Silver Needle Thermotherapy Regulates TRPV1 to Alleviate Endoplasmic Reticulum Stress and Relieve Myofascial Pain

Abstract

Background

Silver needle thermotherapy (SNT) has a remarkable therapeutic effect on myofascial pain syndrome (MPS), but its mechanism of action remains to be clarified. This study aims to explore the effect of SNT on endoplasmic reticulum stress (ERS) in myofascial trigger points (MTrPs) of MPS rats and reveal its potential molecular mechanism.

Methods

The MPS rat model was established. SNT treatment and TRPV1 adeno-associated virus injection were respectively performed on MTrPs of rats in different groups. After successful virus transfection, Hematoxylin-eosin staining, Transmission electron microscope, Western Blotting, Immunofluorescence analysis were performed. Experiments were conducted to observe the ERS of MTrPs and pain changes in each group of rats.

Results

(1) The mechanical withdrawal threshold (MWT) of MPS rats decreased significantly, while SNT treatment could reverse the changes in MWT; (2) In MPS rats, MTrPs muscle fibers showed atrophy, degeneration and disordered arrangement. However, SNT treatment or interference with TRPV1 expression could improve the changes in muscle fibers; (3) SNT treatment or interference with TRPV1 expression can alleviate endoplasmic reticulum (ER) dilation and cystic space widening in MTrPs of MPS rats; (4) SNT treatment or interference with the expression of TRPV1 can inhibit the expressions of TRPV1, p-TRPV1, CaMKII, CHOP and BIP in the MTrPs muscle tissue of MPS rats.

Conclusion

SNT can increase the pain threshold, repair the damaged myofascia, alleviate ERS and pain of MPS rats.

Introduction

Myofascial pain syndrome (MPS) is a common musculoskeletal pain disorder, characterized by the formation of myofascial trigger points (MTrPs) in muscles or fascia.¹ It is estimated that over 100 million American adults suffer from chronic pain, with MPS being the main cause.² At present, there are many methods for treating MPS in clinical practice, but most of them have poor long-term efficacy. Therefore, it is particularly crucial to deeply understand the pain formation mechanism of MPS and identify new therapeutic targets.

It is currently widely believed that the formation of MTrPs is the main cause of MPS,³ but there are still many unknowns regarding its specific nature. In skeletal muscle cells, the sarcoplasmic reticulum (SR) is responsible for storing and releasing Ca²⁺, maintaining intracellular Ca²⁺ homeostasis. Professor Simons’ overall hypothesis holds that the disorder of Ca²⁺ release and reuptake within the sarcoplasmic reticulum is the key to pain formation. This disorder causes an increase in [Ca²⁺] within the sarcoplasmic reticulum, which in turn leads to continuous muscle fiber contraction, the formation of muscle tension zones, and ultimately triggers a local tissue energy crisis, promoting the release of pain-causing substances and inflammatory factors, which leads to pain.⁴

On the other hand, the Transient receptor potential vanilloid 1 receptor (TRPV1), as a non-selective cation channel, is widely expressed in nociceptive receptors.⁵ The activation of TRPV1 can lead to the inflow of extracellular Ca²⁺,⁶ which in turn causes the activation of Ca/calmodulin-dependent protein kinases or CaM kinases(CaMKII) factors, which is considered a potential mechanism of inflammation-mediated hyperalgesia.⁷ In addition, abnormal CaMKII activity is an important factor promoting the increase of intracellular [Ca²⁺], which can disrupt the calcium ion balance within skeletal muscle cells, promote apoptosis of skeletal muscle cells, and ultimately lead to structural damage and functional abnormalities of skeletal muscle.⁸ Skeletal muscle injury can lead to structural damage or functional decline of calcium pumps, thereby triggering Endoplasmic reticulum stress(ERS),⁹ and eventually triggering pathological inflammatory responses, intensifying pain.¹⁰

Studies have shown that muscle injury or the formation of myofascial trigger points can lead to microenvironmental changes such as local tissue acidification (pH can be reduced to below 6.0),¹¹ the release of ATP and inflammatory mediators (bradykinin, prostaglandins, etc), and the upregulation of NGF and GDNF expression.¹² And through multiple pathways such as direct activation, P2X3 receptor synergy, GPC-PKC /PKA phosphorylation and upregulation of membrane expression, it collaboratively activates the highly expressed TRPV1 on harmful afferent fibers (C fibers and Aδ fibers), converting it from the quiescent state to the activated state. The Ca²⁺ influx mediated by the opening of TRPV1 activates CaMKII. After activation, CaMKII feedback phosphorylates TRPV1, forming a positive feedback amplification loop, which leads to an increase in the excitability of the nociceptive receptor, that is, peripheral sensitization.¹³ Continuously activated TRPV1 causes cytoplasmic Ca²⁺ overload; With the imbalance of calcium homeostasis and the increase of calcium processing load in the endoplasmic reticulum, ERS is triggered and the expression of pro-inflammatory factors is upregulated through the PERK-eIF2α-ATF4-CHOP axis,¹¹ further sensitizing local receptors, forming a vicious cycle of “damage - sensitization - inflammation”. The high-frequency signal amplified by this axis is transmitted into the dorsal horn of the spinal cord along Aδ and C fibers, accompanied by the release of CGRP and substance P, activating NMDA receptors^14,15 inducing Long-term potentiation and glial cell activation, and ultimately triggering and maintaining pain and its central sensitization.¹⁶

Silver needle thermotherapy(SNT), originated from the “Nine Needles” in ancient China, exerts significant anti-inflammatory and analgesic effects through mechanisms such as relaxing soft tissues and strongly stimulating pain points compared with traditional acupuncture.¹⁷ Clinically, SNT has been used to treat various pain disorders and has shown an effective rate of over 90% in the treatment of MPS.^18,19 However, although SNT has shown good clinical efficacy, the basic research on its molecular mechanism is still limited and mostly remains at the level of clinical experience.

