The peripheral PI3K/AKT/MLC signalling pathway alleviates myofascial pain in rats by inhibiting abnormal contraction at myofascial trigger points

Abstract

Background:

Myofascial trigger points (MTrPs) for abnormal skeletal muscle contraction are the cause of myofascial pain. The G protein-coupled receptor family and tyrosine kinase receptor family regulate the contraction of vascular smooth muscle through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Phosphorylated myosin light chain (p-MLC) is associated with skeletal muscle contraction. The aim of the current study was to explore the effect and mechanism of the PI3K/AKT/MLC signalling pathway on myofascial pain in rats.

Methods:

A rat model of myofascial pain was established by a blunt strike to the gastrocnemius muscle combined with centrifugal exercise for 8 weeks, followed by recovery for 4 weeks. Different concentrations of the PI3K inhibitor LY294002 (0.01, 0.1, or 1 mg/ml) were subsequently injected into the MTrPs of rats with myofascial pain to observe the effects on the mechanical tenderness threshold at the MTrPs.

Results:

LY294002 (0.1 mg/ml) inhibited myofascial pain at 0.5, 1 and 2 h after injection, and LY294002 (1 mg/ml) inhibited myofascial pain at 0.5, 1, 2 and 4 h after injection. The expression of PI3K increased on the enlarged muscle fibre membrane at MTrPs. LY294002 (1 mg/ml) inhibited the expression of PI3K, p-AKT, and p-MLC and the abnormal contraction of muscle fibres at MTrPs and alleviated nerve fibre compression at MTrPs. Moreover, LY294002 inhibited the expression of Fos in the spinal dorsal horn of rats with myofascial pain.

Conclusions:

These findings suggested that the increased expression of PI3K/p-AKT/p-MLC was related to myofascial pain in rats and that the PI3K inhibitor LY294002 might alleviate myofascial pain in rats by inhibiting abnormal contraction at MTrPs.

Introduction

Myofascial pain syndrome (MPS) involves autonomic, sensory, and motor nervous system dysfunction caused by myofascial trigger points (MTrPs). ¹ Patients with myofascial pain often develop comorbid depressive symptoms, which makes pain symptoms more complicated and refractory. ² MTrPs are abnormal contraction nodules in taut bands (TBs) of muscles during palpation and are highly sensitive to pain. ³ MTrPs contain nociceptors that can cause pain and produce peripheral and central sensitization. ⁴ Although the correlation between myofascial pain and trigger points is still controversial, trigger point-induced myofascial pain is still the most widely accepted theory, and the formation of trigger points may be related to abnormal skeletal muscle contraction.^5,6 Phosphorylated myosin light chain (p-MLC) is associated with skeletal muscle contraction, and the level of p-MLC in muscle fibres at trigger points is increased. ⁷

C-fos is a rapid response gene that is quickly expressed when stimulated. The Fos protein is an expression product of c-fos that regulates cell growth, differentiation, and apoptosis. The expression of Fos increases in nerve cells after pain stimulation, and the number of Fos protein-positive neurons is proportional to the intensity of pain stimulation. ⁸ The expression of Fos in the spinal dorsal horn is related to the development of pain. ⁹ Myofascial pain increases Fos expression in the spinal dorsal horns of rats. ¹⁰

The G protein-coupled receptor family and tyrosine kinase receptor family regulate vascular tension and vascular smooth muscle contraction through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. ¹¹ PI3K induces the contraction of mouse vascular smooth muscle and regulates blood pressure. ¹² PI3K inhibitors have been shown to inhibit the contraction of rat vascular smooth muscles.^13,14 A PI3K inhibitor (LY294002) inhibits vascular smooth muscle contraction by inhibiting calcium ion channels, reducing calcium ion influx. ¹⁵ LY294002 inhibits the contraction of colonic smooth muscle in rats and alleviates constipation. ¹⁶ PI3K is associated with the contraction of mouse bronchial smooth muscle, and inhibiting the PI3K/AKT pathway can alleviate asthma attacks. ¹⁷ We hypothesize that PI3K may cause abnormal contraction of skeletal muscle fibres, leading to myofascial pain. To test our hypothesis, we established a rat model of myofascial pain and observed the effects of different concentrations of the PI3K inhibitor LY294002 on myofascial pain.

