Study design, materials and methods
Twenty-seven female Sprague-Dawley rats were utilized. The OAB model was established via intraperitoneal injection of CYP (75 mg/kg) every three days for three doses. Successful modeling was validated by urodynamic parameters indicating increased non-voiding contractions. Rats were randomly assigned into five groups: blank (n=3), acupuncture (n=6), acupuncture control (n=6), pre-acupuncture (n=6), and pre-acupuncture control (n=6).
Both acupuncture and pre-acupuncture groups received intervention at bilateral BL33, BL35, SP6, and ST36. Electroacupuncture (dense-disperse wave, 1/10 Hz, 1 mA) was applied to BL33 and BL35 for 20 minutes daily, while manual acupuncture was performed at SP6 and ST36. The pre-acupuncture group received 7 days of treatment prior to modeling, whereas the acupuncture group received 14 days of treatment post-modeling. Rats were euthanized for tissue collection at designated endpoints (Figure 1). Bladder histopathology was assessed via HE and toluidine-blue staining. Moreover, immunohistochemistry (IHC) is utilized to visualize the spatial distribution and protein expression levels of adenosine A2a receptors (A2aR), transient receptor potential vanilloid 1 (TRPV1), and protein gene product 9.5 (PGP 9.5) within the bladder tissue. Additionally, urinary nerve growth factor (NGF) was quantified by ELISA, and the protein expression of A2aR and TRPV1 in bladder tissue was further determined by Western-Blot. The intensities of the protein bands were analyzed by ImageJ software.
Statistical analyses were calculated using Student’s t-test and one-way ANOVA. A P-value < 0.05 indicated significance.
Results
Besides increased non-voiding contractions presented in urodynamic, HE staining of bladder tissue demonstrated the submucosal edema, hemorrhage, inflammatory cell infiltration and urothelial abrasion following CYP induction, which confirmed successful OAB modeling. Interestingly, both acupuncture and pre-acupuncture group showed relatively mild histopathological abnormalities compared to controls in HE staining, so did less mast cell infiltration in toluidine-blue staining (Figure 1). Besides, the lower expressions of A2aR, TRPV1 and PGP 9.5 were found in both acupuncture and pre-acupuncture group compared with controls in IHC (Figure 1). Semi-quantitative analysis based on Western-Blot further showed a downregulated expression of A2aR and TRPV1 in acupuncture and pre-acupuncture group compared to controls. Notably, the pre-acupuncture group-maintained expression levels (TRPV1: 0.139; A2aR: 0.119) similar to those of the blank group (TRPV1: 0.145; A2aR: 0.12), effectively attenuating the pathological upregulation observed in the pre-acupuncture control group (TRPV1: 0.162; A2aR: 0.166) (Figure 2). In addition, urinary NGF levels remained stable in the pre-acupuncture group but were significantly elevated in the pre-acupuncture control group, demonstrating a significant inter-group difference (Figure 2).
Interpretation of results
These findings indicate that acupuncture at BL33, BL35, SP6, and ST36 provides significant therapeutic and prophylactic benefits for CYP-induced OAB, likely through the modulation of the bladder sensory signaling pathway.
1. Attenuation of TRPV1 Overexpression: TRPV1 is a pivotal cation channel in bladder hypersensitivity. Our results suggest that acupuncture elevates the sensory threshold by inhibiting TRPV1 overexpression, thereby reducing pathological urgency signals.
2. Regulation of A2aR-Mediated Sensitization: A2aR acts as a critical modulator of afferent nerve activity. The suppression of A2aR expression, particularly in the pre-acupuncture group, suggests that acupuncture blocks the A2aR-TRPV1 sensitizing axis, mitigating detrusor overactivity.
3. Preventive vs. Therapeutic Efficacy: The superior stability of molecular markers in the pre-acupuncture group suggests that early intervention may pre-condition the autonomic and sensory systems, providing a protective buffer against chemical cystitis.