Therefore, we attempted to establish an MPS animal model to explore whether ERS exists in the MPS rat model and treat it with SNT. We observed the effects of acupuncture and thermal effects on the pain threshold and pathological morphology of rats, as well as their relationship with TRPV1 and ERS, in order to provide a theoretical basis for the treatment of MPS with SNT, and promote the modernization of this traditional therapy.

Materials and Methods

Animals and Ethics Statement

Fifty adult male Sprague-Dawley (SD) rats of SPF grade (weighing 220±30g), provided by the Animal Center of Guizhou Medical University, with the certificate number: SCXK (Xiang) 2022–0011. All experiments in this study were conducted strictly in accordance with the requirements of the Laboratory Animal Ethics Committee of Guizhou Medical University (Ethics Approval Number: 2201530). Six rats were randomly selected from all the rats as the Control group (Control) by the random number table method. The remaining rats all received the establishment of the MPS rat model. Excluding four rats that died due to anesthesia or fighting and four rats that failed in model establishment, the remaining 36 rats with successful model establishment were also randomly divided into the following groups by the random number table method: Myofascial pain syndrome group (MPS), Silver needle thermotherapy group (MPS+SNT), Silver Needle Acupuncture group (MPS+SNA), TRPV1 virus intervention group (MPS+ TRPV1-miRNA), TRPV1 virus intervention followed by Silver needle thermotherapy group (MPS+ TRPV1-miRNA +SNT) and TRPV1 virus empty group (MPS + Ctrl-miRNA). There were 6 rats in each group (as shown in Figure 1).

Figure 1.

Schematic diagram of animal grouping and experimental treatment. Six rats were randomly selected from the 50 SPF male SD rats to serve as the Control group using a random number table, and the remaining 44 rats were subjected to MPS model establishment (weeks 1–8). Successfully modeled rats (n = 36) were randomly divided into six groups (n = 6 each) and received respective interventions at week 12 and week 16. All rats were sacrificed at week 16. For abbreviations, see the Abbreviations section below.

At the end of the experiment, rats were deeply anesthetized via isoflurane inhalation (induction at 5%, maintenance at 2–3%, oxygen flow rate of 1–2L/min in a sealed transparent induction chamber) until the corneal reflex disappeared and complete muscle relaxation was achieved. After confirming deep anesthesia, euthanasia was performed by overdose isoflurane inhalation (concentration>5%) combined with thoracotomy, and death was confirmed by palpation of cardiac arrest. The operator monitored the animals throughout the procedure to ensure no recovery of consciousness. This euthanasia protocol complies with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 edition) regarding euthanasia using inhalant anesthetics.

Instruments

The silver needle thermal conductivity inspection instrument (Shanghai Shuxin Technology Development Co., Ltd., YRX160256), self-made blunt impactor (provided by the pain teaching and research section of Guizhou Medical University), animal experiment treadmill (Tianjin Beirui Future Technology Co., Ltd., DWPT type), Von Frey filament (Stoelting, Wood Dale), upright fluorescence microscope (ZEISS, Axio Imager), dehydrator (Leica, TP1020), embedding machine (Leica, EG1150H) Paraffin sectioning machine (Leica, CM2245). Chemiluminescence Imaging System (CLINX, Chemi Scope6200), Multi-function Microplate Reader (BIOTeK, StbergyH4), Desktop Frozen Microcentrifuge (BECKMAN, AllgraX-15R), Oven (Tianjin Tester Instrument Co., Ltd., WGL-125B), Histochemical Pen (Biosharp, BC003), Front Placement Microscope (Leica, DM2500), Laser Confocal Microscope (Zeiss, LSM710), Electronic Analytical Balance (Changshu Shuangjie Electronics Co., Ltd., JJ124-BC), Ultrasonic Breaker (Bandelin, HD2070), Vertical Protein Electrophoresis Three-Piece Set (BIO-RAD, 1658033), Transmission Electron Microscopy (JEOL, JEM-1400FLASH).

Reagents

Adeno-associated virus AAV9-TRPV1-RNAi, provided by Shanghai Jikexin Technology Co., LTD., with a concentration of 1.36E+ 13V g/mL. TRPV1 Antibody (Affinity Biosciences, TRPV1 antibody-DF10320), phosphorylated transient receptor potential vanilloid type1 (p-TRPV1) antibody was purchased from Affinity Biosciences. Binding Immunoglobulin Protein (BIP) and C/EBP Homologous Protein (CHOP) were purchased from Shenyang Wanlei Biotechnology Co., LTD., and CaMKII antibody was purchased from Wuhan Sanying Biotechnology Co., LTD. And modified hematoxylin-eosin (HE) staining Kit (Beijing Solaibao Technology Co., LTD., G1121), etc.

Experimental Methods

MPS Animal Model Replication

Referring to the research method of Huang Qiangmin et al²⁰ Rats were anesthetized with isoflurane, an inhalation anesthetic for animals. The right medial femoral muscle of the rats was fixed and marked, and the percussor was freely dropped from a height of 20cm to strike the marked area. The next day, the rats were made to run on a −16° running platform at a speed of 16m/s for 90 minutes. During running, use noise and sticks to drive away the rats to ensure they are in good exercise condition. The above intervention measures were carried out once a week for a total of 8 weeks, followed by only regular feeding for the next 4 weeks (as shown in Figure 1).

Silver Needle Thermotherapy

After anesthetizing rats with isoflurane, the right medial femoral muscle of the rats was located and MTrPs were palpated, which were marked as the treatment area for the silver needle. After disinfection, silver needles were respectively inserted at the starting and ending points of the right medial femoral muscle to pierce the tension zone of the rat muscle, with the tip reaching the bone surface. According to the experimental design of this study, the MPS+SNT and MPS+TRPV1-miRNA+SNT group received heating treatment at a temperature of 110°C for 15 minutes. In the MPS+SNA group, only silver needles were inserted without any heating treatment. After the treatment is completed, the silver needle is slowly removed. The puncture site is routinely disinfected with 75% alcohol and covered with a sterile dressing. Wait for the rats to wake up.

