Study On The Mechanism Of Deactivation Effect Of Acupressure On Trigger Points Based On The Regulation Of Intracellular Calcium By Ry

Objective:Myofascial trigger points（MTr Ps）are points of excessive stress in skeletal muscle,which manifest on palpation as highly sensitive nodules in the muscle tension zone,and are in essence abnormal contractions of muscle nodules.Pressing intervention can relieve muscle tension,spasm or contracture,and exert the effect of muscle relaxation and release.During muscle contraction and diastole,sarcoplasmic reticulum Ry R and related regulatory proteins have important roles in the regulation of intracellular calcium ions.In this study,we mainly investigated:（1）the deactivating effects of different sites of pressing intervention on MTr Ps;（2）the deactivating effects of different strengths of pressing intervention on MTr Ps;（3）the effects of pressing intervention on intracellular calcium ion concentration and calcium regulatory proteins in myocytes.Methods:Study 1:Sixty SPF-rated male SD rats were randomly divided into a blank group of 10 rats and 50 rats participating in modeling,and the MTr Ps model was established by blunt strikes combined with centrifugal exercise,with the striking position being the left medial thigh muscle.After successful modeling,those who met the evaluation criteria were randomly divided into model group,local press group,contralateral press group and distal press group,10 rats in each group.The blank group and the model group were not intervened,and the rest of the press groups were intervened with the homemade press stimulator for 14 d.The electrophysiological changes of MTr Ps were recorded by electromyography before and after the intervention,and the changes of soft tissue tension of MTr Ps were detected by soft tissue tonometry,and the changes of local mechanical pain threshold of MTr Ps were detected by mechanical pressure pain threshold tester.At the end of the intervention,the left medial thigh muscle or MTr Ps tissue was taken to observe the pathological morphology by HE staining,and the SP and CGRP contents were measured by immunohistochemistry and ELISA.Study 2:Sixty SPF-rated male SD rats were randomly divided into a blank group of 10rats and 50 rats participating in modeling,and the MTr Ps model was established by blunt strikes combined with centrifugal exercise,with the striking position being the left medial thigh muscle.After successful modeling,those who met the evaluation criteria were randomly divided into model group,light press group,medium press group and heavy press group,10 rats in each group.The blank group and the model group were not intervened,and the remaining press groups were intervened with homemade press stimulators for 14 d.Electrophysiological changes of MTr Ps were recorded by electromyography before and after the intervention,soft tissue tension changes of MTr Ps were detected by soft tissue tonometry,and local mechanical pain threshold changes of MTr Ps were detected by mechanical pressure pain threshold tester.At the end of the intervention,the tissue of the left medial thigh muscle or MTr Ps was taken and the pathological morphology was observed by HE staining,and the levels of COX-2,PGE2 and BK were detected by ELISA.Study 3:Sixty SPF-rated male SD rats were randomly divided into a blank group of 10rats and 50 rats participating in modeling,and the MTr Ps model was established by blunt strikes combined with centrifugal exercise,with the striking position being the left medial thigh muscle.After successful modeling,those who met the evaluation criteria were randomly divided into model group,press group,press+agonist group and press+solvent group,10 rats in each group.The blank group and the model group were not intervened,and the remaining three groups were intervened with homemade pressor stimulator for 14 d.The press+agonist group was injected with PKA agonist 8-Bromo-c AMP solution,and the press+solvent group was injected with equal amount of saline.Electrophysiological changes of MTr Ps were recorded by electromyography,soft tissue tension changes of MTr Ps were detected by soft tissue tonometry,and mechanical pain threshold changes were detected by mechanical pressure pain threshold tonometry before and after the intervention.At the end of the intervention,the left medial thigh muscle or MTr Ps tissue was taken to observe the pathological morphology by HE staining,P-Ry R1 expression by immunohistochemistry,Ca²⁺-ATPase and c AMP content by ELISA,PKA,P-Ry R1,FKBP12,PDE4D and