基于cAMP/PKA/P-RyR1与胞内钙离子探讨按法干预大鼠肌筋膜触发点的机制

doi:10.12114/j.issn.1007-9572.2024.0054

中国全科医学 ›› 2026, Vol. 29 ›› Issue (18): 2513-2522.DOI: 10.12114/j.issn.1007-9572.2024.0054

• 论著 • 上一篇

基于cAMP/PKA/P-RyR1与胞内钙离子探讨按法干预大鼠肌筋膜触发点的机制

蒋全睿¹^,², 冯祥², 刘丹², 李桃², 杨舟², 李江山², 刘小卫², 李武²^,^*()

1．530001　广西壮族自治区南宁市，广西中医药大学针灸推拿学院
2．410208　湖南省长沙市，湖南中医药大学针灸推拿与康复学院

收稿日期:2025-02-19 修回日期:2025-10-17 出版日期:2026-06-20 发布日期:2026-05-21
通讯作者: 李武
作者贡献：
蒋全睿负责实验实施、论文撰写；蒋全睿、冯祥负责实验数据分析处理；蒋全睿、冯祥、刘丹、李桃负责指标检测；杨舟、刘小卫、李武负责数据和文章的校对；李江山、刘小卫、李武负责实验设计。
基金资助:
国家自然科学基金资助项目(82174526，82274676，82374613); 湖南省自然科学基金资助项目(2023JJ30458); 湖南省科技创新计划项目(2022RC1221)

Effects of Pressing Intervention on Myofascial Trigger Points in Rats Based on cAMP/PKA/P-RyR1 and Intracellular Ca²⁺ Content

JIANG Quanrui¹^,², FENG Xiang², LIU Dan², LI Tao², YANG Zhou², LI Jiangshan², LIU Xiaowei², LI Wu²^,^*()

1. College of Acupuncture & Moxibustion and Tui-Na, Guangxi University of Chinese Medicine, Naning 530001, China
2. College of Acupuncture-Moxibustion, Tuina and Rehabilitation, Hunan University of Chinese Medicine, Changsha 410208, China

Received:2025-02-19 Revised:2025-10-17 Published:2026-06-20 Online:2026-05-21
Contact: LI Wu

