After being acclimated to laboratory conditions for 1 week, young and aging female rats (12 rats from each group) were sacrificed by CO₂ asphyxiation. Their bladders were isolated and placed in Krebs solution containing 118 mmol/l NaCl, 4.75 mmol/l KCl, 1.18 mmol/l MgSO₄, 1.18 mmol/l KH₂PO₄, 24.8 mmol/l NaHCO₃, 10.0 mmol/l C₆H₁₂O₆, and 2.5 mmol/l CaCl₂ (pH 7.40). After removing the vertex of each bladder, the mucosa, and the organization below the mucosa, the body of the bladder was longitudinally opened. An 8×3 mm detrusor strip was harvested and suspended in a bath (cat. no. ML0126/10; ADInstruments Pty, Ltd., Bella Vista, Australia) containing Krebs solution at 37°C and continuously supplied with a mixture of O₂ (95%) and CO₂ (5%). The detrusor strip was connected to a tension sensor (cat. no. MLT0210/D; ADInstruments Pty, Ltd.), which was linked to a biological signal amplifier (cat. no. ML221; ADInstruments Pty, Ltd.). The tension generated by the detrusor strip was measured and recorded using an eight-channel biological function recording system (cat. no. ML870; ADInstruments Pty, Ltd.). Prior to the experiment, the detrusor strips were stabilized for 30 min in a bath containing Krebs solution.

Following adjusting the detrusor strip to a tension of 1.0 g, the strip was stabilized for 30 min. Thereafter, KCl was added in the bath to a final concentration of 40 mmol/l before reaching an equilibrium state in 30 min, and the strip tension was recorded (20–22). According to the cumulative concentration effect experiment (23), ISO, which is a β-AR agonist, was cumulatively added into the bath to final concentrations of 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ and 10⁻⁴ M, with a 5-min interval for each concentration, and the tension under each ISO concentration was recorded. The process was repeated thrice, and the mean reading was taken. In addition, the detrusor strip was washed and stabilized with Krebs solution at 37°C prior to each experiment. The following formula was used for the calculations of the effect of maximum relaxation (E_max):

E_max=(NC-NX)/NCx100%, where NC is the tension prior to adding ISO, NX the tension after adding agonist and E_max is the percentage of the tension under the maximum ISO concentration and initial tension.

In addition, the intrinsic activity (IA) was calculated using the following formula:

IA=E_max(experimental group)/E_max (control group)

Furthermore, 50% effective concentration (EC₅₀₎ was calculated based on the cumulative concentration effect curve determined by Powerlab in LabChart version 8 software (ADInstruments Pty, Ltd.) and was presented as the following equation: PD₂=−log (EC₅₀). All data were presented using Powerlab software.

The method used was the same as the test for the role of β-AR in detrusor strip relaxation. The E_max, IA, and PD₂ values of the β₃-AR agonist (BRL37344) were determined.

The detrusor strip was adjusted to a tension of 1.0 g and stabilized for 30 min in the bath. KCl was added to a final concentration of 40 mmol/l, which was stabilized for 30 min. Afterwards, the detrusor strip tension was recorded (24). The β₃-AR antagonist SR59230A was added into the bath to concentrations of 0, 10⁻⁷, 10⁻⁶ and 10⁻⁵ M. Under each concentration of SR59230A, the ISO was cumulatively added into the bath to concentrations of 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ and 10⁻⁴ M, with a 5-min interval for each ISO concentration, and the tension of each ISO concentration was recorded. The strip was washed and stabilized with Krebs solution at 37°C between each concentration of SR59230A. The PD₂ value of ISO under each concentration of SR59230A was determined by Powerlab software, and a concentration-effect curve was obtained.

The detrusor strip was adjusted to a tension of 1.0 g, and the cumulative concentration effect experiment on ISO was performed. After the maximum relaxant effect was obtained, the strip was washed and incubated in Krebs solution at a temperature of 37°C until equilibrium was reached. SR59230A was added at 10⁻⁶ M into the bath, and the cumulative concentration effect experiment of ISO was repeated. E_max, IA, PD₂ and PA₂ values were then compared. In the following formula (25) PA₂ denotes the intensity of competitive antagonist:

PA₂=−log [X]

X=10^{−PD2 (After)}/10^{−PD2 (Before)}

After being acclimated to laboratory conditions for 1 week, young rats were marked as a blank control (n=12), whereas aging rats were randomly divided into 4 groups (n=12 in each), including the model group (administered water) and the groups that were administered with low, middle and high dosages of SQW. Water and SQW was administrated orally, once daily for 6 weeks.