PP1expression by WB,and intracellular Ca²⁺concentration by fluorescence staining.Results:Study 1:Compared with the blank group,the model group had lower mechanical pain thresholds and increased local SP and CGRP content（P<0.05）,indicating an increase in pain-causing substances and significant punctate pressure pain;soft tissue tension was elevated（P<0.05）,indicating higher local tissue stiffness;HE staining microscopically showed that the increased volume myocytes might be contracted nodules and some of the significantly reduced volume myocytes might be atrophied or stretched In addition,connective tissue hyperplasia and inward displacement of myocyte nuclei were also found;electromyography revealed the presence of spontaneous potentials,and the frequency and amplitude were significantly higher than those of the blank group（P<0.05）,suggesting the presence of spontaneous electrical activity.The above indicates the formation of contractile nodules,significant pressure pain,increased local tissue hardness,and the presence of spontaneous electrical activity after MTr Ps modeling.Compared with the model group,pressing intervention at three different sites could increase mechanical pain thresholds（P<0.05）and decrease SP and CGRP content,soft tissue tension,and spontaneous electrical potential frequency amplitude（P<0.05）,suggesting that they all could cause MTr Ps"punctate pressure pain","skeletal muscle tension bands"and"spontaneous electrical activity",i.e.,they could deactivate MTr Ps.The differences in mechanical pain threshold and soft tissue tension before and after intervention were greater in the local press group than in the contralateral press group and the distal press group（P<0.05）,and the SP and CGRP content and spontaneous potential frequency amplitude were smaller than in the contralateral press group and the distal press group（P<0.05）,and the morphological improvement was significant.The difference between the mechanical pain threshold and soft tissue tension before and after intervention in the contralateral press group and the distal press group was not statistically significant（P>0.05）,and the SP,CGRP content,and spontaneous potential frequency amplitude were smaller than those in the distal press group（P<0.05）,which suggested that the deactivation effect of MTr Ps local press stimulation>MTr Ps contralateral limb press stimulation>Taixi point area press stimulation.Study 2:Compared with the blank group,the model group showed lower mechanical pain thresholds（P<0.05）and increased levels of BK,COX-2 and PGE2（P<0.05）,suggesting increased pain-causing substances and significant pressure pain in the model group;soft tissue tension was elevated（P<0.05）,indicating higher local tissue stiffness;morphological observation of HE staining showed that the increased volume myocytes may be contracted nodules,and some The morphological observation of HE staining showed that the enlarged myocytes might be contracted nodules and some of the significantly reduced myocytes might be atrophied or elongated myocytes,in addition to connective tissue hyperplasia and inward displacement of myocyte nuclei.The above indicates the formation of contractile nodules,significant pressure pain,increased local tissue hardness,and the presence of spontaneous electrical activity after modeling.Compared with the model group,mechanical pain thresholds increased（P<0.05）and BK,COX-2 and PGE2 levels decreased（P<0.05）in the medium-press and heavy-press groups,indicating that both could relieve pain;soft tissue tension decreased（P<0.05）,indicating that both could reduce local tissue stiffness of MTr Ps;morphological HE staining showed partial recovery of cell morphology and partial reduction of connective tissue hyperplasia The decrease in the frequency and amplitude of spontaneous potentials in electromyography（P<0.05）indicated that both of them could reduce the spontaneous physiological activity.There was no significant difference between the above indexes in the light press group and the model group（P>0.05）.The above suggests that both medium and heavy force pressing intervention could deactivate MTr Ps,while light force pressing intervention had no such effect.The difference in mechanical pain threshold and soft tissue tension before and after intervention was greater in the heavy pressing group than in the medium pressing group（P<0.05）,and the levels of BK,COX-2 and PGE2 were smaller than in the medium press group（P<0.05）,and the morphological observations were better than in the medium press group,and the frequency