Electroacupuncture-induced Changes in Motor Protein Pathways in Postpartum Stress Urinary Incontinence

Summary

Electroacupuncture improved urodynamic parameters in a rat model of postpartum stress urinary incontinence and was associated with modulation of motor protein–related pathways. These findings suggest potential molecular mechanisms underlying its therapeutic effects.

Abstract

Postpartum stress urinary incontinence (SUI) significantly impacts women's quality of life, yet the underlying molecular mechanisms of effective treatments remain elusive. This study aimed to investigate the therapeutic effects and molecular pathways of electroacupuncture (EA) in a rat model of SUI. We established the SUI model using vaginal balloon dilation (VBD) and randomized subjects into model and EA-treated groups. Functional recovery was assessed by urodynamic testing, which showed that EA significantly increased leak point pressure (LPP) and maximum bladder capacity (MBC). Histopathological analysis using hematoxylin and eosin and Masson’s trichrome staining revealed improved muscle architecture and a 42.3% reduction in collagen deposition, along with suppressed inflammatory responses. Quantitative proteomics identified 2,907 differentially expressed proteins (DEPs), with the STRING database highlighting motor protein pathways as central targets. Specifically, EA selectively downregulated core proteins, including MYL1, TNNI2, MYLPF, TNNT3, and TNNT1. These findings, validated by western blot and qPCR, suggest that EA alleviates SUI by modulating muscle contractility and remodeling the pelvic floor, providing insight into potential mechanisms underlying targeted SUI therapy.

Introduction

Stress urinary incontinence (SUI) is a common pelvic floor disorder that affects 15–35% of postpartum women^1,2, significantly impacting their quality of life and mental health. In addition to the physical symptoms, postpartum SUI creates a substantial psychosocial burden; many women experience embarrassment, reduced self-esteem, and social withdrawal. This condition can diminish feelings of femininity and lead to reluctance to engage in various activities, which may worsen feelings of isolation and contribute to postpartum depression³. SUI also presents economic challenges due to the costs associated with continence products and healthcare visits. While treatment options such as pelvic floor training and surgical interventions exist, their limitations in efficacy and the risk of recurrence highlight the need for more effective and less invasive therapies that target the underlying causes of postpartum SUI^4,5. The pathogenesis of SUI is multifactorial, involving pelvic floor muscle dysfunction, degradation of connective tissues, and neuromuscular injury. Mechanical trauma during vaginal delivery is a primary contributor to postpartum SUI^6,7. Additionally, dysregulation of the remodeling of the extracellular matrix—marked by collagen fiber depletion and abnormal elastin metabolism—compromises pelvic support structures⁸. Emerging evidence suggests that impaired contractility of the striated urethral sphincter muscles is a critical factor driven by aberrant expression of muscle-specific proteins, including motor proteins that regulate actin-myosin interactions⁹.

Electroacupuncture (EA), a modern adaptation that integrates traditional acupuncture with electrical stimulation, enhances therapeutic efficacy by modulating neural circuits and promoting tissue repair processes^10,11,12,13. EA has shown success in managing pain, neurodegenerative diseases, and urological disorders, including overactive bladder and chronic prostatitis. Its effects are mediated through dual mechanisms: (1) neuromodulation, which involves activation of somatic sensory nerves and central pain-inhibitory pathways¹⁴, and (2) tissue-level regulation, promoting angiogenesis, reducing inflammation, and restoring extracellular matrix homeostasis^15,16,17. Notably, EA has improved pelvic floor muscle function in SUI patients, as demonstrated by increased urethral closure pressure and enhanced electromyographic activity. However, the molecular pathways underlying EA’s therapeutic effects on SUI—particularly its role in regulating muscle contractility-associated proteins—remain inadequately understood.

During urine storage, the sustained contraction of the urethral sphincter is maintained through the precise regulation of motor proteins and their regulatory subunits, which orchestrate actin-myosin interactions in striated muscle^18,19,20. In the resting state, inhibitory subunits such as troponin I2 (TNNI2)—a fast-twitch skeletal muscle-specific isoform—bind to actin filaments, stabilizing the troponin-tropomyosin complex to block myosin-binding sites and enforce muscle relaxation^21,22. Concurrently, myosin light chain 1 (MYL1), a regulatory subunit modulating myosin ATPase activity, remains unphosphorylated under low calcium conditions, which limits the efficiency of cross-bridge cycling²³. Upon neural activation during physical stress, calcium influx triggers conformational changes in the troponin complex: calcium binds to troponin C (TNNC), displacing TNNI2 from actin, retracting tropomyosin, and exposing myosin-binding sites. Simultaneously, calcium/calmodulin-dependent myosin light chain kinase (mLCK) phosphorylates MYL1, enhancing myosin ATPase activity and facilitating the formation of forceful actin-myosin cross-bridges. Structural subunits, such as troponin T1 (TNNT1), ensure the proper alignment of the troponin complex on actin, enabling synchronized responses to calcium signals^24,25. In stress urinary incontinence (SUI), pathological overexpression of inhibitory subunits (e.g., TNNI2) prolongs the actin-myosin blockade, while dysregulation of MYL1 and TNNT1 disrupts myosin assembly and calcium sensitivity, collectively impairing sphincter contractility^5,26. These molecular anomalies correlate with histological hallmarks of SUI, including disorganized muscle fibers and reduced urethral resistance. Restoring the physiological expression of TNNI2, MYL1, and TNNT1 thus emerges as a therapeutic priority, as it recalibrates the contraction-relaxation equilibrium, enhancing the sphincter’s adaptive capacity to generate closure pressure during stress events.