Local Injection of Adeno-Associated Virus at the MTrP Site

At 12 weeks, rats in the MPS+TRPV1-miRNA group and the MPS+TRPV1-miRNA+SNT group were anesthetized with isoflurane, and the AAV9-TRPV1-RNAi viral stock was aspirated using a 5ul microsyringe. Three sites were selected around the MTrPs for injection, with 3μL injected at each site, totaling 9μL. Negative control virus (CON539) was injected simultaneously into the MPS + Ctrl-miRNA group. Rest for 4 weeks and wait for virus transfection (as Figure 1).

Detection of Mechanical Withdrawal Threshold (MWT)

This study used distal hind paw MWT to assess hyperalgesia caused by proximal muscle injury. This method is well-established. It is based on two concepts: secondary hyperalgesia and referred pain. Both are core features of MPS and central sensitization.^21,22 From a neurophysiological perspective, the hind paw and thigh muscles of rats share overlapping spinal segmental innervation. The L4 segment is primarily involved.²³ Central sensitization expands the receptive fields of spinal dorsal horn neurons. As a result, nociceptive input from the proximal quadriceps femoris muscle can manifest as mechanical hyperalgesia in the hind paw.²⁴ Therefore, hind paw MWT mainly reflects central amplification of muscle-derived nociceptive information,²⁵ it does not reflect local peripheral pathological changes. Sluka et al demonstrated this mechanism. They repeatedly injected pH 4.0 saline into the gastrocnemius muscle. This induced long-term and widespread reductions in both muscle and paw MWT. Their findings confirmed two points. First, central sensitization does not depend on persistent nociceptive input. Second, hind paw MWT is a reliable indicator of muscle-derived central pain.^26–28 Numerous studies have also shown that hind paw MWT is a predominantly centrally mediated measure.^29–31 Thus, it is suitable for evaluating central sensitization in MPS. Therefore, this study used the right hind paw MWT to assess pain changes in MPS rats.

MWT was measured respectively 1 day before modeling (d0), 4 weeks after modeling (4w), 8 weeks after modeling (8w), 12 weeks after modeling (12w), 4 weeks after local injection of adeno-associated virus (16w), and 2 weeks after treatment (14w or 18w). According to the “Up&Down” method introduced by Dixon,³² a series of Von Frey fiber filaments were used to stimulate the skin in the middle of the right foot of rats. The fiber filaments needed to be bent to a “C” or “S” shape and maintained for 6–8 seconds. It was observed and recorded whether the rats had foot retraction and foot licking reactions. The absence of foot retraction and foot licking reactions is recorded as a negative reaction “O”, while the presence of reactions is recorded as a positive reaction “X”, resulting in a sequence arranged in “O” or “X”. The threshold was calculated based on the formula:

where,

G represents the threshold for 50% of animals to produce paw withdrawal behavior, Xf is the log value of the final fiber filament, k is the fixed table value of different sequences, and δ is the mean difference of each fiber filament after taking the logarithm, which is about 0.224.

Sample Collection

The medial femoris muscle on the right side of the rat was exposed. It was cut and stripped at the starting and ending points of the muscle. According to the experimental design, it was divided into three parts. One part was rinsed with 0.9% frozen sodium chloride solution and placed in a cryotube for storage at −80°C for Western blotting analysis and detection. One copy was fixed in 2% glutaraldehyde and stored at 4°C for transmission electron microscopy detection. One sample was soaked in 4% paraformaldehyde solution and stored at room temperature for HE staining and immunofluorescence staining.

Hematoxylin-Eosin Staining (HE)

Muscle tissue samples were immersed in 4% paraformaldehyde solution and fixed for 24 hours. Paraffin embedding, sectioning, xylene dewaxing, gradient ethanol rehydration, hematoxylin counterstaining, hydrochloric acid ethanol differentiation after complete return to blue, eosin staining gradient ethanol dehydration, xylene transparency, neutral resin sealing, observation of muscle fiber morphology and arrangement under an optical microscope. The cross-sectional area of muscle fibers in rat MTrPs was quantified using ImageJ software across different groups.

Western Blotting

The fresh tissue samples were taken out, and the lysis solution was added according to the weight volume ratio of 1:10, and the lysis solution was fully ground. After ultrasonication, it was allowed to stand for 30min, and then centrifuged at 12000 r/min and 4°C for 20min. The supernatant was taken to be measured. The protein concentration was determined by BCA method, and the denatured protein was added according to 50μg and 15mL. After separation by SDS-PAG gel electrophoresis, it was transferred to PVDF membrane, blocked with TBST solution containing 5% skim milk powder for 2h, and added with primary antibody at 4°C overnight. The next day, TBST was washed 10min×3 times, incubated with secondary antibody at room temperature for 1h, and TBST was washed 10min× 3 times. The ECL luminescent liquid was added dropwise to expose and take photos, and the absorbance of the strip was analyzed by ImageJ software.

Transmission Electron Microscope(TEM)

The muscle was trimmed into 1× 1×1mm³ pieces, which were pre-fixed with 3% glutaraldehyde and then re-fixed with 1% osmium tetroxide. After acetone was dehydrated step by step, the muscle was embedded and sliced by ultra-thin slicer. The thickness of the slice was about 60–90nm. First stained with uranium acetate for 10–15min, and then stained with lead citrate for 1–2min. After staining, TEM was used for observation.

Immunofluorescence Analysis(IF)

Muscle tissue samples were immersed in 4% paraformaldehyde solution for 24 hours, paraffin-embedded, sliced, dewaxed in xylene, rehydrated with gradient ethanol, and heated in a microwave oven by 10mM citrate buffer (PH6.0) for antigen repair. Subsequently, 10% goat serum was used to seal at room temperature for 1h, TBST was washed for 10min×3 times, and TRPV1 and CaMKII primary antibodies from different sources were added to incubate overnight at 4°C. The next day, the samples were taken out and rewarmed for 30min, TBST was washed for 10min×3 times, and fluorescent secondary antibodies corresponding to different species of primary antibodies were added, and incubated for 1h at room temperature in the dark. TBST was washed for 10min×3 times, and the DAPI-containing sealing agent was added dropwise for sealing treatment, and then the images were observed and captured under a fluorescence microscope. Fluorescence intensity in different groups was quantified using ImageJ software.