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摘要/Abstract

摘要： 背景按法干预触发点是公认有效的缓解疼痛的治疗方法，这体现了中医推拿的舒筋止痛作用，但是目前其作用机制尚未完全阐明。目的探讨按法对肌筋膜触发点的作用是否与环磷酸腺苷（cAMP）/蛋白激酶A（PKA）/雷诺丁受体1（RyR1）对胞内Ca2+的调节有关。方法将60只SD大鼠按照随机数字表法分为无需造模的空白组大鼠10只和参与触发点造模的大鼠50只，造模大鼠采用钝性击打造模，造模后将符合模型评价标准者随机分配入模型组、按法组、按法+激动剂组与按法+溶剂组。按法组采用按法刺激仪器干预，按法+激动剂组通过腹腔注射8-Bromo-cAMP溶液20 mg/kg，按法+溶剂组注射等量0.9%氯化钠溶液。检测各组大鼠机械痛阈值、软组织张力、肌电图检测。末次肌电图检测结束后麻醉并处死大鼠，收集左侧大腿内侧肌的肌肉紧张带或结节区域，酶联免疫吸附试验检测cAMP含量，蛋白质印迹法检测PKA、P-RyR1和12kDa FK506结合蛋白（FKBP12）蛋白表达，免疫组化法检测磷酸化雷诺丁受体1（P-RyR1），荧光染色法检测胞内Ca2+浓度。结果最终纳入造模成功大鼠40只，模型组、按法组、按法+激动剂组和按法+溶剂组各10只。干预前模型组、按法组、按法+激动剂组和按法+溶剂组机械痛阈值低于空白组（P<0.05），干预后模型组、按法+激动剂组机械痛阈值低于空白组，按法组、按法+激动剂组和按法+溶剂组机械痛阈值高于模型组，按法+激动剂组机械痛阈值低于按法组、按法+溶剂组（P<0.05）。干预前后组内比较结果显示，干预后按法组、按法+激动剂组、按法+溶剂组机械痛阈值高于干预前（P<0.05）。干预前模型组、按法组、按法+激动剂组和按法+溶剂组软组织张力D0.2低于空白组（P<0.05），干预后模型组、按法+激动剂组软组织张力D0.2低于空白组（P<0.05），按法组、按法+激动剂组、按法+溶剂组软组织张力D0.2高于模型组（P<0.05），按法+激动剂组、按法+溶剂组软组织张力D0.2高于按法+激动剂组（P<0.05）。干预前后组内比较结果显示，干预后按法组、按法+溶剂组软组织张力D0.2高于干预前（P<0.05）。肌电图检测结果显示，模型组、按法组、按法+激动剂组、按法+溶剂组肌电图振幅与频率均高于空白组（P<0.05），按法组、按法+激动剂组、按法+溶剂组肌电图振幅与频率均低于模型组（P<0.05），按法+激动剂组肌电图振幅与频率均高于按法组、按法+溶剂组（P<0.05）。酶联免疫吸附试验结果显示，模型组、按法+激动剂组cAMP高于空白组（P<0.05），按法组、按法+激动剂组、按法+溶剂组cAMP低于模型组（P<0.05），按法+激动剂组cAMP高于按法组、按法+溶剂组（P<0.05）。蛋白质印迹法结果显示，模型组、按法+激动剂组PKA、P-RyR1和FKBP12高于空白组（P<0.05），按法组、按法+溶剂组PKA、P-RyR1和FKBP12低于模型组（P<0.05），按法+激动剂组PKA、P-RyR1和FKBP12高于按法组、按法+溶剂组（P<0.05）。免疫组化染色结果显示，模型组、按法+激动剂组P-RyR1 IOD高于空白组（P<0.05），按法组、按法+溶剂组P-RyR1 IOD低于模型组（P<0.05），按法+激动剂组P-RyR1 IOD高于按法组、按法+溶剂组（P<0.05）。免疫荧光检测结果显示，模型组、按法组、按法+激动剂组、按法+溶剂组平均荧光强度高于空白组（P<0.05），按法组、按法+激动剂组、按法+溶剂组平均荧光强度低于模型组（P<0.05），按法+激动剂组平均荧光强度高于按法组、按法+溶剂组（P<0.05）。结论触发点局部按法干预可能通过抑制cAMP/PKA/P-RyR1，促使胞内Ca2+浓度下降，从而舒展异常收缩肌节缓解疼痛。

关键词: 肌筋膜疼痛综合征, 肌筋膜扳机点痛, 推拿疗法, 环磷酸腺苷/蛋白激酶A/雷诺丁受体1信号通路

Abstract:

Background

Pressure manipulation at trigger points is a recognized and effective therapeutic approach for pain relief, reflecting the muscle-relaxing and analgesic effects of traditional Chinese tuina. However, its underlying mechanism of action has not yet been fully elucidated.

Objective

To investigate whether the effect of pressure manipulation on myofascial trigger points is associated with the regulation of intracellular Ca²⁺ by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/ryanodine receptor 1 (RyR1) pathway.

Methods

Sixty Sprague-Dawley rats were assigned, according to a random number table, to a blank group not subjected to modeling (n=10) and a modeling cohort subjected to trigger-point modeling (n=50). Rats in the modeling cohort underwent blunt-strike modeling. After modeling, rats that met the model evaluation criteria were randomly allocated to the model group, pressure manipulation group, pressure manipulation+agonist group, and pressure manipulation+vehicle group. The pressure manipulation group received intervention using a pressure manipulation stimulation device. The pressure manipulation+agonist group received intraperitoneal injection of 8-Bromo-cAMP solution at 20 mg/kg, whereas the pressure manipulation+vehicle group received an equivalent volume of 0.9% sodium chloride solution. Mechanical pain threshold, soft-tissue tension, and electromyography were assessed in each group. After the final electromyographic assessment, the rats were anesthetized and euthanized. The taut bands or nodular regions of the left medial thigh muscle were collected. cAMP content was measured using enzyme-linked immunosorbent assay. Protein expression levels of PKA, phosphorylated RyR1 (P-RyR1), and 12-kDa FK506-binding protein (FKBP12) were determined by Western blotting. P-RyR1 was detected by immunohistochemistry, and intracellular Ca²⁺ concentration was assessed by fluorescence staining.