Methods

Experimental animals

The Animal Care and Use Committee of Shandong University approved the experiments. Six-week-old male and female Sprague‒Dawley (SD) rats (weighing 200–250 g) were used in this study. Three rats were included per cage, and the rats were housed under controlled relative humidity (20%–30%) and a 12-h light‒dark cycle at room temperature (24°C). The rats had free access to food and water. All the animal experiments were performed in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Measurement of mechanical hyperalgesia

In this study, we used the same animal model as that reported by Huang et al.^18,19 The rats were randomly divided into five groups: control+NS, MTrPs+NS, and MTrPs+PI3K inhibitor LY294002 (MCE, 0.01, 0.1 and 1 mg/ml). The investigators were blinded to the treatment groups during the behavioural and histological assessments. The rat model of active MTrPs was established via a blunt strike on the left gastrocnemius muscle (GM) and eccentric exercise for 8 weeks followed by a 4-week recovery period. On the first day of each week, the striking point on the left GM of the rats in all the groups was marked and struck by a hand-made stick device (made by the Sports Medicine Teaching and Research Office of Shanghai Sport University, Shanghai, China) dropped from a height of 20 cm with a kinetic energy of 2.352 J. Every second day of each week, the rats were run on a treadmill (Sans Biotechnology Co., Ltd., Nanjing, Jiangsu, China) for 1.5 h at a −16° downwards angle and a speed of 16 m/min. The general health of the rats during the procedures, such as body weight, condition of the struck limb, and movement ability, was monitored weekly.

A Randall-Selitto instrument (Shandong Provincial Institute of Science and Technology, Jinan, Shandong, China) equipped with a round head probe (tip diameter: 8 mm) was used to measure mechanical hyperalgesia as a hindlimb withdrawal threshold in response to mechanical stimulation of the left hind limb. The rats were acclimated for 7 days in a laboratory environment prior to the start of the study. For the behavioural experiments, rats were constrained in a cylinder with their left hind limbs exposed, and the Randall-Selitto probe was applied to the left GM. In the Randall-Selitto test, the pressure was automatically increased until the rat retracted its left hind leg. Seven measurements were taken at 2-min intervals. The maximum and minimum values were removed, and the average of the remaining values was taken as the withdrawal threshold. To identify the active MTrPs, we palpated and marked hard contracture nodules in the taut band (TB) of the left GM with a reduced mechanical tenderness withdrawal threshold.

Nest-building behaviour test

The sensitivity of the nest-building behaviour test to spontaneous pain has been reported in various studies.^20,21 The nest-building behaviour of the rats in the control and MTrPs groups was evaluated by assessing nest quality after 6 h and 24 h of exposure to clean sawdust bedding and thirty-two pieces of rectangular tasteless paper of the same size (5 × 7 cm). After 6 h and 24 h, the nest-building ability was rated on a 5-point scale according to the degree of papers torn and the integrity of the nest construction: (1) papers not noticeably touched; (2) papers partially torn; (3) papers mostly shredded but often no identifiable nest site; (4) an identifiable but flat nest; and (5) a near perfect nest. ²²

Injection of a PI3K inhibitor

After successful modelling, the effects of the intramuscular injection of a PI3K inhibitor (LY294002) on the left hind limb withdrawal threshold in response to mechanical stimulus were explored in rats with MTrPs at week 13. LY294002 (MCE, 0.01, 0.1 and 1 mg/ml) was intramuscularly administered to the hard contracture nodules in the TB of the MTrPs group (30 µl×3 points per rat). The mechanical withdrawal threshold was measured with a Randal-Selitto apparatus at 0.5, 1, 2, 4, 6, 8 and 24 h after intramuscular administration. The effects of the vehicle (0.9% saline, NS) were also examined by administering the same volume as that of the inhibitors.