Statistically Treated

SPSS 17.0 statistical software was used for statistical analysis. The data were expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance was used for comparison among multiple samples, and LSD-t test was used for pairwise comparison of means between groups. p<0.05 was considered statistically significant.

Results

TRPV1 Protein is Highly Expressed in the MTrPs of MPS Rats

Firstly, to verify whether the MPS rat model was successfully established, this study detected the changes in MWT in rats. As shown in Figure 2a, during the modeling period, the MWT of rats decreased significantly compared with that before modeling and reached stability at 12 weeks.

Figure 2.

The changes in MWT and TRPV1 expression in MTrPs of MPS rats at different periods during modeling. (a) The MWT of MPS rats during modeling was determined before modeling (d0), at 4 weeks of modeling (4w), at 8 weeks of modeling (8w), and at 12 weeks of modeling (12w). Compared with d0, **p<0.01, ***p<0.001; (b) HE staining images of MTrPs muscle tissues in the Control group and MPS group rats, scale 100μm; (c) Statistical analysis of muscle fiber cross-sectional area in Control and MPS rats, compared with Control group, ***p<0.001; (d and e) The band map of TRPV1 protein and the quantitative analysis results of protein in MTrPs of MPS rats, *p<0.05; (f) TRPV1 immunofluorescence staining plots of MTrPs in the Control group and MPS group rats, scale: 50μm; (g) Quantitative analysis of TRPV1 mean fluorescence intensity, compared with Control group, **p<0.01.

In addition, two experienced clinicians palpated the medial femoral muscle on the right side of the rats. The results showed that compared with the rats in the Control group, obvious nodules could be palpated in the MPS rats. Subsequently, the sampling was completed, and HE staining was performed on the MTrPs muscle tissue to observe the changes in muscle structure (Figure 2b). The cross-sectional structure of the muscles in the Control group was uniform, closely and regularly arranged, while the MTrPs muscle fibers in the MPS group were uneven elliptical structures and atrophy and degeneration occurred. As shown in Figure 2c, the cross-sectional area was significantly larger in the Control group than in the MPS group. The above results suggest that the MPS rat model has been successfully established.

To further explore the pathogenesis of MPS, we also conducted Western Blotting (Figure 2d and e) and IF (Figure 2f and g) experiments to detect the expression of TRPV1 in the MTrPs muscle tissue of MPS rats. The experimental results suggest that the expression of TRPV1 in MTrPs of MPS rats is significantly higher than that in the Control group.

Endoplasmic Reticulum Stress Exists in the MTrPs Muscle Tissue of MPS Rats

To assess whether ERS exists in the MTrPs muscle tissue of MPS rats, we first used a TEM to observe the microstructure of the MTrPs muscle tissue (as shown in Figure 3a). The results indicated that the myofibrils in the muscle tissue of the Control group rats were neatly arranged, the muscle segment structure was clear, the morphology and structure of the sarcoplasmic reticulum were normal, and it is evenly distributed among the muscle fibers. In contrast, in the MTrPs muscle tissues of the MPS group rats, the sarcoplasmic reticulum mostly showed severe dilation, with significantly widened capsule cavities, and only a small portion of the sarcoplasmic reticulum exhibited moderate dilation.

Figure 3.

The TEM results as well as the expression of CHOP and BIP proteins of MTrPs muscle tissues in MPS rats. (a) TEM of MTrPs of rats in Control group and MPS group, scale bars: 2 μm (8000×) and 500 nm (30,000×); (b and c) Western Blotting The expression protein bands and corresponding quantitative analysis results of CHOP and BIP protein in MTrPs of the rats in the Control group and the MPS group, **p<0.01, ***p<0.001.

In addition, in this study, the expression levels of ERS marker proteins (BIP, CHOP) in MTrPs of MPS rats were detected through Western Blotting experiments. As shown in Figure 3b and c, the expression levels of CHOP and BIP proteins in the muscle tissue of MPS rats were significantly higher than those in the Control group.

The above results indicate that RES does exist in the MTrPs muscle tissue of MPS rats.

SNT Relieves the Pain Behavior of MPS Rats by Inhibiting the Expression of TRPV1 and Repairs the Damaged Muscle Fibers at the MTrPs of MPS Rats

To explore the effect of SNT hyperthermia on the pain threshold of MPS rats, two groups were set up in this study, namely Silver needle thermotherapy group (MPS+SNT) and Silver Needle Acupuncture group (MPS+ SNA), and the MWT of the two groups of rats was measured 2 weeks after treatment. As shown in Figure 4a and b, compared with MPS rats, the MWT of rats in the MPS+SNT group was significantly increased, while there was no significant change in the MPS+SNA group.

Figure 4.

The effect of SNT on the expression of TRPV1 in the MTrPs muscle tissue and on the pain of MPS rats. (a) The right hind paw MWT of rats in each group was measured before treatment (w12), 2 weeks after treatment (w14). Compared with the w12, **p<0.01; ns, not significant (p > 0.05); (b) The left hind paw MWT of rats in each group was measured before treatment (w12), 2 weeks after treatment (w14). Compared with the w12, ****p<0.0001; ns, not significant (p > 0.05); (c) HE staining Local muscle tissue staining map of MTrPs in four groups of rats, scale: 100μm; (d) Statistical analysis of muscle fiber cross-sectional area in four groups, compared with Control group, **p<0.01; compared with MPS group, ^##p<0.01; (e) Immunofluorescence staining of TRPV1 in MTrPs muscle tissues of four groups of rats, scale: 50μm. (f) Quantitative analysis of TRPV1 mean fluorescence intensity, compared with Control group, ****p<0.0001; compared with MPS group, ^##p<0.01; compared with MPS+SNT group, ^{^^}p<0.01; (g and h) Western Blotting was used to detect the TRPV1 protein band and quantitative analysis results in MTrPs of four groups of rats. Compared with the Control group, *p<0.05, **p<0.01; compared with the MPS group, ^#p< 0.05; compared with the MPS+SNT group, ^{^}p<0.05.

