Electroacupuncture Alleviates Bladder Overactivity via Inhabiting Bladder P2X3 Receptor

Electroacupuncture (EA) has been widely applied for overactive bladder, but the mechanism of its action remains to be clarified. This study was aimed to investigate EA regulating the effect of purinergic signaling in the OAB of rats. Electroacupuncture (continuous wave, 30 Hz, 1 mA) was applied to stimulate the Ciliao point (BL32) and the Huiyang point (BL35) of rats. Results showed that when the P2X3 receptor in bladder peripheral level and the spinal cord central level was involved in the bladder micturition reflex of the afferent signaling, intravenous administration P2X3 antagonist AF-353 can significantly inhibit urination in naive rats and OAB of rats and increase bladder volume and micturition pressure. EA stimulation alleviated bladder overactivity significantly and after the P2X3 receptor was blocked, the EA effect was weakened. EA stimulation can effectively reduce the P2X3 mRNA and protein expression in OAB of rats, spinal cord (L6-S1), and DRG (L6-S1) and can significantly reduce the number of positive P2X3 cells in OAB of rats, spinal cord (L6-S1), and DRG (L6-S1). These findings suggest that EA stimulation could alleviate bladder overactivity, and the function is closely related to the inhabited P2X3 receptor in the bladder.


Introduction
Overactive bladder (OAB) is defined as a symptom complex comprising urinary urgency, with or without urgency incontinence, usually with frequency and nocturia, in the absence of other local or metabolic factors that would account for the symptoms [1]. Many studies have proved that electroacupuncture stimulation could alleviate bladder overactivity, improve urine dynamics condition, and increase the bladder capacity [2][3][4]. e effect of acupuncture on bladder activity has been confirmed, but the underlying mechanisms are incompletely understood.
Purinergic signaling is an important contributor to normal urine storage and voiding and further implicated in bladder functional disorders related to interstitial cystitis/ painful bladder syndrome, neurogenic bladder secondary to spinal cord injury, lower urinary tract symptoms, bladder outlet obstruction, diabetes, and aging [5][6][7][8][9][10][11]. In the bladder, P2X receptors act on the detrusor muscle in a motor role, whereas P2X 3 receptors are thought to have a sensory role. is sensory function comprises both a physiological action in normal bladder filling and emptying cycles and a nociceptive action in pathological states. It has been shown that in bladder sensory neurons, P2X 3 receptors are expressed and function as homomeric or heteromeric (P2X2/3) channels, as indicated by the use of selective antagonists [12,13].
ere was evidence of increased P2X 3 expression in the urothelium of the OAB group [14]. e role of the P2X 3 receptors in urgency and the pathophysiology of OAB has become the focus of intense interest. erefore, the present study was performed in rats to examine the following: (1) the effects of the P2X 3 receptor in the bladder peripheral system contributor to bladder physiological action and pathological overactivity; (2) whether electroacupuncture on bladder overactivity is related to the P2X 3 receptor mediation pathway.

Animals and Study Design.
All experiments were performed on 40 female Sprague Dawley rats weighing 200-300 g, obtained from the Experimental Animal Center, Shanghai University of Traditional Chinese Medicine. e rats were maintained under standard laboratory conditions with a 12-h light/12-h dark cycle and free access to food pellets and tap water. e room temperature was maintained at 22 ± 1°C and relative humidity at 45% to 50%. All rats were acclimatized for at least 3 days before the experiments. All experiments were performed complying with international ethical standards in the care and use of animals and were approved by the Institutional Animal Care Committee of Shanghai University of Traditional Chinese Medicine.

Acupuncture Treatment.
We followed the methods of Feng et al. [15], in which conscious rats were treated by acupuncture. One acupuncture needle (diameter, 0.22 mm) was positioned almost vertically underneath the periosteum about 5 mm lateral to the midline of the S 2 (BL32, Ciliao), and the other acupuncture needle (diameter, 0.22 mm) was positioned almost vertically underneath the periosteum about 5 mm lateral to the midline of the coccyx (BL35, Huiyang), bilateral symmetry. It was stimulated electrically at both right and left points with a frequency of 30 Hz and intensity of 1 mA. e experiment was divided into in vivo experiment and in vitro experiment. In in vivo experiment, acupuncture was performed for 1 minute each time, and in in vitro experiment, acupuncture was performed for 3 days.

