduced(P<0.05),and the mitochondria crists were deformed or vacuolated,and the sarcoplasmic reticulum was blurred.2.The contents of ATP,muscle glycogen and ATPase increased in the Pressing group and lidocaine group(P<0.05),while the contents of lactic acid,CGRP,PGE2,SP and BK decreased,with statistically significant differences(P<0.05).Compared with lidocaine group,the content of ATP and muscle glycogen in the Pressing group increased(P<0.05),and the increase of muscle glycogen content was statistically significant(P<0.05),but the increase of ATP content was not statistically significant(P>0.05).Lactic acid content decreased,but there was no statistical significance(P>0.05).The content of PGE2 and bradykinin increased,and the difference was statistically significant(P<0.05).SP and CGRP contents only decreased numerically,and there was no statistical significance(P>0.05).Under electron microscope,the myofibrils in the Pressing group and the lidocaine group were similar to those in the blank group.The myofibrils were arranged in a more orderly manner,with a contrast of light and dark bands.The Z line was clear and continuous,and mitochondria were distributed between the myofibrils near the Z line.In the Pressing group,mitochondria gathered under the sarcoplasmic membrane,the number of which increased(P<0.05),and the shape and size were basically normal,with elongated rod-shaped and oval shape or fused shape.Only a few showed swelling or crest structure disorder,no obvious edema of cytoplasm,and the sarcoplasmic reticulum was clearly visible.Compared with the lidocaine group,the number of mitochondria was slightly increased numerically in the Pressing group,but there was no statistical significance(P>0.05).Conclusion: The Pressing Manipulation can increase ATP,muscle glycogen and ATPase content,improve mitochondrial function,remove lactic acid and sensitizer,and promote muscle fiber damage repair of MTRPs.The effect is similar to lidocaine,but it is stronger than lidocaine in improving tissue energy crisis of MTrPs.Part ThereObjective: To observe the effects of the Pressing Manipulation on AMPK pathway related proteins in MTrPs tissues,and whether AMPK inhibitors can inhibit the therapeutic effect of massage on MTrPs tissues and improve the energy metabolism of MTrPs tissues,and to explore the intracellular molecular biological mechanism of Pressing Manipulation.Methods: 60 SD rats were randomly divided into blank group,model group,Pressing group,Pressing+inhibitor group and Pressing+NS(normal saline)group with 12 rats in each group.The rat model of MTrPs was established by blunt shock combined with centrifugal motion.The Pressing group was treated with Pressing Manipulaton,7.5min each time,once every other day,for a total of 7 times.The Pressing+inhibitor group was intraperitoneally injected AMPK inhibitor Compound C,once every other day,for3 times at 0,2 and 4 days after modeling.The specific usage and dosage of Compound C were as follows: normal saline was mixed with a concentration of 10mg/ m L solution,and the injection dose was 25mg/kg.And then receving Pressing Manipulaton,the treatment time points were as follows: 1,3,5,7,9,11,13 days after modeling,7 times in total.Pressing + NS group: after modeling,saline was intraperitoneally injected for 3times at the same time point as the method +AMPK inhibitor group,with an injection dose of 2.5 m L /kg,and then MTrPs was receved Pressing Manipulaton for 7 times at the same time point as the Pressing+inhibitor group.The general condition of rats was observed,local temperature was measured by infrared thermal imaging,pain threshold was measured by von-frey,local soft tissue tension was measured by soft tissue tensiometer,electromyography was collected,and the changes of muscle fiber morphology and structure were observed by HE staining light microscope.The changes of ATP,lactic acid,muscle glycogen and ATPase in MTrPs tissues were observed by high performance liquid method,the changes of local sensitization substances(CGRP,PGE2,SP and BK)were detected by ELISA,and the ultrastructure of muscle fibers and the number,morphology and structure of mitochondria were observed by electron microscope.The changes of P-AMPK,PGC-1α and GLu T4 were detected by WB.Results:1.The performance of rats in the Pressing+inhibitor group was similar to that in the model group,with symptoms such as mental depression,decreased activity,decreased diet and water intake,poor hair luster and yellow color,and left hind limb weakness and claudication.Thed pain threshold decreased,soft tissue tension of myofascial trigger poits increased(P<0.05);The electromyogram showed the peak potential with increased amplitude.Under light microscope,muscle fibers were thickened,enlarged and nucleated inward,and the cross sectional area of muscle fibers increased(P<0.05).Under electron microscopy,the myofibrils were disordered,the light and dark bands were blurred,the Z-line was wavy,the hoof-knot tissue and endoplasmic reticulum were hyperplasia,and the distribution of mitochondria was irregular.The number of mitochondria was reduced and deformed(P<0.05),the fracture of mitochondrial crest was reduced or vacuolated,and the sarcoplasmic reticulum was blurred.2.The protein expression levels of p-AMPK,PGC-1α and GLu T4 and p-AMPK/AMPK ratio of MTrPs in model group were significantly decreased(P<0.05);Compared with model group,the protein expression levels of p-AMPK,PGC-1α and Glu T4 and the p-AMPK/AMPK ratio of Pressing group and Pressing+NS group were significantly increased(P<0.05);Compared with Pressing group,the contents of ATP,muscle glycogen and ATPase in Pressing+inhibitor group were decreased(P<0.05),while the content of lactic acid was increased(P<0.05),while the protein expression levels of p-AMPK,PGC-1α and GLu T4 and p-AMPK/AMPK ratio were significantly decreased(P<0.05).Conclusion: AMPK pathway was inhibited in local tissues of MTrPs model rats,and AMPK pathway related proteins could be activated by Pressing Manipulation to promote mitochondrial biogenesis and glucose uptake.AMPK inhibitors can inhibit the improvement of symptom of MTrPs rats by Pressing Manipulation,and inhibit the therapeutic effect of Pressing Manipulation on MTrPs and the improvement of tissue energy crisis in MTrPs rats,suggesting that the cellular molecular biological mechanism of Pressing Manipulation is related to the activation of AMPK pathway. |
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