We conducted HE staining to evaluate the morphological changes of muscle tissues in different groups. In the MPS+SNT group, a small number of muscle fibers showed mild atrophy and degeneration, and the overall morphology was similar to that of the Control group. This indicates that SNT can repair the damaged muscle fibers at the MTrPs of MPS rats. Although the muscle fiber structure of the MPS+SNA group was improved to some extent compared with the MPS group, the degree was relatively limited (see Figure 4c). Similarly, quantitative analysis of HE-stained muscle cross-sectional areas revealed that MPS rats exhibited a significantly reduced cross-sectional area, which was significantly restored following SNT treatment. However, no significant improvement was observed in the muscle fiber cross-section after SNA treatment alone (Figure 4d).

We also detected the expression level of TRPV1 in the MTrPs muscle tissues of rats in different groups through immunofluorescence detection. It was found that compared with the MPS and MPS+SNA groups, TRPV1 was significantly decreased in the MPS+SNT group of rats. However, there was no statistically significant difference in the expression level of TRPV1 between the MPS group and the MPS+SNA group (as demonstrated in Figure 4e and f).

In addition, Western Blotting experiments was conducted on the expression of TRPV1 in the MTrPs muscle tissues of four groups of rats in this study. The results showed that compared with the MPS group, the expression of TRPV1 in the MPS+SNT group was decreased and much lower than that in the MPS+SNA group. However, there was no statistical significance between the MPS+SNA group and the MPS group (as presented in Figure 4g and h).

SNT Mediates TRPV1 Alleviates Endoplasmic Reticulum Stress in MTrPs of MPS Rats, Thereby Reducing Pain

To explore the effect of SNT on ERS in rat MTrPs, we collected muscle tissues of MTrPs in different groups of rats and conducted transmission electron microscopy observations. The results indicated that compared with the MPS group, the myofibrils of rats in the MPS+SNT group were arranged more neatly, and most of the sarcoplasmic reticulum showed moderate dilation and mild widening of the cystic cavity space. In contrast, the MPS+SNA group presented with severe dilation of the sarcoplasmic reticulum and significant widening of the cystic space (as shown in Figure 5a).

Figure 5.

The effect of SNT on the expression of CHOP, BIP and the results of TEM in the MTrPs of MPS rats.(a)TEM of the MTrPs of rats in each group, scale bars: 2 μm (8000×) and 500 nm (30,000×). (b and c) Western Blotting was used to detect BIP and CHOP protein expression protein bands and corresponding quantitative analysis results in the MTrPs of rats in each group. Compared with the Control group, *p < 0.05, **p < 0.01, ***p<0.001; compared with the MPS group, ^#p<0.05, ^##p<0.01; compared with the MPS+SNT group, ^{^}p<0.05.

In addition, this study also detected the expression levels of ERS marker proteins (BIP, CHOP) in MTrPs of rats in different groups. The results are shown in Figure 5b and c, compared with the MPS group, the expression levels of BIP and CHOP in the MPS+SNT group were decreased, while there was no statistically significant difference in the expression in the MPS+SNA group.

The above results indicate that SNT may alleviate the ERS level of MTrPs in MPS rats and alleviate the expression of TRPV1.

AAV9-TRPV1-RNAi Alleviates Endoplasmic Reticulum Stress by Down-Regulating the Expression of TRPV1/CaMKII, and Reduces Hyperalgesia in MPS Rats

AAV9-TRPV1-RNAi Reduces the Phosphorylation Level of TRPV1 and the Expression of CaMKII, and Alleviates Hyperalgesia in MPS Rats

To verify that SNT alleviates pain in MPS rats by regulating the expression of TRPV1, in this study, TRPV1 adeno-associated virus was injected into MTrPs of MPS rats to down-regulate the expression of TRPV1. Then, the therapeutic effects of TRPV1 down-regulation and SNT on pain in MPS rats were compared.

As shown in Figure 6a and b, compared with the MPS group and the MPS+Ctrl-miRNA group, the MWT of MPS+SNT group and MPS+TRPV1-miRNA group was significantly increased, while the results of the MPS+TRPV1-miRNA+SNT group only slight relief.

Figure 6.

Effects of AAV9-TRPV1-RNAi local injection on MWT and expression of TRPV1, p-TRPV1 and CaMKII in MTrPs of MPS rats. (a) The right hind paw MWT of rats in each groups was measured before intervention (w12), after intervention (w16 or w18), **p<0.01, ****p<0.0001; ns, not significant (p > 0.05). (b) The left hind paw MWT of rats in each groups was measured before intervention (w12), after intervention (w16 or w18), *p<0.05, ****p<0.0001; ns, not significant (p > 0.05). (c) HE staining of muscle tissue of MTrPs in rats of each groups, scale: 100μm. (d) Statistical analysis of muscle fiber cross-sectional area in each groups, compared with Control group, **p<0.01; compared with MPS group, ^##p<0.01, ^###p<0.001; compared with MPS+SNT group, ^{^}p<0.05. (e and f) Immunofluorescence staining of TRPV1 in MTrPs muscle tissues of each groups of rats, scale: 50μm, and quantitative analysis of TRPV1 mean fluorescence intensity, compared with Control group, ****p<0.0001; compared with MPS group, ^####p<0.0001; compared with MPS+SNT group, ^{^^^^}p<0.0001; compared with MPS+TRPV1-miRNA group, ⁺⁺⁺⁺p<0.0001. (g and h) Western Blotting was used to detect TRPV1, p-TRPV1, CaMKII protein expression bands and quantitative analysis results in the MTrPs of each group rats. Compared with the Control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; compared with the MPS group, ^#p<0.05, ^####p<0.0001; compared with the MPS+SNT group, ^{^}p<0.05, ^{^^}p<0.01, ^{^^^^}p<0.0001; compared with the MPS+TRPV1-miRNA+SNT group, ⁺⁺p<0.01, ⁺⁺⁺p<0.001, ⁺⁺⁺⁺p<0.0001; compared with the MPS+Ctrl-miRNA group, ^ΔΔp<0.01, ^ΔΔΔΔp<0.0001.