Results

A total of 40 successfully modeled rats were ultimately included, with 10 rats each in the model group, pressure manipulation group, pressure manipulation+agonist group, and pressure manipulation+vehicle group. Before intervention, the mechanical pain thresholds in the model group, pressure manipulation group, pressure manipulation+agonist group, and pressure manipulation+vehicle group were lower than those in the blank group (P<0.05). After intervention, the mechanical pain thresholds in the model group and pressure manipulation+agonist group remained lower than those in the blank group; the thresholds in the pressure manipulation group, pressure manipulation+agonist group, and pressure manipulation+vehicle group were higher than those in the model group; and the threshold in the pressure manipulation+agonist group was lower than those in the pressure manipulation group and pressure manipulation+vehicle group (P<0.05). Within-group comparisons before and after intervention showed that the mechanical pain thresholds in the pressure manipulation group, pressure manipulation+agonist group, and pressure manipulation+vehicle group were higher after intervention than before intervention (P<0.05).

Conclusion

Local manual stimulation of trigger points may alleviate pain through the downregulation of intracellular Ca²⁺ concentrations mediated by inhibition of the cAMP/PKA/P-RyR1 pathway, resulting in the relaxation of abnormally contracted muscle nodules.

Key words: Myofascial pain syndromes, Myofascial trigger point pain, Tui Na therapy, cAMP/PKA/P-RyR1 pathway

蒋全睿,冯祥,刘丹,等. 基于cAMP/PKA/P-RyR1与胞内钙离子探讨按法干预大鼠肌筋膜触发点的机制[J]. 中国全科医学, 2026, 29(18): 2513-2522. DOI: 10.12114/j.issn.1007-9572.2024.0054.
JIANG Quanrui,FENG Xiang,LIU Dan, et al. Effects of Pressing Intervention on Myofascial Trigger Points in Rats Based on cAMP/PKA/P-RyR1 and Intracellular Ca²⁺ Content[J]. Chinese General Practice, 2026, 29(18): 2513-2522. DOI: 10.12114/j.issn.1007-9572.2024.0054.

图/表 11

表1 各组大鼠干预前后触发点局部机械痛阈值比较（±s，N）

Table 1 Comparison of trigger point local mechanical pain threshold before and after intervention in each group

组别	只数	干预前	干预后	t_配对值	P值
空白组	10	29.1±1.4	29.8±2.0	1.041	0.325
模型组	10	12.9±1.0^a	13.4±2.9^a	0.706	0.498
按法组	10	13.3±1.4^a	28.2±2.7^b	19.869	<0.001
按法+激动剂组	10	13.4±1.5^a	20.6±4.2^abc	7.226	<0.001
按法+溶剂组	10	12.6±1.6^a	27.9±2.6^bd	21.547	<0.001
F值		261.413	54.044
P值		<0.001	<0.001

表2 各组大鼠干预前后触发点软组织张力D0.2比较（±s，mm）

Table 2 Comparison of soft tissue tension D0.2 at trigger point in each group before and after intervention

组别	只数	干预前	干预后	t_配对值	P值
空白组	10	3.089±0.166	3.096±0.105	0.120	0.907
模型组	10	2.564±0.130^a	2.570±0.119^a	0.190	0.854
按法组	10	2.565±0.118^a	3.135±0.139^b	10.575	<0.001
按法+激动剂组	10	2.630±0.127^a	2.762±0.172^abc	7.226	<0.001
按法+溶剂组	10	2.590±0.101^a	3.079±0.187^bd	8.111	<0.001
F值		30.109	28.580
P值		<0.001	<0.001

图1 各组大鼠触发点组织肌电图

Figure 1 Electromyography of trigger point tissue of rats in each group

表3 各组大鼠触发点组织肌电图振幅与频率比较（±s）

Table 3 Comparison of amplitude and frequency of electromyography of trigger point tissues in each group