Protocol

All procedures were approved by the Animal Ethics Committee of Guangzhou Miles Biotechnology Co., Ltd., approval number is IACUC-MIS2023045.

Establishment of the SUI Rat Model
A total of 20 female Sprague-Dawley (SD) rats, aged 6–8 weeks, were utilized to create a model of pelvic floor injury, simulating childbirth. To induce vaginal balloon dilation (VBD), the rats were anesthetized via intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). A deflated 10 Fr Foley catheter was carefully inserted into the mid-vaginal canal, and then the balloon was inflated with 3 mL of sterile saline. The balloon was maintained in place for 4 hours to replicate the prolonged mechanical stretching of pelvic floor muscles and compression of the pudendal nerve associated with labor. The modeling period was 7 days. On the 8th day, 6 animals were randomly selected for urodynamic testing.

The successful establishment of the model was verified through urodynamic testing and the measurement of serum lactate dehydrogenase (LDH) levels, which serve as indicators of muscle injury. After confirming successful modeling, SUI rats were randomly divided into a sham-operated group and a treatment group, with five rats in each group. At the same time, five normal SD rats were provided as a control group. In the sham group, acupuncture needles were inserted at the acupoints BL23 (located 6 mm lateral to the L2 spinous process) and GB30 (located posterior to the hip joint) without any electrical stimulation. In the electroacupuncture group, the same acupoints were stimulated using an electroacupuncture device, following the standard clinical treatment method. The electroacupuncture device was set to a 1 Hz sparse-dense waveform at 2 V. Each intervention lasted 30 min per day over 10 consecutive days. All animals were monitored daily and housed under standard laboratory conditions. At the end of the experiment, urodynamic testing was performed on the animals in each group. Subsequently, each group of rats was anesthetized with a mixture of 3% isoflurane and 1.0 L/min oxygen. After the rats lost consciousness, the isoflurane concentration was adjusted to 5%, and samples were collected after the rats died from respiratory failure.

Urodynamic tests
Following isoflurane-induced anesthesia, animals were positioned in supine posture and secured. A midline lower abdominal incision exposed the bladder¹⁸. An epidural catheter was implanted through a 2 mm diameter fistula at the bladder dome, advanced 0.5–1.0 cm into the lumen, and secured with an 11-0 suture via purse-string technique. The catheter was connected to a pressure monitoring system and perfused with 37 °C methylene blue-stained saline at 10 mL/h via microsyringe pump, with concurrent intravesical pressure monitoring. Maximum bladder capacity was calculated as the product of the infusion duration (the interval between infusion initiation and the first urethral fluid droplet) and the flow rate. Leak point pressure (LPP) was determined by recording the peak intravesical pressure immediately after the abrupt release of manually applied abdominal compression. All parameters were acquired using a urodynamic analysis system, with triplicate measurements per metric. All samples were randomly coded and analyzed in a single-blind manner by three independent operators, each performing a single measurement. The results from the three independent measurements were used for statistical analysis.

Measure the serum lactate dehydrogenase (LDH) level
On postoperative day 7, approximately 1 mL of whole blood was obtained from the tail vein of each rat. The collected samples were left at room temperature for 1 h to allow clot formation, after which they were centrifuged at 2,000 × g for 15 min. Approximately 0.5 mL of supernatant was carefully collected as serum. Serum lactate dehydrogenase (LDH) levels were quantitatively determined using a D-lactate dehydrogenase assay kit according to the manufacturer's protocols.

Proteomic Profiling and Bioinformatics Analysis
Quantitative proteomic analysis was conducted on urethral smooth muscle tissues isolated from model and treatment groups. Proteins were identified from mass spectrometry raw data using Proteome Discoverer 2.4 with a 1% false discovery rate threshold. Bioinformatic analysis of differentially expressed proteins (DEPs) was performed using R-based packages: differential expression analysis was conducted using the limma package (|log2FC| > 1, adjusted p < 0.05), gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the clusterProfiler package, with enriched terms/pathways considered significant at adjusted p < 0.05 and q-value < 0.2; hierarchical clustering was visualized using the pheatmap package. A protein-protein interaction (PPI) network was constructed using the STRING database (v11.5, confidence score > 0.7).

Hematoxylin-eosin staining
Rat smooth muscle tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Serial sections (5 µm thickness) were prepared using a microtome and stained using a standardized H&E staining kit. Morphological evaluation was performed using bright-field microscopy at 40×–400× magnification, focusing on the structural integrity of muscle tissue, inflammatory cell infiltration, and matrix remodeling.