CYP-Induced OAB Operation and Cystometry.
Rats were induced by intraperitoneal injecting with CYP (Sigma-Aldrich) (150 mg/kg). Cystometry was performed under 48 h after CYP injection, and rats were anesthetized by intraperitoneal injection of 20% urethane (0.5 ml/100 g). One end of the polyethylene catheter (diameter 0.42 mm) was inserted into the lumen through a small incision at the dome of the bladder for infusion of saline (0.9% NaCl, 0.2 ml/min), and the other was connected to a pressure transducer for recording intravesicular pressure. Another catheter was inserted into the right femoral artery and femoral vein, respectively, for recording the blood pressure and drug administration.

Hematoxylin-Eosin
Staining. Bladder samples were fixed with 4% paraformaldehyde overnight and stained with hematoxylin and eosin as previously described [16].

RNA Extraction and Quantitative RT-PCR.
e rats were euthanized under anesthesia of 20% urethane (0.5 ml/ 100 g), and then their abdomen were opened and urinary bladder were aseptically isolated and immediately frozen in liquid nitrogen and placed immediately to store at −80°C until use. Each sample was collected in 0.5 mL ice-cold Trizol (Invitrogen, Carlsbad, CA, USA). After its homogenization and sonification, RNA was extracted with chloroform, precipitated with isopropyl alcohol, and dissolved in 40 μl RNAse-free DEPC. e RNA concentration and purity were analyzed by a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE), with the spectral absorption at 260 and 280 nm. For cDNA synthesis, oligo (dT) primers, 1 μg of each total RNA sample, and the RevertAid ™ First Strand cDNA Synthesis Kit (Fermentas, Ontario, Canada) was used, following the guidelines of the manufacturer. cDNA samples were placed on ice and stored at −20°C until further use. Prior to the analysis, 10 μl of each cDNA sample was diluted with 90 μl of MilliQ water.
qPCRs were performed with the Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Quantitative RT-PCR was carried out in 20 μL buffer solution containing 1 μL of diluted cDNA sample, 10 μL 2 × SYBR green II qRT-PCR kit (Toyobo, Osaka, Japan), 1 μL of each primer (5 μM), and 8 μl of MilliQ water. e primer sequences are listed in Table 1. Cycling conditions were 10 min 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. After cycling, a melting protocol was performed with 15 sec at 95°C, 1 min at 60°C, and 15 sec at 95°C, to control for product specificity. e fold change (FC) in target gene cDNA relative to the selected endogenous control gene was determined as follows: Ct values were defined as the number of the PCR cycles at which the fluorescence signals were detected.
2.6. Western Blotting. Total protein was extracted from the frozen tissues, and protein concentration was quantified using the BCA protein assay kit (Beyotime Biotechnology Co., Haimen, Jiangsu, China). Samples (30 μg total protein per loading) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and electrotransfered onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). e membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with the primary antibodies recognizing β-actin (mouse monoclonal, 1: 5000, Abcam, Cambridge, UK) and P2X 3 (rabbit polyclonal, 1 : 1000, Abcam, Cambridge, UK) at 4°C overnight. en, the membranes were incubated with a horseradish peroxidase-conjugated antimouse (1: 5000, Abcam, Cambridge, UK) or goat anti-rabbit HRP (1: 4000, Abcam, Cambridge, UK). e signal was visualized with the ECL Plus reagent (GE healthcare, Buckinghamshire, UK) and exposed onto the X-ray film (Kodak, Rochester, NY, USA). Protein ratios were calculated based on densitometrical quantification of scanned films in Image J software.

Data Analysis.
All data were expressed as mean ± SEM. Statistical significance was tested using one-way analysis of variance (ANOVA), with values of P < 0.05 considered significant.

OAB Model Evaluation.
After 48 h of CYP intraperitoneal injection, the rat bladder wall was thick, swollen, and hyperemia. Under the microscope, we can observe the exfoliation of bladder epithelial cells, the edema of tissue, and the infiltration of inflammatory cells. Detrusor cells were larger, which showed a change in the vacuole model and the colour of the cytoplasm was deeper ( Figure 1). In urodynamics, OAB rats bladder detrusor showed an unstable contraction wave. Compared with naive rats (0.22 ± 0.01 mL, n � 20), the bladder volume was significantly reduced in OAB rats (0.10 ± 0.01 mL, n � 20) (P < 0.001). Compared with naive rats (36.01 ± 1.10 mmHg, n � 20), the bladder micturition pressure was significantly decreased in OAB rats (20.84 ± 0.78 mmHg, n � 20) (P < 0.001).
e OAB model was made successful ( Figure 2).