Electromyography

Electromyography (EMG) reflects muscle contractile function. ²³ The EMG signals of the rats in the control+NS, MTrPs+NS, and MTrPs+LY294002 (1 mg/ml) groups were recorded. The rats were anaesthetized by an intraperitoneal injection of 3 ml/kg 10% chloral hydrate and fixed on a board. TBs were marked in the left gastrocnemius muscle (GM). Two needle electrodes were connected to the TB of the gastrocnemius muscle, and one needle electrode was connected to the tail of the rat as a reference electrode. EMG signals were examined with an EMG device (ME0960313, Haishen United Medical Equipment Co., Ltd., Suzhou). The EMG signals were recorded for 5 min and analysed by group.

Haematoxylin–eosin staining

One hour after injection of LY294002 (1 mg/ml), the rats were anaesthetized with chloral hydrate (10%, 3 ml/kg, i.p.), sterilized, and intracardially perfused with physiological saline, and the left GM near the injection site was quickly extracted. One part of the muscle was fixed in 4% paraformaldehyde for 24 h and subsequently used for haematoxylin‒eosin (HE) and immunohistochemical staining, and the other part of the muscle was stored in liquid nitrogen for Western blot analysis. The L_4‒5 spinal cords of the rats were quickly extracted for immunohistochemical staining. Muscle samples from the rats were fixed with formalin. The samples were subsequently processed via a series of routine procedures, including dehydration, paraffin embedding, and sectioning. Sections with a thickness of 4 μm were attached to glass slides, dewaxed and stained with HE. The sections were then dehydrated in a concentration gradient of ethanol (70%–100%) and xylene and covered with cover slips. The sections were observed with an optical microscope equipped with a digital camera (Olympus, Tokyo, Japan).

Immunohistochemical staining

For immunohistochemical (IHC) staining, a streptavidin–biotin labelling method was used. Paraffin-embedded sections were heated at 68°C for 2 h, separated in xylene and rehydrated with a graded ethanol series at room temperature. The antigens were subsequently retrieved by microwaving the sections with citrate buffer (pH = 6) for 15 min. After being washed twice in phosphate-buffered saline (PBS), the sections were placed in a wet chamber and incubated in 3% hydrogen peroxide for 10 min. After being washed three times with PBS, the tissue sections were incubated with normal goat serum at 37°C for 30 min, followed by incubation with an anti-PI3K antibody (Santa Cruz, sc-166365, 1:100) and an anti-UCHL1/PGP9.5 antibody (Proteintech, 14730-1-AP 1:200) at 4°C overnight. The spinal cord sections were stained with an anti-Fos antibody (Proteintech, 26192-1-AP 1:200). After the sections were washed three times in PBS, they were incubated with biotin-labelled goat anti-rabbit serum at 37°C for 30 min (Beijing Zhongshan Golden Bridge Biotechnology, sp. 9000) and then incubated with horseradish peroxidase-labelled streptavidin complex at 37°C for 30 min. After washing three times in PBS, the sections were stained with 0.05% diaminobenzidine (DAB) and haematoxylin. Finally, the sections were dehydrated and covered. The negative control group was incubated with PBS instead of the primary antibody. Images were captured with a BX53 microscope (Olympus, Tokyo, Japan) with an Imaging Micropublisher camera (Abingdon, Virginia, USA).

