Study On The Mechanism Of Osteoking On Myofascial Pain Syndrome Based On CAMP/PKA/PGC1α Signaling Pathway

Objective:The characteristics and mechanisms of the intervention of Osteoking in myofascial pain syndrome（MPS）are still unclear.This study will establish myofascial pain syndrome model to clarify the pharmacological effect of Osteoking in MPS,At the same time,we further explored the mechanism of Osteoking in the treatment of MPS through Systems biology prediction combined with holistic animal experimental validation methods.Methods:SD rats were randomly divided into normal group,model group,low,medium,and high dose groups of Osteoking(OK 0.66ml·kg^-1,1.31ml·kg^-1,2.63ml·kg^-1),and positive drug Celecoxib group(CE 21mg·kg^-1).The normal group rats did not receive any stimulation.The model group and medication group rats were established with chronic MPS models by hitting the left lower limb medial thigh muscle combined with exercise fatigue.During the modeling period,Osteoking was administered for intervention,Simultaneously Celecoxib was also administered in the same manner.SMALGO claw pressure analgesis to determine the tenderness threshold of the trigger point in MPS rats;The Von Frey acupuncture tactile measurement kit detects the mechanical pain sensitivity threshold of MPS rats;Acetone stimulation of the soles of rats to evaluate the response of MPS rats to acetone induced cold pain stimulation;At weeks 8 and 10 of the model,the spontaneous electrical state and convulsive response of the muscle tissue at the trigger point in MPS rats were measured using an electromyography device;Using Cat Walk gait analyzer to detect gait changes in MPS rats;HE staining was used to observe the changes of muscle histopathology morphology in the pain trigger points of MPS rats;The toxic side effects of the Osteoking were evaluated,According to the trend of body weight and the coefficient of dirty brain;By using network pharmacology methods,a"drug effector molecule-disease gene"interaction network is constructed to predict the key targets and related molecular mechanisms of the"energy metabolism inflammation immune imbalance network"of Osteoking-MPS;Using ELISA method to detect interleukin-1 in serum and muscle tissue of trigger pointsβ（IL-1β）、tumor necrosis factorα（TNF-α）Content/activity of substance P（SP）,bradykinase（BK）,Na-K-ATPase,Ca²⁺pump(Ca²⁺ATPase),calcium ion(Ca²⁺),lactate dehydrogenase（LDH）,c AMP,protein kinase A（PKA）,glutathione（GSH）,malondialdehyde（MDA）,superoxide dismutase（SOD）;Western blot detection of PKA、PGC-1α、TFAM protein expression level and IкBα、NF-κB p65 phosphorylation protein expression level of the muscle tissue at the trigger point in MPS rats;Immunofluorescence detected the intensity of positive expression of p-NF-κB p65of the muscle tissue at the trigger point in MPS rats.Results:（1）Compared with the normal group,in the 8 and 10 weeks,the MPS model group had abnormal spontaneous potential,and the positive rate of local convulsion response and EMG signal detection in the muscle tissue of the trigger point was 100%,indicating that the MPS model had been successfully established;Compared with the model group,at 10 weeks,the positive rates of electromyographic signal detection and local convulsive response in the medium and high-dose groups of Osteoking were significantly reduced（p<0.05）,indicating that intervention with Osteoking can alleviate spontaneous discharge and convulsive response in MPS rats.Compared with the normal group,the model group rats showed a significant decrease in the pressure pain threshold and mechanical pain threshold at the trigger point after 1-10 weeks of modeling,while the cold pain hypersensitivity score increased significantly after 4-10 weeks of modeling,and the differences were statistically significant（p<0.05,p<0.01,or p<0.001）;Compared with the model group,the pain threshold at the trigger point of MPS rats was significantly increased in the medium and high-dose groups of Osteoking after 4-10 weeks of administration,and the mechanical pain sensitivity threshold was significantly increased after 1-10 weeks of administration.The cold pain hypersensitivity score of MPS rats in the high-dose group of Osteoking was significantly reduced after 4-10 weeks of administration,and the differences were statistically significant（p<0.05,p<0.01,or p<0.001）.The gait analysis results showed that compared with the normal group,the model group rats had a decrease in the pressure of the modeled footprints when walking naturally through the channel,and the standing time,swaying time,and Duty Cycle of the rats were significantly