Effects of AF-353 on EA Stimulation.
In the naive rats (n � 4), after intravenous administration AF-353 (0.1 mL 300 μM), the bladder volume (0.27 ± 0.01 mL) was significantly large than before (0.17 ± 0.01 mL) (P < 0.001) and the micturition pressure (27.20 ± 1.25 mmHg) was no obviously different than the control (25.10 ± 1.63 mmHg) (P � 0.212). When the bladder activity got back to the normal, EA was treated and the bladder volume (0.28 ± 0.01 mL) and the micturition pressure (26.88 ± 1.30 mmHg) were significantly increased compared with that of the control (P < 0.001). In EA treatment after intravenous administration, the bladder volume (0.27 ± 0.01 mL) was significantly increased compared with the baseline (P < 0.001) but was significantly decreased compared with only EA treatment (P < 0.001) and was no significantly different compared with only intravenous administration (P � 0.06). e micturition pressure (26.80 ± 1.15 mmHg) was no significantly different compared with only intravenous administration and only EA treatment. In OAB rats (n � 4), after intravenous administration AF-353 (0.1 ml 300 μM), the bladder volume (0.18 ± 0.01 mL) was significantly larger than before (0.06 ± 0.00 ml) (P < 0.001) and the micturition pressure (25.39 ± 1.16 mmHg) was significantly higher than before (23.61 ± 0.60 mmHg) (P � 0.048). When the bladder activity got back to the normal, EA was treated and the bladder volume (0.18 ± 0.01 mL) and the micturition pressure (25.92 ± 1.17 mmHg) were significantly increased compared with the control (P < 0.001). In EA treatment after intravenous administration, the bladder volume (0.17 ± 0.01 mL) and the micturition pressure (25.27 ± 1.12 mmHg) were significantly increased compared with the control (P < 0.001, P � 0.044), but there was no significant difference compared with only intravenous administration and only EA treatment (P � 0.096 P � 0.268, P � 0.617, P � 0.051) ( Figure 5).