Western blot analysis

Muscle tissue near the injection site on the left GM was quickly removed and stored in liquid nitrogen. The muscle samples were lysed in lysis buffer (protease and phosphatase inhibitors were added to the lysis buffer). The lysed homogenate was subsequently centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were collected, diluted in 5× sample buffer and denatured at 100°C for 10 min. The proteins were subsequently separated via 10% SDS‒PAGE and transferred to PVDF membranes, which were blocked with 5% skim milk powder for 1.5 h at room temperature and incubated at 4°C overnight with the following primary antibodies: anti-PI3K (Santa Cruz, sc-166365, 1:200), anti-p-AKT (Affinity, AF0016, 1:1000), anti-GAPDH (Proteintech, 10494-1-AP, 1:5000), and anti-p-MLC (Affinity Biosciences, AF8618, 1:1000). The membranes were washed with Tris-buffered saline with Tween-20 (TBST) and incubated for 1.5 h with a secondary antibody (1:5000) at room temperature. The blots were developed with a chemiluminescent reagent (Millipore).

Measurement of the cross-sectional area

We randomly selected one IHC section from each rat and then randomly selected three microscope fields (400×). The cross-sectional areas of all the fibres in each field were measured and averaged.

Statistical analyses

The sample size for each group was estimated by PASS15.0. Through a two-tailed test with a statistical power of 90% and α = 0.05, the calculated sample size for each group was determined to be five rats. In the behavioural experiments of this study, there were 8–10 rats per group, and in the histological experiments, there were six rats per group. The data are expressed as the mean ± standard deviation (SD). The data were analysed with SPSS 22.0 statistical software. Statistical analysis between two groups was performed with an independent-samples t test. Statistical comparisons of more than two groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. The results described as significant are based on the criterion of p < 0.05.

Results

Muscle histology

On the basis of the microscopic analysis of haematoxylin and eosin (H&E)-stained tissue, the muscle fibres in the control rats were uniform in size, polygonal, and regular in arrangement in the cross-sectional space (Figure 1(A)a). Large round muscle cells (contraction knots) were observed in cross-sections of muscle fibres at MTrPs (white arrows in Figure 1(A)c). In longitudinal sections of muscle fibres at MTrPs, the muscle fibres were regular and uniform in thickness (Figure 1(A)b). The continuous expansion of pyramidal muscle fibres was observed in longitudinal sections (white arrows in Figure 1(A)d). At MTrPs, the muscle fibre space of the cross-sections and longitudinal sections increased, and obvious inflammatory cell infiltration was observed. In muscle fibres at MTrPs, the central nuclei of cells (black heads in Figure 1(A)c and Figure 1(A)d) were observed.

Figure 1.

(A) Light microscopy images of muscle fibres stained with haematoxylin and eosin (200×). control group: a (cross section) and b (longitudinal section). MTrPs group: c (cross section), d (longitudinal section). Large round muscle cells in cross sections of MTrPs (white arrows in Figure 1(A)c). Pyramidal muscle fibres in longitudinal sections of MTrPs (white arrows in Figure 1(A)d). Central nuclei of cells (black arrows in Figure 1(A)c and Figure 1(A)d). (B) Time course of the mechanical withdrawal thresholds measured with a Randall-Selitto apparatus in the control and MTrPs groups. The mechanical withdrawal thresholds of the control and MTrPs groups were measured with a Randall-Selitto apparatus before and after the strike. The mechanical withdrawal thresholds of the MTrPs group decreased from weeks 3 to 8 and remained low until week 12. n = 10 in the control and MTrPs groups. (C) Nest-building behaviour test of the rats in the control and MTrPs groups. The nest-building scores of the rats in the MTrPs group were lower than those in the control group after 6 h and 24 h of exposure to clean sawdust bedding and thirty-two pieces of rectangular tasteless paper of the same size (5 × 7 cm). n = 10 in the control and MTrPs groups. Statistical analysis of the control group and the MTrPs group was performed via an independent-samples t test, degrees of freedom (dfs) = 18, −2-scores<2; *P<0.05, **P<0.01 compared with the control group.

Mechanical hyperalgesia in MTrPs

Mechanical withdrawal thresholds measured with a Randall-Selitto apparatus in rats with MTrPs (M group) were significantly lower than those in control rats from weeks 3 through 12. The pain thresholds continued to decline from week 3 to week 8 and remained low through week 12. The threshold for control rats did not differ from the baseline threshold (Figure 1(B)).