and amplitude of spontaneous potentials were smaller than in the medium press group（P<0.05）,suggesting that the deactivation effect of heavy force pressing intervention was greater than that of medium force pressing intervention.Study 3:Compared with the blank group,there were characteristic changes in pathomorphology in the model group,with frequent spontaneous potentials visible on electromyography with significantly higher frequency and amplitude（P<0.05）,increased soft tissue tension（P<0.05）,and decreased mechanical pain threshold（P<0.05）,suggesting contractile nodule formation,significant pressure pain,increased tissue stiffness,and significant spontaneous electrical activity in the model group.The mean intensity of calcium staining fluorescence,c AMP,PKA,and P-Ry R1 content were increased（P<0.05）,PDE4D,FKBP12 and Ca²⁺-ATPase content were decreased（P<0.05）,and PP1 content was not significantly altered（P>0.05）,suggesting that It is possible that a decrease in local PDE4D content increases c AMP-dependent PKA phosphorylation of Ry R1,and no significant change in PP1 dephosphorylation leads to an increase in P-Ry R1 and a decrease in FKBP12 leading to an increase in calcium release,while a decrease in Ca²⁺-ATPase expression leads to a decrease in calcium recycling,resulting in an increase in intracellular calcium ion concentration.Compared with the model group,the press group showed improved pathomorphology,decreased EMG frequency and amplitude,soft tissue tension（P<0.05）,and increased mechanical pain thresholds（P<0.05）,suggesting a decrease in contractile nodal stiffness,relief of pressure pain,and diminished spontaneous electrical activity in the pressor group.The mean intensity of calcium staining fluorescence,c AMP,PKA,and P-Ry R1 content decreased（P<0.05）,and PDE4D,FKBP12 and Ca²⁺-ATPase content increased（P<0.05）while PP1 expression was not significantly altered（P>0.05）;suggesting that possibly c AMP-dependent PKA phosphorylation of Ry R1 was diminished and PP1 dephosphorylation was not significantly altered after the pressor intervention.It is suggested that PDE4D expression increased and c AMP content decreased after the intervention according to the method,c AMP-dependent PKA phosphorylation of Ry R1 was weakened,and PP1dephosphorylation was not significantly changed,prompting a decrease in P-Ry R1,an increase in FKBP12 and thus a decrease in calcium release,as well as an increase in Ca2+-ATPase expression leading to an increase in calcium recycling and ultimately a decrease in intracellular calcium ion concentration.The cell morphology and arrangement of the press+agonist group were not significantly different from that of the model group.Compared with the by-product group,soft tissue tension,EMG frequency and amplitude increased,mechanical pain threshold decreased in the press+agonist group（P<0.05）,calcium staining fluorescence mean intensity,c AMP,PKA,P-Ry R1 content increased（P<0.05）,FKBP12 content decreased（P<0.05）,PDE4D,Ca²⁺-ATPase and PP1 content did not change significantly（P<0.05）,and the above indexes were not significantly changed in the pressor+solvent group（P>0.05）.It was suggested that PKA agonist could inhibit the regulatory effect of pressing intervention on the above indexes,while its solvent saline had no such inhibitory effect.The above suggests that local pressor stimulation of MTr Ps may relieve the contractile nodules of MTr Ps and relieve pain by downregulating c AMP,PKA,and P-Ry R1 and upregulating PDE4D,FKBP12,and Ca2+-ATPase,which in turn induce a decrease in intracellular[Ca²⁺]i.Conclusion:1.MTr Ps local,Taixi acupoint area and MTr Ps contralateral limb area pressing intervention can all deactivate MTr Ps.With this deactivation effect,MTr Ps pressing intervention is better than MTr Ps contralateral limb area pressing intervention,and MTr Ps contralateral limb area pressing intervention is better than Taixi acupoint area pressing intervention.2.Within a certain range of force values,both medium force and heavy force MTr Ps local pressing intervention can deactivate MTr Ps,and the effect of heavy force MTr Ps local pressing intervention on MTr Ps deactivation is better than that of medium force MTr Ps local pressing intervention.The light force MTr Ps local pressing intervention could not deactivate MTr Ps.3.Local pressing intervention of MTr Ps may relieve the contractile nodules and pain of MTr Ps by downregulating c AMP,PKA,and P-Ry R1 levels and upregulating PDE4D,FKBP12 and Ca²⁺-ATPase levels to induce a decrease in intracellular[Ca²⁺]i.