Note: i Control group, ii MPS group, iii MPS+SNT group, iv MPS+TRPV1-miRNA group, v MPS+TRPV1-miRNA+SNT group, vi MPS+Ctrl-miRNA group.

Moreover, the HE staining results of MTrPs muscle tissues in different groups were consistent with the changing trends of their MWT. In the MPS+SNT and MPS+TRPV1-miRNA groups, only a small number of muscle fibers in the muscle tissues of rats had slight atrophy and degeneration, and the morphology was close to that of the Control group. However, the structure of the MPS+TRPV1-miRNA+SNT group was disordered (Figure 6c and d).

To further explore the mechanism, we detected the expression levels of TRPV1, p-TRPV1 and CaMKII in the MTrPs muscle tissues of rats in different groups through immunofluorescence staining and Western Blotting. The results showed that compared with the Control group, the expressions of TRPV1 in the MPS group, MPS+Ctrl-miRNA group and MPS+TRPV1-miRNA+SNT group were significantly upregulated, while the expressions in the MPS+SNT group and the MPS+TRPV1-miRNA group decreased (as shown in Figure 6e–h). Meanwhile, Western blotting results showed that p-TRPV1 and CaMKII were significantly increased in the MPS group and the MPS+Ctrl-miRNA group, whereas their levels decreased after SNT or TRPV1-miRNA treatment. It is worth noting that in the MPS+TRPV1-miRNA+SNT group, the expression of p-TRPV1 was increased compared with the MPS+SNT group (as shown in Figure 6g and h).

In addition, we also observed the co-localization of TRPV1 and CaMKII in the MTrPs muscle tissue through immunofluorescence technology. Co-localization of TRPV1 and CaMKII was analyzed by pixel intensity scatter plots and fluorescence intensity profiles. More importantly, co-localization was quantified using Manders’ M1 coefficient, which represents the fraction of TRPV1 immunofluorescence overlapping with CaMKII. The results showed that Manders’ M1 coefficient increased in the MPS group, MPS+TRPV1-miRNA+SNT group and MPS+Ctrl-miRNA group, while decreased in the MPS+SNT group and MPS+TRPV1-miRNA group (as shown in Figure 7).

Figure 7.

Co-localization of TRPV1 and CaMKII in the MTrPs of rats in each group. (a) Co-localization of TRPV1 and CaMKII in MTrPs muscle tissues of each groups of rats, scale: 50μm. (b) quantitative analysis of the fraction of TRPV1 immunofluorescence overlapping with CaMKII in MTrPs muscle tissues of each groups of rats, compared with Control group, **p<0.01; compared with MPS group, ^#p<0.05, ^##p<0.01; compared with MPS+SNT group, ^{^}p<0.05; compared with MPS+TRPV1-miRNA group, ⁺p<0.05, ⁺⁺p<0.01. (c) Pixel intensity scatter plots of each groups of rats. (d) Fluorescence intensity profiles of each groups of rats.

Note: i Control group, ii MPS group, iii MPS+SNT group, iv MPS+TRPV1-miRNA group, v MPS+TRPV1-miRNA+SNT group, vi MPS+Ctrl-miRNA group.

The above results suggest that under the condition of TRPV1 virus intervention, SNT may be an additional damage to rats, reactivating TRPV1 in the later stage of treatment and leading to an increase in its phosphorylation level.

Local Injection of AAV9-TRPV1-RNAi Can Improve Endoplasmic Reticulum Injury in MPS Rats

In this study, the expression of TRPV1 in MTrPs of MPS rats was down-regulated by local injection of AAV9-TRPV1-RNAi, and the expression levels of ERS marker proteins (CHOP, BIP) were determined to explore the role of TRPV1 in ER injury in MPS rats. The results showed that compared with the MPS group, AAV9-TRPV1-RNAi significantly reduced the expression levels of CHOP and BIP in MTrPs. Furthermore, although down-regulation of CHOP expression was observed in the MPS+TRPV1-miRNA+SNT group, there was no statistically significant difference in BIP levels compared with the MPS group (as shown in Figure 8a and b).

Figure 8.

Effect of AAV9-TRPV1-RNAi local injection on the expression of CHOP and BIP in the MTrPs and the results of TEM of each group rats. (a and b) Western Blotting was used to detect the expression of BIP and CHOP protein in the MTrPs of each group of rats and the corresponding quantitative analysis results. Compared with the Control group, ***p<0.001, ****p<0.0001; compared with the MPS group, ^#p<0.05, ^##p<0.01, ^####p<0.0001; compared with the MPS+SNT group, ^{^}p<0.05, ^{^^^}p<0.001, ^{^^^^}p<0.0001; compared with the MPS+TRPV1-miRNA group, ⁺⁺p<0.01, ⁺⁺⁺⁺p<0.0001. (c) TEM of the MTrPs of rats in each group, scale bars: 2 μm (8000×) and 500 nm (30,000×).

Note: i Control group, ii MPS group, iii MPS+SNT group, iv MPS+TRPV1-miRNA group, v MPS+TRPV1-miRNA+SNT group, vi MPS+Ctrl-miRNA group.

In addition, the transmission electron microscopy results of MTrPs muscle tissues in different groups of rats showed that in the MPS+TRPV1-miRNA+SNT group, some sarcoplasmic reticulum still showed mild dilation, and the capsule cavity space was slightly widened. In contrast, in the MPS+TRPV1-miRNA group, the majority of the sarcoplasmic reticulum structures were normal, with only a very small number showing mild dilation and increased cystic cavity volume (as shown in Figure 8c).

The above results suggest that intervening in the expression of TRPV1 can effectively improve the muscle tissue structure and ER injury of MTrPs in MPS rats.