Nest-building behaviour test

The nesting building scores of the rats in the MTrPs group were lower than those in the control group after 6 h and 24 h of exposure to clean sawdust bedding and thirty-two pieces of rectangular tasteless paper of the same size (Figure 1(C)).

Electromyography

The frequencies and amplitudes of EMG signals from the left gastrocnemius muscle of the rats in the MTrPs+NS group were greater than those from the control+NS group. LY294002 (1 mg/ml, 1 h after intramuscular injection at the trigger points) reduced the frequencies and amplitudes of EMG signals at the trigger points of the rats (Figure 2).

Figure 2.

(A) EMG signals of the control+NS, MTrPs+NS, and MTrPs+LY294002 (1 mg/ml) groups. (B and C) The frequencies and amplitudes of EMG signals from the left gastrocnemius muscle of the rats in the MTrPs+NS group were greater than those in the control+NS group, **P<0.01. The frequencies and amplitudes of EMG signals from the left gastrocnemius muscle of the rats in the MTrPs+LY294002 (1 mg/ml) group were lower than those of the rats in the MTrPs+NS group, ^##P<0.01. n = 8 in the control+NS, MTrPs+NS, and MTrPs+LY294002 (1 mg/ml) groups. Statistical analyses of the control+NS, MTrPs+NS, and MTrPs+LY209002 (1 mg/ml) groups were performed via one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test; between-groups degrees of freedom (dfBs) = 2, within-groups degrees of freedom (dfWs) = 21, −2.

PI3K expression was upregulated on muscle cell membranes at trigger points

Immunohistochemical staining of PI3K in the GM of the control group and the MTrPs group revealed that PI3K expression was higher on muscle cell membranes at MTrPs in the MTrPs group than in the control group (Figure 3).

Figure 3.

The mean optical density of PI3K on muscle cell membranes at MTrPs in the MTrPs group (400×). The negative control group was incubated with PBS instead of PI3K ((a) and (b)). The expression of PI3K was measured on muscle cell membranes at the MTrPs in the MTrPs group (d) and was significantly greater than that in the control group (c) (white arrows: large round muscle cells; black arrows: PI3K on muscle cell membranes at the MTrPs). n = 6 rats in the MTrPs and control groups. **p < 0.01. Statistical analysis of the control group and the MTrPs group was performed via an independent-samples t test, degrees of freedom (dfs) = 10, −2-scores<2.

LY294002 reversed the pain behaviours induced by MTrPs and inhibited the expression of PI3K and p-AKT in MTrPs

The PI3K inhibitor LY294002 (0.01 mg/ml, 0.1 mg/ml or 1 mg/ml) was injected into the trigger sites of the rats with myofascial pain. LY294002 (0.1 mg/ml) increased the mechanical pain thresholds of MTrPs at 0.5, 1, and 2 h after injection. LY294002 (1 mg/ml) increased the mechanical pain thresholds of MTrPs at 0.5, 1, 2, and 4 h after injection. The analgesic effect of LY294002 peaked at 1 h (Figure 4(A)). The expression of PI3K and p-AKT was assessed at 1 h after the intramuscular injection of LY294002. Compared with that in the control group, the expression of PI3K and p-AKT at MTrPs was greater, and LY294002 (0.01 mg/ml, 0.1 mg/ml and 1 mg/ml) decreased the expression of PI3K at MTrPs; additionally, LY294002 (0.1 mg/ml and 1 mg/ml) decreased the expression of p-AKT at MTrPs (Figure 4(B)).

Figure 4.