Discussion
is study demonstrated that the P2X 3 receptor which in bladder peripheral level was involved in the bladder micturition reflex of the afferent signaling, and intravenous administration P2X 3 antagonist AF-353 can significantly inhibit naive rats and OAB rats bladder urination and increase bladder volume and micturition pressure. EA stimulation suppressed bladder overactivity significantly, and after the P2X 3 receptor function was blocked by AF-353 intravenous administration, the EA effect was weakened.
Purinergic signaling is a term that relates to adenosine triphosphate (ATP) binding to its receptor (purinergic receptors such as P2X and P2Y subtypes). is pathway has been implicated in bladder functional disorders related to interstitial cystitis/painful bladder syndrome, neurogenic bladder secondary to spinal cord injury, lower urinary tract symptoms, diabetes, and aging. Purinergic signaling occurs at multiple sites, including the central nervous system, peripheral motor and sensory nerves, detrusor smooth muscle,  Figure 6: (a) Relative expression level of the P2X 3 mRNA in the bladder. (b) Relative expression level of the P2X 3 protein in the bladder. * Versus the control rats group, P < 0.05. △Versus the OAB rats group, P < 0.05. and bladder urothelium [8]. Purinergic signaling is increasingly appreciated as an important contributor to the normal bladder functions of urine storage and voiding. e translation of disease and injury to abnormal sensory and motor function, resulting in lower urinary tract symptoms, is in part mediated by the interaction of adenosine 5′-triphosphate and related purines with specific purinergic receptors [17].    Evidence-Based Complementary and Alternative Medicine e role of purinergic signaling in the regulation of urinary bladder function was established when published reports demonstrated that P2X 2 /P2X 3 knockout animals had significantly altered bladder function. P2X 3 receptors are preferentially found on nerve fibres and are involved in bladder afferents signaling and pain transduction [18,19].
ere was evidence of increased P2X 3 expression in the urothelium of the BOO group [20] locking the purinergic component which may be helpful in addition to antimuscarinics in blocking BOO-induced detrusor overactivity [21]. A study suggested that intravenous injection of the P2X 3 antagonist AF-353 decreased the frequency of sensory field potentials evoked by activation of bladder noxious pathways and decreased the frequency of nonvoiding contractions in rats with neurogenic bladder hyperactivity [22]. Our studies were consistent with this. e results showed that intravenous administration of AF-353 could significantly inhibit naive rats and OAB rats' bladder urination and increase bladder volume and micturition pressure. e acupuncture points of stimulation in this study were the classical points BL32 and BL35 corresponding to sacral vertebrae S 2 level and coccyx, which belongs to the Bladder Meridian of Foot Taiyang in traditional Chinese medicine.
e clinical studies showed that acupuncture stimulation to the sacral vertebrae increases bladder capacity and suppresses overactive bladder [23,24]. In our previous clinical studies, we found that electroacupuncture with continuous wave, 30 Hz, 1 mA stimulation could suppress bladder unstable contraction frequency and decrease detrusor maximum pressure in the bladder filling period thereby suppressing bladder overactivity. erefore, we used the same therapy through the experiment.  In this study, we have found that EA stimulation alleviated bladder overactivity significantly, and after P2X 3 receptor function was blocked by AF-353 intravenous administration, the EA effect was weakened. Immunohistochemical studies showed that P2X 3 receptors were expressed in the rat bladder cytoplasm and cell membrane, the detrusor muscle layer, nerve fiber bundles, and bladder transitional epithelial, especially in the cortex, and the quantity of the P2X 3 receptor in OAB rats was significantly increased compared with naive rats and was less distributed in EA rats. RT-PCR and western blotting studies showed that the expression of P2X 3 mRNA and protein in OAB rats was significantly higher than naïve rats but could be reduced by EA treatment.
ese results implied that P2X 3 receptors were closely associated with bladder sensory afferent, and EA regulation relied on P2X 3 receptors mediated. EA treatment may influence bladder ATP release, lead to bladder cells P2X3 receptor function change and then change bladder sensory and motor functions and finally inhibit bladder overactivity. On the sacral spinal micturition center system, immunohistochemical studies showed that P2X 3 receptors were expressed in L 6 -S 1 spinal cord and DRG and highly expressed in a small diameter of DRG cells, and the quantity of the P2X 3 receptor in OAB rats was significantly increased compared with naive rats and was less distributed in EA rats. RT-PCR and western blotting studies showed that the expression of P2X 3 mRNA and protein in the L6-S1 spinal cord of OAB rats was significantly higher than naïve rats but could be reduced by EA treatment, and P2X 3 protein expression in L 6 -S 1 DAG of OAB rats was significantly higher than naive rats but could be reduced by EA treatment. ese results implied that EA regulation relied on P2X 3 receptors mediated in the sacral spinal micturition center system. e possible ways are (1) the EA signals may be uploaded to the brain central first and then descending inhibit ATP release of the presynaptic membrane, influencing the expression of P2X 3 receptors in the spinal cord, reducing the bladder sensory signals transmission, finally inhibiting bladder overactivity. (2) EA signals directly affect the spinal dorsal horn neurons, influence the release of ATP, and lead to the combination change between ATP and P2X 3 receptors on spinal cord dorsal horn neurons and then affect intracellular signaling pathways, finally inhibiting bladder overactivity.
Overactive bladder has a negative impact on quality of life. Electroacupuncture may potentially improve these symptoms. e present findings provide a scientific foundation to clinical treatment and point selection for the treatment of overactive bladder.

Conclusions
A systematic profile of the effects of EA effect on bladder overactivity and the P2X 3 receptor mediation pathway was shown in the present study.
e results show that P2X 3 receptor which in bladder peripheral level and the spinal cord central level was involved in the bladder micturition reflex of the afferent signaling, blocking P2X 3 receptor function which could inhibit bladder overactivity and EA stimulation which could significantly reduce the P2X 3 protein expression in OAB rats bladder, spinal cord (L6-S1), and DRG (L6-S1). EA effect could alleviate bladder overactivity by the inhabited P2X 3 receptor in the bladder.

Data Availability
All the individual participant data collected during the trial after publication will be available for anyone who wishes to access the data immediately following publication in accordance with principles.

Conflicts of Interest
e authors declare that they have no conflicts of interest to this work.