increased.The Swing speed,maximum contact area,and maximum contact intensity were significantly reduced,and the differences were statistically significant（p<0.05）;Compared with the model group,all dose groups of Osteoking and the Celecoxib group could increase the foot print pressure of MPS rats.The Cat Walk gait parameter measurement results showed that the high dose group of Osteoking significantly reduced the standing time,swing time,and Duty Cycle,while the swing speed,maximum contact area,and maximum contact intensity were significantly increased,the differences were statistically significant（p<0.05）.The HE andresults showed that compared with the normal group,the model group rats showed contracted nodules and disordered arrangement of muscle fibers in the muscle tissue of the trigger point;Compared with the model group,each dose group of Osteoking can improve the pathological morphology of the muscle tissue at the trigger point of MPS rats and reduce the expression of Bax in the muscle tissue at the trigger point;The results of body weight and visceral brain coefficient suggest that the Osteoking has no significant toxic or side effects.（2）The network prediction results show that Osteoking acts as a key target for the"energy metabolism-inflammatory immune imbalance network"related to the occurrence and development of MPS,and may participate in the activation of c AMP/PKA/PGC1αsignaling pathway in MPS rats;Further mechanism validation was performed,The ELISA results showed that,compared with the normal group,the In the model group of rats,Na^+-K⁺ATPase,Ca²⁺ATPase,SOD activity,Ca²⁺、GSH、c AMP and PKA content were significantly decreased,LDH activity,MDA,IL-1β,TNF-α,SP and BK content were significantly increased,and the difference was statistically significant（p<0.05,p<0.01,or p<0.001）;Compared with the model group,the high-dose group of Osteoking showed a significant increase in Na-K-ATPase,Ca²⁺ATPase,SOD activity,Ca²⁺、GSH、c AMP and PKA content in the serum and muscle tissue of the trigger point,while the LDH activity and MDA content were significantly reduced.The content of IL-1β,TNF-α,BK,and SP in serum and the trigger point muscle tissue decreased significantly in the medium and high dose groups of Osteoking,and the differences were statistically significant（p<0.05,p<0.01,or p<0.001）.Among them,the high-dose group of Osteoking was the most significant,and the therapeutic effect was better than the positive drug celecoxib.Western blot results showed that compared with the normal group,In the model group,the expression levels of PKA,PGC 1αand TFAM in the muscle tissue of the trigger point reduced significantly（p<0.05,p<0.01）,and the expression levels of p-IκBα/IκBαand p-NF-κB p65/NF-κB p65 were significantly increased（p<0.001）;Compared with the model group,the expression levels of PKA,PGC-1αand TFAM protein in the muscle tissue of the trigger point in the medium and high-dose groups of Osteoking were significantly increased（p<0.05,p<0.01 or p<0.001）,the expression levels of p-IκBα/IκBα、p-NF-κB p65/NF-κB p65 protein in the medium and high-dose groups of Osteoking were significantly reduced（p<0.05,p<0.001）,The high dose groupof Osteoking had the best effect.Celecoxib group could increase the expression levels of PKA and PGC-1αin the muscle tissue of the trigger point（p<0.05）,and reduce the expression levels of p-IκBα/IκBαand p-NF-κB p65/NF-κB p65 protein（p<0.05,p<0.001）.The effect intensity was less than Osteoking of the medium and high dose group.The immunofluorescence results showed that compared with the normal group,Strength of the positive expression of the p-NF-κB p65 protein expression in the muscle tissue of the trigger point were significantly increased（p<0.001）;Compared with the model group,Osteoking of the high dose group significantly reduced Strength of positive p-NF-κB p65 protein expression in the muscle tissue of the trigger point（p<0.01）.Conclusions:Osteoking upregulates c AMP/PKA/PGC1αSignal pathway activates mitochondrial transcription factor TFAM,promotes mitochondrial energy metabolism of Muscle cell,Simultaneously lowering NF-κB signaling pathway inhibits the release of inflammatory factors and pain mediators,thereby promoting anti-inflammatory and analgesic effects,It can promote the repair of muscle fiber damage at Myofascial trigger point,improve abnormal myoelectric activity and twitching reaction,and effectively treat MPS without obvious toxic and side effects,The curative effect is superior to Celecoxib.The relevant research results provide experimental basis for expanding its clinical indications and guiding rational clinical medication.