(A) Effect of LY294002 on the mechanical withdrawal thresholds of the rats in the MTrPs group. LY294002 (0.1 mg/ml) increased the mechanical withdrawal threshold in the MTrPs group at 0.5, 1, and 2 h after muscle injection. LY294002 (1 mg/ml) increased the mechanical withdrawal threshold of the rats in the MTrPs group at 0.5, 1, 2, and 4 h after muscle injection. *p < 0.05, **p < 0.01 compared with the MTrPs+NS group. n = 8 rats in each group. Statistical analyses of the control+NS, MTrPs+NS, and MTrPs+LY209002 (0.01 mg/ml, 0.1 mg/ml, and 1 mg/ml) groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, between-groups degrees of freedom (dfBs) = 4, within-group degrees of freedom (dfWs) = 35, −2<z-scores<2. (B) LY294002 inhibited the expression of PI3K and p-AKT, which had increased in the MTrPs group. The expression levels of PI3K and p-AKT were measured 1 h after the injection of LY294002 or NS. Compared with those in the control group, the expression of PI3K and p-AKT in the MTrPs group was significantly greater, **p < 0.01. LY294002 (0.01 mg/ml) inhibited the expression of PI3K in the MTrPs of the rats; additionally, LY294002 (0.1 mg/ml, 1 mg/ml) inhibited the expression of PI3K and p-AKT, ^##p < 0.01. n = 6 rats in each group. Statistical analyses of the control+NS, MTrPs+NS, and MTrPs+LY209002 (0.01 mg/ml, 0.1 mg/ml, and 1 mg/ml) groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, between-groups degrees of freedom (dfBs) = 4, within-group degrees of freedom (dfWs) =25, −2<z-scores<2.

LY294002 inhibited the abnormal contraction of muscle fibres at MTrPs and inhibited the expression of Fos in the spinal dorsal horn of rats with myofascial pain

Using PGP9.5 to locate nerve fibres at the MTrPs in rats, we found that abnormal contraction knots in muscle fibres at MTrPs compressed nerve fibres, potentially leading to pain. LY294002 (1 mg/ml) inhibited the abnormal contraction of muscle fibres and relieved nerve fibre compression, which might be the mechanism by which LY294002 relieves pain (Figure 5(A)). P-MLC caused skeletal muscle contraction. The expression of p-MLC in muscle fibres at MTrPs was elevated, whereas LY294002 (1 mg/ml) inhibited the expression of p-MLC at trigger sites in rats (Figure 5(B)). The expression of Fos in the spinal cord (L_4–5) of rats with MTrPs increased. LY294002 (1 mg/ml) inhibited the expression of Fos in the spinal cord (L_4–5) of rats with MTrPs (Figure 5(C)).

Figure 5.

LY294002 inhibited the abnormal contraction of muscle fibres at MTrPs and inhibited Fos expression in the spinal dorsal horn of rats with myofascial pain. (A) Contraction knots compressed nerve fibres at MTrPs (400×). Nerve fibres were labelled with PGP9.5 (white arrows: contraction knots; black arrows: nerves). The mean cross-sectional area of muscle cells in the MTrPs group was greater than that in the control group. The mean cross-sectional area of muscle cells in the LY294002 (1 mg/ml) group was smaller than that in the MTrPs group. n=6 rats in each group. (B) The expression of p-MLC was measured at MTrPs in the MTrPs group and was significantly greater than that in the control group. LY294002 (1 mg/ml) inhibited the expression of p-MLC at MTrPs. n=6 rats in each group. (C) The expression of Fos in the spinal cord of rats with MTrPs increased (200×). LY294002 (1 mg/ml) inhibited the expression of Fos in the spinal cord of rats with MTrPs. n=6 rats in each group. **P<0.01, ^##P<0.01. Statistical analyses of the control+NS, MTrPs+NS, and MTrPs+LY209002 (1 mg/ml) groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, between-groups degrees of freedom (dfBs) = 2, within-group degrees of freedom (dfWs) = 15, −2.