Discussion

This study explored the effects of SNT on ERS and pain behavior in MPS rats, with particular attention paid to the role of TRPV1 in this process. Our data suggest that TRPV1, CaMKII, and ERS are interrelated in the pathogenesis of MPS pain. Furthermore, the thermal effect of SNT may play a key role in regulating the TRPV1/CaMKII signaling pathway, a discovery that provides a new perspective for understanding the analgesic mechanism of SNT.

TRPV1, as a key factor in pain conduction, is significantly expressed in the MTrPs of the MPS rat model. This phenomenon suggests that TRPV1 may mediate the occurrence of mechanical hyperalgesia of MPS rats. As a member of the transient receptor potential family, TRPV1 conducts harmful mechanical stimuli in various pain diseases and plays an important role in the development of mechanical hypersensitivity reactions.³³ TRPV1 is widely distributed in the central and peripheral nervous systems of rats and mice.³⁴ Notably, there are tissue differences in the sublocalization of TRPV1 within cells: in skeletal muscle cells, TRPV1 is only located on the sarcoplasmic reticulum^35,36 and regulates the excitation and contraction processes of muscles by influencing Ca²⁺ homeostasis.³⁵ Previous studies have shown that TPPV1 is also expressed in the small muscle afferent nerve fibers of the dorsal root ganglion. Direct injection of the TRPV1 agonist capsaicin can activate muscle nociceptive receptors. The use of drugs to block TRPV1 can alleviate exercise-induced muscle hyperalgesia in rats.^37,38 These findings are consistent with our research results, that is, down-regulating the expression of TRPV1 by local injection of TRPV1 adeno-associated virus can significantly alleviate mechanical hyperalgesia in MPS rats.

In addition, our research has found that SNT may improve the structural damage of MTrPs muscle fibers by regulating the phosphorylation level of TRPV1. As one of the main conduction channels mediating peripheral pain, the regulatory mechanisms of TRPV1’s activity are complex and diverse. For instance, endogenous ligand regulatory pathways can directly influence the activation, sensitization or desensitization processes of channels.^39,40 When stimulated by external factors, the TRPV1 channel opens, allowing cations such as Ca²⁺ to flow into the cell.⁴¹ This process is associated with an increase in intracellular Ca²⁺ concentration and the activation of downstream signaling pathways, including CaMKII.⁴² However, whether TRPV1 directly activates CaMKII via Ca²⁺ signaling requires further investigation.

CaMKII is a member of the calmodulin-dependent protein kinase family. The activation of CaMKII enhances the excitability of neurons and plays an important role in inflammatory pain.⁷ Studies have found that abnormal CaMKII activity can disrupt the Ca²⁺ balance within skeletal muscle cells, leading to apoptosis of skeletal muscle cells and ultimately causing structural damage and functional abnormalities in skeletal muscle.⁷ Meanwhile, studies have also shown that CaMKII is an important factor promoting the increase of intracellular Ca²⁺. Active CaMKII can bind to the sites on TRPV1, leading to the continuous activation of TRPV1 channels, affecting the entry of Ca²⁺ into the sarcoplasmic reticulum, regulating the concentration of Ca²⁺ within the sarcoplasmic reticulum, inducing ERS, and increasing inflammatory signal transduction, thereby causing pain.⁴³ Furthermore, studies have confirmed that under continuous thermal stimulation or prolonged exposure to the activated state, the TRPV1 channel will show a desensitization state. After entering the desensitization state, the neurosensory organs become no longer sensitive to harmful stimuli, including capsaicin.⁴⁴ Due to the desensitization or inactivation of the TRPV1 channel, the inflow of Ca²⁺ is reduced, resulting in a decrease in intracellular Ca²⁺ concentration. This reduction can inhibit Ca²⁺-dependent signaling pathways, reduce the amplification of pain signals, and also affect the activity of CaMKII.⁴⁵ The activation of CaMKII depends on the formation of the Ca²⁺/CaM complex. Therefore, a decrease in Ca²⁺ concentration may lead to a decline in CaMKII activity, thereby reducing the transmission of pain signals.⁴⁶

Furthermore, as the occurrence of ERS is closely related to abnormal regulation of Ca²⁺, this study speculates that desensitization or inactivation of the TRPV1/ CaMKII channel may help restore normal ER function and reduce ERS.

The results show that SNT interferes with CHOP and BIP protein expression in MTrPs of MPS rats, regulates ERS, improves MTrPs muscle tissue disorder, and effectively alleviates pain in MPS rats, suggesting that its analgesic effect may be partially mediated by ERS relief.

The pathological role of ERS in chronic musculoskeletal pain has received increasing attention: persistent nociceptive input from MTrPs induces intracellular Ca²⁺ overload, disrupts ER calcium homeostasis, and triggers the UPR through the three major sensors—PERK, IRE1α, and ATF6.⁴⁷ CHOP and BIP are key markers of ERS activation. The former promotes apoptosis and inflammation, while the latter is the master regulator of UPR initiation.⁴⁸ Both are potential targets for chronic pain treatment.

Although direct studies on the mechanism of SNT-mediated ERS regulation remain limited, evidence regarding electroacupuncture (EA) modulation of ERS provides an important theoretical framework. Studies have shown that EA can alleviate neuropathic pain by inhibiting ERS in the central and peripheral nervous systems.⁴⁹ For example, 2 Hz EA can suppress BIP-IRE1α-mediated UPR in the ACC,⁵⁰ attenuating downstream p38/JNK signaling and neuronal sensitization.⁵¹ This suggests that thermal and mechanical stimuli associated with acupuncture may share molecular mechanisms in ERS regulation.

The thermal component is a distinctive feature of SNT that differentiates it from traditional acupuncture. The needle tip temperature of SNT is typically 40–45°C. This moderate thermal stimulation may exert a hormetic effect rather than exacerbating ERS. The potential mechanisms include: transient activation of HSP70 and HSP90, enhancing protein folding capacity;^52,53 modulation of SERCA activity, restoring intracellular Ca²⁺ homeostasis;⁴⁸ and inhibition of the PERK-eIF2α-ATF4-CHOP pro-apoptotic axis—the downregulation of CHOP observed in this study serves as supporting evidence.

