恒古骨伤愈合剂对肌筋膜疼痛综合征大鼠的干预作用

Intervention of Osteoking in Rats with Myofascial Pain Syndrome

目的:明确恒古骨伤愈合剂对肌筋膜疼痛综合征(MPS)模型大鼠的干预作用,并从抗炎角度初步探索其缓解慢性疼痛的药理机制。方法:60只SD大鼠设立分组,分别为正常组、模型组、恒古骨伤愈合剂低、中、高剂量组(0.66、1.31、2.63 mL·kg^(-1)),塞来昔布组(21 mg·kg^(-1)),通过打击结合离心运动法建立MPS大鼠模型,造模同时灌胃给予恒古骨伤愈合剂和塞来昔布,SMALGO爪压力测痛仪检测大鼠激痛点压痛阈值,Von-Frey针和丙酮刺激法分别检测足底机械痛敏阈值和冷痛敏刺激反应;模型8周和10周,肌电图仪测定激痛点自发放电状态及抽搐反应;CatWalk步态分析仪检测大鼠步态变化;酶联免疫吸附测定法检测血清及激痛点中P物质(SP)、缓激肽(BK)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)的表达水平;蛋白免疫印迹法(Western blot)检测MPS大鼠激痛点中核转录因子-κB(NF-κB)抑制蛋白α(IκBα)、磷酸化(p)-IκBα、NF-κB p65、p-NF-κB p65的蛋白表达水平;免疫荧光法检测激痛点中p-NF-κB p65的阳性表达情况。结果:与正常组比较,模型组大鼠在8周和10周激痛点肌电信号和局部抽搐反应检测阳性率均为100%,激痛点压痛阈值、机械痛敏阈值明显降低,冷痛超敏反应评分明显升高,步态异常,血清和激动点IL-1β、TNF-α、SP、BK、p-IκBα、p-NF-κB p65蛋白表达水平和p-NF-κB p65入核阳性表达强度均显著升高(P<0.01);与模型组比较,恒古骨伤愈合剂中、高剂量组肌电信号检测和局部抽搐反应阳性率明显降低(P<0.05),恒古骨伤愈合剂中、高剂量组压痛阈值、机械痛敏阈值明显升高,恒古骨伤愈合剂高剂量组冷痛超敏反应评分显著降低(P<0.01),恒古骨伤愈合剂高剂量组站立时间、摇摆时间和步行周期明显升高,摇摆速度、最大接触面积、最大接触强度明显降低(P<0.05),且恒古骨伤愈合剂中、高剂量组p-IκBα/IκBα、p-NF-κB p65/NF-κB p65蛋白表达水平均明显降低(P<0.05,P<0.01),恒古骨伤愈合剂高剂量组p-NF-κB p65入核阳性表达强度显著降低(P<0.01)。结论:恒古骨伤愈合剂能有效缓解MPS的激痛点疼痛,改善步态,其机制与下调NF-κB p65炎症信号通路降低血液和激痛点组织中炎症因子和疼痛介质的表达有关。

Objective:To clarify the intervention effect of Osteoking(OK)in rats with myofascial pain syndrome(MPS)and preliminarily explore the pharmacological mechanism of OK in relieving chronic pain from the perspective of anti-inflammatory disease.Method:The 60 SD rats were divided into normal group,model group,low,medium,and high dose OK groups(0.66,1.31,2.63 mL·kg^(−1)),and positive celecoxib group(21 mg·kg^(−1)).The MPS rat model was established by beating combined with the centrifugal exercise method,and the OK and celecoxib were given at the same time.SMALGO paw pressure pain manometer detected the shock pain point tenderness threshold of rats,and the Von-Frey needle and acetone stimulation method detected the mechanical hyperalgesia threshold and cold hyperalgesia stimulation response respectively.Eight weeks and 10 weeks after modeling,the spontaneous discharge state and convulsion response of MPS rats were determined by electromyograph(EMG)instrument.The gait changes of MPS rats were detected using a CatWalk gait analyzer.The expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),substance P(SP),and bradykinin(BK)were measured by enzyme-linked immunosorbent assay(ELISA).The protein expression levels of nuclear transcription factor-κB(NF-κB)inhibiting proteinα(IκBα),phosphorylates(p)-IκBα,NF-κB p65,and p-NF-κB p65 were detected in MPS rats by Western blot.The positive expression of p-NF-κB p65 was detected by immunofluorescence.Result:Compared with the normal group,the model group shows 100%positive rates for EMG signal and local convulsions response at both the 8th and 10th weeks.The tenderness threshold and mechanical hyperalgesia threshold are significantly reduced.Cold hyperalgesia score is significantly increased,and gait is abnormal.The expression levels of serum and trigger points IL-1β,TNF-α,SP,BK,p-IκBα,and p-NF-κB p65,as well as the positive expression intensity of p-NF-κB p65 are significantly increased(P<0.01).Compared with the model group,the positive rate of EMG detection and local convulsion response is significantly reduced in the medium and high dose OK groups(P<0.05).The tenderness threshold and mechanical hyperalgesia threshold increase significantly in the medium and high dose OK groups,and the cold hyperalgesia score is significantly reduced in the high dose OK group(P<0.01).The standing time,swing time,and walking period are significantly increased.The swing speed,maximum contact area,and maximum contact intensity are significantly decreased in the high dose OK group(P<0.05).Moreover,the protein expression levels of p-IκBα/IκBαand p-NF-κB p65/NF-κB p65 are significantly reduced in the medium and high dose OK groups(P<0.05,P<0.01).The positive expression intensity of p-NF-κB p65 is significantly decreased in the high dose OK group(P<0.01).Conclusion:The mechanism of OK in relieving the pain in trigger points of MPS and improving gait abnormalities is related to the downregulation of the NF-κB p65 inflammatory signaling pathway to reduce the expression of inflammatory factors and pain mediators in blood and trigger point tissue.