Discussion

The present study demonstrated the following: (1) the mechanical tenderness threshold at the MTrPs of the rat GM increased, and LY294002 reduced the mechanical tenderness at the MTrPs; (2) the average diameter and cross-sectional area of muscle fibres at the trigger points increased, and LY294002 reduced the average diameter and cross-sectional area of muscle fibres at MTrPs, inhibiting the abnormal contraction of muscle fibres; (3) the expression of PI3K, p-AKT and p-MLC at MTrPs increased, and knots generated by abnormal contraction at MTrPs compressed nerve fibres, leading to pain, whereas LY294002 (1 mg/ml) reduced the expression of PI3K, p-AKT, and p-MLC at trigger points, inhibited the abnormal contraction of muscle fibres, alleviated nerve compression, and alleviated myofascial pain; and (4) the expression of Fos in the spinal dorsal horn of rats with myofascial pain increased, and LY294002 inhibited the expression of Fos in rats with myofascial pain.

MPS is a common type of muscle pain that is characterized by the presence of MTrPs.^24–26 MPS is caused by dysfunction of the myofascial system, often accompanied by dysfunction of the autonomic nervous system. ²⁷ The myofascial system includes contractile muscles and connective tissue (nerves, blood vessels, and lymph), which form a fascia that surrounds and permeates into the muscles.^28–30 The pathological mechanism of MTrPs-induced myofascial pain is still unclear, and the possible hypotheses are as follows: (1) energy crisis: sustained muscle contraction compresses blood vessels and consumes a large amount of energy to produce acidic metabolites; ²⁵ (2) peripheral sensitization: more nerve fibres at MTrPs receive pain signals, leading to pain sensitivity; ³¹ and (3) inflammatory response: inflammatory substances at MTrPs cause metabolic disorders and lead to pain. ³² Our model reflects chronic myofascial pain after eight consecutive weeks of muscle injury. In this study, after 4 weeks of rest, the rats in the MTrPs group had very few inflammatory cells near trigger points, indicating that the inflammatory response had dissipated. These findings suggest that the impact of the inflammatory response on myofascial pain is relatively small.

Research has shown that EphB1, a member of the tyrosine kinase receptor family, is highly expressed in the muscles of patients with myofascial pain and rats with myofascial pain. The EphrinB1/EphB1 signalling system is associated with myofascial pain in humans and rats. ³³ ERK causes abnormal contraction of muscle fibres at MTrPs of the GM in rats, leading to an increase in the average cross-sectional area of muscle fibres and causing myofascial pain. An ERK inhibitor reduced the average cross-sectional area of muscle fibres at MTrPs in rats with myofascial pain and lowered the mechanical tenderness pain threshold. Previous studies have identified important receptors and signalling pathways that cause abnormal contractions at MTrPs but lack evidence on how muscle fibres with abnormal contractions at MTrPs cause pain. Our findings suggested that muscle contraction knots at MTrPs compressed nerve fibres, leading to pain; additionally, we observed that the PI3K inhibitor LY294002 suppressed abnormal muscle fibre contraction at MTrPs and alleviated nerve fibre compression by inhibiting the PI3K/p-AKT/p-MLC pathway, which might be the mechanism by which LY294002 relieves pain (Figure 6). Research has revealed significant pathway‒pathway interactions between the PI3K/AKT signalling pathway and the cardiac muscle contraction pathway. ³⁴ Interpathway interactions may also exist between the PI3K/AKT pathway and the skeletal muscle contraction pathway. The PI3K inhibitor LY294002 might also inhibit abnormal contraction of muscle fibres at trigger points in rats in this study by suppressing other pathways that not only interact with PI3K/AKT but are also related to skeletal muscle contraction, such as the MEK-ERK pathway.^7,35 The PI3K/AKT pathway affects smooth muscle contraction in mouse bladder myocytes by increasing the release of Ca²⁺. ³⁶ PI3K signalling increases the Ca²⁺ concentration in human skeletal muscle cells, which may lead to skeletal muscle contraction. ³⁷ Suppression of the PI3K/AKT signalling pathway reduces acetylcholine receptor formation in rat skeletal muscle cells and inhibits skeletal muscle cell contraction. ³⁸ Furthermore, the PI3K/AKT signalling pathway affects energy metabolism in skeletal muscle, which may cause an energy crisis and trigger myofascial pain.^39–40 Moreover, Ca²⁺ and acetylcholine receptors in skeletal muscle cells are involved in the contraction of muscle fibres. ⁴¹ The abnormal contraction of muscle fibres at MTrPs may be related to the increased release of Ca²⁺ or the activation of acetylcholine receptors in muscle fibres through the PI3K/AKT pathway. However, the mechanism by which PI3K/AKT induces the abnormal contraction of muscle fibres at MTrPs and causes myofascial pain needs to be investigated further.