Of particular importance, this study further revealed that co-localization signals of TRPV1 and CaMKII in MTrPs muscle tissue were significantly elevated when MPS rats exhibited pain. Conversely, SNT downregulated the expression and co-localization of both TRPV1 and CaMKII, suggesting that SNT may modulate ERS via the TRPV1-CaMKII axis. The molecular logic underlying this axis is as follows: TRPV1 activation mediates Ca²⁺ influx, leading to CaMKII activation, subsequent disruption of ER Ca²⁺ homeostasis, and ultimately ERS triggering.^11,54 Therefore, SNT may restore ER calcium homeostasis and attenuate UPR through the inhibition of TRPV1 phosphorylation and CaMKII activity. This hypothesis is supported by evidence that TRPV1 antagonists and CaMKII inhibitors reduce ERS markers in various pathological models.^55,56

In conclusion, SNT may affect the ERS level of MTrPs by regulating the expression of TRPV1/CaMKII, and alleviate mechanical hyperalgesia in MPS rats. Given that SNT combines the dual effects of acupuncture and hyperthermia, this study suggests that the main therapeutic effect of this therapy on MPS rats is likely to stem from its thermal effect, that is, through long-term hyperthermia, it promotes the down-regulation of TRPV1 and its phosphorylation levels.

The thermal effect of SNT may have a direct impact on the TRPV1 channel. On the one hand, thermal effects may cause temporary desensitization of TRPV1, reducing the inflow of Ca2+ and thereby lowering the activation of CaMKII. On the other hand, hyperthermia may promote blood circulation, improve local tissue oxygenation and nutrient supply, help restore ER function, reduce ERS, improve structural damage to muscle fibers, and ultimately relieve pain in MPS rats.

In summary, this study demonstrates that SNT may effectively alleviate ERS and pain in MPS rats by modulating the TRPV1/CaMKII signaling pathway, thereby providing a multimodal therapeutic strategy for MPS. Moving forward, our research will further explore the clinical translational potential of SNT to provide additional insights into comprehensive treatment strategies. Nevertheless, this study has certain limitations.

We primarily focused on the effects of SNT on TRPV1 and ERS expression; however, SNT may also exert therapeutic effects through other yet-to-be-elucidated mechanisms. The animal model employed in this study may not fully recapitulate the complexity of human MPS. Future investigations are warranted to further explore the diverse mechanisms underlying SNT and to validate these findings in clinical practice. Additionally, this study did not directly assess dynamic changes in ER Ca²⁺ concentration following SNT intervention; future studies could integrate ER Ca²⁺ imaging with branch-specific pharmacological modulation of the UPR to further clarify the precise mechanisms by which SNT regulates ERS. Moreover, the present study is correlational in nature and did not confirm a direct interaction between TRPV1 and CaMKII; future research should employ targeted mechanistic experiments to establish causality.

Additionally, this study did not resolve the cell type-specific expression patterns of TRPV1 and CaMKII; future work should employ dual immunofluorescence, single-cell RNA sequencing, or laser capture microdissection combined with qPCR to refine their functional localization. Second, AAV9 exhibits broad tropism, and locally injected AAV9-TRPV1-RNAi may be taken up by nerve terminals and retrogradely transported to dorsal root ganglion somata. Consequently, the observed analgesia might partly reflect peripheral neuronal TRPV1 knockdown rather than direct sarcoplasmic reticulum targeting. Although intramuscular focal injection and a Ctrl-miRNA control were used to mitigate this confounder, complete exclusion of neuronal transduction could not be guaranteed. Future studies should utilize muscle-specific promoters, tissue-restricted viral tracing, or muscle-cell–conditional TRPV1 knockout rats, together with calcium imaging, to directly validate the regulatory role of miRNA-TRPV1 in sarcoplasmic reticulum calcium homeostasis.

Conclusions

SNT may exert its analgesic effect through thermal inhibition of TRPV1 phosphorylation and CaMKII activity, with concomitant downregulation of ERS marker proteins (CHOP and BIP) in MTrPs of MPS rats, attenuation of ER damage in MTrPs muscle tissue, amelioration of the pathological status of damaged myofascia, and alleviation of mechanical hyperalgesia in MPS rats. This study provides experimental evidence and mechanistic insights for the clinical application of SNT in the treatment of MPS. While these findings are consistent with a potential regulatory axis, the causal relationship between TRPV1-mediated CaMKII activation and ERS modulation remains to be established through more targeted mechanistic studies.

Funding Statement

This work was supported by Guizhou Province Science and Technology Plan Project (Grants No: Qianke He Foundation-ZK[2023]Key 044), Guizhou Province Science and Technology Plan Project (Grants No: Qianke He Foundation-ZK[2024]Key 036), Guizhou Province Science and Technology Plan Project (Grants No: Qianke He Foundation-ZK[2023]general 389), Guizhou Province Science and Technology Plan Project (Grants No:Qianke He Foundation-ZK[2023]general 370) and Guizhou Province Science and Technology Plan Project (Grants No: Qianke He Foundation-ZK[2024]general 211).

Abbreviations

SNT, Silver needle thermotherapy; MPS, Myofascial pain syndrome; MTrPs, myofascial trigger points; MWT, The mechanical withdrawal threshold; TRPV1, Transient receptor potential vanilloid 1 receptor; CaMKII, Ca/calmodulin-dependent protein kinases or CaM kinases; ER, Endoplasmic reticulum; ERS, Endoplasmic reticulum stress; p-TRPV1, Phosphorylated Transient Receptor Potential Vanilloid Type 1; BIP, Binding Immunoglobulin Protein; CHOP, C/EBP Homologous Protein; HE, Hematoxylin-eosin staining; TEM, Transmission electron microscope; SD, Sprague-Dawley.

Data Sharing Statement

Materials related to this study can be obtained from Dr. Xinyao Chen (m18610166824@163.com) with a reasonable request.

Ethics Approval and Consent to Participate

All experimental procedures involving animals were authorized by the Institutional Animal Care and Utilization Committee.

Disclosure

The authors report no conflicts of interest in this work.