Masson's Trichrome staining
According to the experimental protocol approved by the ethics committee, we collected samples from the animals on the tenth day of treatment. Rat muscle tissues were fixed in 4% paraformaldehyde and paraffin-embedded. Serial sections (5 µm thickness) were cut using a rotary microtome. Deparaffinized sections were stained with Masson's trichrome. Stained sections were imaged under a bright-field microscope at 100× magnification.

Western blotting
Total proteins were extracted from rat smooth muscle tissue by mechanical disruption in RIPA lysis buffer maintained at low temperature and supplemented with protease inhibitors. The lysates were clarified by centrifugation at 10,000 × g for 15 min at 4 °C, and the protein-containing supernatant was collected for analysis. Protein concentration was determined with a bicinchoninic acid (BCA) assay using bovine serum albumin as the calibration standard. Equal amounts of protein (30 µg) were loaded onto 10% or 12% SDS–polyacrylamide gels and separated under reducing conditions. After electrophoresis, proteins were transferred onto PVDF membranes with a pore size of 0.22 µm. Membranes were incubated in 3% BSA for 1 h at room temperature to block nonspecific binding sites, followed by overnight incubation at 4 °C with primary antibodies recognizing GAPDH, MYL1, TNNI2, TNNT1, TNNT3, and MYLPF. After washing with TBST three times, HRP-labeled secondary antibodies were applied for 2 h at room temperature. Signal detection was performed using a chemiluminescent substrate, and images were captured with a luminescence imaging system. Quantification of protein expression was carried out by densitometric analysis, with GAPDH serving as the internal reference.

Quantitative real-time PCR (qPCR)
Tissue samples were lysed using a total RNA extraction reagent, and RNA was isolated following the manufacturer’s instructions provided with the RNA extraction kit. Gene-specific primers were designed using dedicated software (Table 1) and their specificity was verified through BLAST analysis (≥18 bp homology, Tm = 58–62 °C). RNA was then reverse transcribed using a reverse transcription kit, and the expression of the target gene was detected using a SYBR Green qPCR detection kit.

Statistical analysis
Statistical comparisons between two groups were performed using an independent-samples t-test, whereas differences among multiple groups were evaluated using one-way ANOVA followed by Bonferroni post hoc analysis. Data are expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). A value of p < 0.05 was considered indicative of statistical significance.

Results

Urodynamic parameters in rats with vaginal balloon dilation injury
Bladder function, including urinary continence and maximum bladder capacity, was evaluated pre- and post-modeling. Urodynamic analyses revealed statistically significant deteriorations in both maximum bladder capacity (MBC) and leak point pressure (LPP) following vaginal balloon dilation (VBD) compared to baseline (P < 0.05). The MBC decreased from 2.34 ± 0.21 mL to 0.75 ± 0.32 mL (Table 2 and ...

Discussion

SUI, a common urological disorder affecting postpartum women, is pathologically linked to pelvic floor support structure damage during pregnancy and delivery^7,27,28. Characterized by involuntary urine leakage during activities that increase intra-abdominal pressure (e.g., coughing, sneezing, or exercise), SUI significantly impairs patients' physical and psychological well-being, as well as their quality of life

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm PVDF membranes	Millipore	ISEQ00010
2× UniTaq SYBR qPCR Master Mix (ROX-)	TIANYAbiotech	P1504
4% paraformaldehyde	GSbioth	GS539A
BCA assay kit	Dingguo Biotech	BCA02
bright-field microscopy	MSHOT	ml31
D-lactate dehydrogenase assay kit	Beyotime	P0392S
ECL Prime reagent	Cytiva	RPN2232
Electrophoresis apparatus	WIX	miniPro4
Fully Automated Chemiluminescence Image Analysis System	FUJIFILM	LAS-3000
GAPDH antibody	Affinity Biosciences	T0004
HRP-conjugated secondary antibodies	Affinity Biosciences	S0001,S0002
ImageJ 1.54 software	NIH
Masson's trichrome staining kit	GSbioth	GS800A
MYL1 antibody	Cloud-Clone	PAB105Ra01
MYLPF antibody	Affinity Biosciences	DF9048
name	company	catalog Number
PowerLab Chart v5.2.1	ADInstruments
Primer Premier 5.0 software	PREMIER Biosoft
PrimeScript RT Master Mix	TAKARA	RR036A
Proteome Discoverer 2.4 software	Thermo Fisher Scientific
Rapid semi-dry rotary film apparatus	Bio-Rad	788BR04132
Real-time fluorescence qPCR quantitative system	TIANLONG	TL100
RIPA lysis buffer	Beyotime	P0013B
RNA extraction kit	Aidlab	RN70
rotary microtome	Leica	RM2235
SDS-PAGE gels	Beyotime	P0052A,P0053A
standardized H&E staining kit	GSbioth	GS700A
TBST buffer	biosharp	BL346A
TNNI2 antibody	Cloud-Clone	PAD230Ra01
TNNT1 antibody	Cloud-Clone	PAD231Ra01
TNNT3 antibody	Cloud-Clone	PAD233Hu01
total RNA extraction reagent	biosharp	BL258A
Urodynamic testing system	Techman	BL-420