Figure 6.

The proposed mechanism by which the PI3K inhibitor LY294002 relieves MPS-related pain.

We observed the effect of intramuscular injection of the PI3K inhibitor LY294002 on myofascial pain in rats. It is possible that a small amount of PI3K inhibitor LY294002 may act on vascular, gastrointestinal, and airway smooth muscles through the blood circulation of skeletal muscle, but this did not affect our study of myofascial pain in rats after skeletal muscle sampling. We found that LY294002 (0.01 mg/ml) inhibited the expression of PI3K but did not inhibit the expression of p-AKT or mechanical tenderness at MTrPs. The possible reasons were as follows: (1) the pharmacological effects of LY294002 (0.01 mg/ml) were insufficient to inhibit the expression of p-AKT and p-MLC and did not inhibit abnormal contractions at MTrPs; (2) although the expression level of PI3K was inhibited by LY294002 (0.01 mg/ml), there was still a high expression level of PI3K at MTrPs, leading to increases in the expression levels of p-AKT and p-MLC, causing abnormal contraction of trigger muscle fibres, compression of nerve fibres, and sensitivity to pain; and (3) other signalling molecules also act on p-AKT, causing pain at MTrPs. LY294002 (0.1 mg/ml) inhibited the expression of PI3K and p-AKT and alleviated mechanical tenderness at MTrPs for 2 h. LY294002 (1 mg/ml) inhibited the expression of PI3K, p-AKT, and p-MLC, alleviated the abnormal contraction of muscle fibres at MTrPs, relieved nerve fibre compression, and prolonged the duration of mechanical tenderness to 4 h. The weakening of the pharmacological effects of LY294002 and other signalling pathways leading to the abnormal contraction of muscle fibres at MTrPs resulted in the recovery of the mechanical tenderness threshold at MTrPs.

The central axonal branch of muscle nociceptors projects to the spinal cord through the dorsal horn. Mechanical stimuli detected by the peripheral terminals of afferent muscle fibres are transmitted to the spinal dorsal horns and, subsequently, to higher nervous centers. ⁴² Fos expression in the spinal dorsal horns of rats is induced by gastrocnemius muscle pain. ⁴³ Compression stimulation of the sciatic nerve increases the expression of the pain marker Fos in the spinal dorsal horns. ⁴⁴ In this model, muscle contraction knots at MTrPs compressed peripheral nerve fibres. Mechanical compression stimuli of these peripheral nerve fibres by muscle contraction knots at MTrPs in this model were transmitted to the spinal dorsal horns and increased Fos expression in the spinal dorsal horn. We observed the effect of intramuscular injection of the PI3K inhibitor LY294002 on Fos expression in the spinal dorsal horn of rats with myofascial pain. After intramuscular injection, LY294002 primarily targeted skeletal muscles, thereby avoiding the interference of systemic drug administration on the expression of Fos in the spinal dorsal horn. We found that intramuscular injection of LY294002 inhibited the compression of peripheral nerve fibres by muscle contraction knots at MTrPs, which reduced the expression of Fos in the spinal dorsal horns of the rats in this model.

Our research has several limitations. Owing to the less rigorous study design, we did not assess the effects of LY294002 (0.01 mg/ml and 0.1 mg/ml) on p-MLC expression or the abnormal contraction of muscle fibres at MTrPs. Additionally, we did not explore how p-AKT caused an increase in p-MLC expression in MTrPs, which occurred possibly through its action on calcium ion channels on the muscle fibre cell membrane.