组别	只数	振幅（μV）	频率（次/min）
空白组	10	0	0
模型组	10	143.4±34.8^a	145.6±20.4^a
按法组	10	39.2±15.4^ab	9.3±3.7^ab
按法+激动剂组	10	96.0±18.5^abc	101.9±27.9^abc
按法+溶剂组	10	44.0±23.1^abd	7.6±5.1^abd
F值		66.855	179.632
P值		<0.001	<0.001

表4 各组大鼠触发点组织cAMP含量比较（±s，nmol/L）

Table 4 Comparison of cAMP content in trigger point tissues of rats in each group

组别	只数	cAMP
空白组	8	6.36±0.81
模型组	8	13.01±0.66^a
按法组	8	7.37±0.55^b
按法+激动剂组	8	10.58±1.67^abc
按法+溶剂组	8	7.07±0.72^bd
F值		67.383
P值		<0.001

图2 各组大鼠触发点组织PKA、P-RyR1、FKBP12蛋白条带图注：a为PKA，b为P-RyR1，c为FKBP12；A为空白组，B为模型组，C为按法组，D为按法+激动剂组，E为按法+溶剂组。

Table 2 Protein bands of PKA，P-RyR1，FKBP12 in trigger point tissue of rats in each group

表5 各组大鼠触发点组织PKA、P-RyR1和FKBP12相对表达量比较（±s）

Table 5 Comparison of the relative expression levels of PKA，P-RyR1 and FKBP12 in trigger point tissues of rats in each group

组别	只数	PKA	P-RyR1	FKBP12
空白组	5	0.367±0.134	0.229±0.068	0.516±0.124
模型组	5	0.829±0.198^a	0.851±0.217^a	0.244±0.074^a
按法组	5	0.452±0.183^b	0.285±0.117^b	0.588±0.155^b
按法+激动剂组	5	0.744±0.062^ac	0.618±0.188^ac	0.327±0.065^ac
按法+溶剂组	5	0.429±0.118^bd	0.314±0.190^bd	0.553±0.092^bd
F值		9.918	12.908	9.999
P值		<0.001	<0.001	<0.001

图3 各组大鼠触发点组织P-RyR1免疫组化染色图（×200）

Figure 3 P-RyR1 immunohistochemical staining of trigger point tissue of rats in each group

表6 各组大鼠触发点组织P-RyR1免疫组化积分光密度值比较（±s）

Table 6 Comparison of P-RyR1 immunohistochemical integral optical density in trigger point tissues of rats in each group

组别	只数	IOD
空白组	3	12 555±2 914
模型组	3	76 405±7 740^a
按法组	3	22 018±4 679^b
按法+激动剂组	3	65 911±11 756^ac
按法+溶剂组	3	23 607±5 730^bd
F值		47.960
P值		<0.001

图4 各组大鼠触发点组织肌细胞钙离子荧光染色图（×400）

Figure 4 P-RyR1 immunohistochemical staining of trigger point tissue of rats in each group

表7 各组大鼠触发点组织肌细胞钙离子平均荧光强度（±s）

Table 7 The average fluorescence intensity of calcium ions in muscle cells at trigger point in each group

组别	只数	平均荧光强度
空白组	8	30.27±3.30
模型组	8	106.90±4.39^a
按法组	8	53.53±11.90^ab
按法+激动剂组	8	83.62±11.58^abc
按法+溶剂组	8	57.87±8.35^abd
F值		92.594
P值		<0.001

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基于cAMP/PKA/P-RyR1与胞内钙离子探讨按法干预大鼠肌筋膜触发点的机制

Effects of Pressing Intervention on Myofascial Trigger Points in Rats Based on cAMP/PKA/P-RyR1 and Intracellular Ca²⁺ Content

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基于cAMP/PKA/P-RyR1与胞内钙离子探讨按法干预大鼠肌筋膜触发点的机制

Effects of Pressing Intervention on Myofascial Trigger Points in Rats Based on cAMP/PKA/P-RyR1 and Intracellular Ca2+ Content

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Effects of Pressing Intervention on Myofascial Trigger Points in Rats Based on cAMP/PKA/P-RyR1 and Intracellular Ca²⁺ Content