Rats were administered anesthesia intraperitoneally using 10% ethyl carbamate (0.8 g/kg), and their bladders were emptied. A bladder piezometric and water infusion tube that was connected with a WZ-50C6 microinjection pump [cat. no. WZ-50C6; Smiths Medical Instrument (Zhejiang) Co., Ltd., Hangzhou, Zhejiang Province, China] was inserted into the urethra of the rats. The abdominal pressure tube was inserted 4 cm into the rectum, and the bladder piezometric tube, microinjection pump and abdominal pressure tube were connected to the urodynamic detector (cat. no. 94-R01-BT; Delphis; Laborie, Mississauga, ON, Canada) (26).

Normal saline was infused into the bladder at a speed of 0.5 ml/min, and the infusion was stopped if leaking from the urethra was observed. BP, maximum voiding pressure (MVP), FUL, MUP and MUCP were then measured. In addition, the post-void residual (PVR) was recorded using a 1 ml injector to collect the remaining urine in the bladder. The MBC, voiding volume (VV), EV and bladder capacity (BC) were calculated using the following formula:

MBC=infusion time × infusion speed

VV=MBC-PVR

EV=VV/MBC

BC=MBC/BP

Normal saline was again infused into the bladder, and the epigastrium was gently pressed via the Valsalva method (27) when the injection volume in the bladder reached half of the MBC. The pressure was recorded as the abdomen leak point pressure (ALPP) if leaking from the urethra was observed. Furthermore, the entire process was repeated twice, and the mean of each parameter was determined.

Following the urodynamic test, rats were sacrificed by CO₂ asphyxiation, and the detrusor strips were harvested and used for the β-AR function test detailed above.

Following sacrifice, 40 mg of bladder tissue was isolated to quantify β₃-AR mRNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. Briefly, samples were homogenized, incubated at room temperature for 5 min, and 0.2 ml chloroform per 1 ml TRIzol was added. Following centrifugation at 12,000 × g for 10 min (4°C), the supernatants were removed to a new tube and a 2-fold volume of isopropanol was added to precipitate the mRNA. Subsequent to freeze centrifugation, the mRNA precipitation was dissolved in RNase-free water. The mRNA concentration was measured at an absorbance of 260 nm, and the purity was evaluated via the A₂₆₀/A₂₈₀ ratio (BioSpec-nano; Shimadzu Corp., Kyoto, Japan). mRNA samples (1 µg) were then used for the RT reaction using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The RT reaction was performed at 42°C for 1 h and 70°C for 5 min. In addition, a Maxima SYBR-Green/ROX qPCR Master Mix kit (Thermo Fisher Scientific, Inc.) was used for qPCR, according to the manufacturer's instructions. The primers used were as follows: β₃-AR forward, 5′-GGGCCACATTGGCGCTGACT-3′ and reverse, 5′-TGGGTGTCCCGACTGT-3′; and GAPDH forward, 5′-TGCTGGGGCTGGCATTGCTC-3′ and reverse, 5′-CCCCAGGCCCCTCTGTTGT-3′. In addition, the cycle profile was set to 95°C for 15 sec; 60°C for 30 sec; 72°C for 15 sec and for 40 cycles. Data were then analyzed using the 7500 Real-Time PCR System software version 2.0.6 (ABI 7500; Applied Biosystems; Thermo Fisher Scientific, Inc.), and the results were quantified using the 2^−ΔΔCq method (28).

Data are presented as the mean ± standard deviation and were analyzed using a one-way analysis of variance for multiple experiment groups. All statistical analyses were performed with SPSS 19.0 software (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

As shown in Table I, significantly lower E_max, IA and PD₂ values were observed in aging rats compared with young controls. Given that PD₂ represents the affinity between an agonist and a receptor, this result indicated the lower sensitivity and affinity of the detrusor receptor to the β-AR agonist in aging rats.

As shown in Table II, the E_max, IA and PD₂ of the β₃-AR agonist in the model group were significantly lower than those in the young control group, indicating that the sensitivity or the number of β₃-AR was significantly decreased in aging rats.

As the concentrations of the β₃-AR antagonist increased, the PA₂ values of ISO markedly increased, whereas the PD₂ values decreased (Table III). This observation indicated the blocking effect of the β₃-AR antagonist on ISO-induced relaxation in the detrusor strip.

As shown in Table IV, in the presence of SR59230A, E_max, IA, and PD₂ of ISO in aging rats were significantly lower than those of ISO in young controls. However, PA₂ was significantly higher in the aging rats in comparison to the value in the young controls. These results indicated that less β₃-AR antagonist was recruited to reach the same antagonistic level in aging rats, as compared with young rats.

As shown in Table V, significantly lower E_max, IA and PD₂ values were observed in aging rats in the model group compared with the young control group. Following administration, the middle and high doses of SQW significantly reversed this decrease.

As shown in Table VI, compared with young controls, a significant decrease in E_max, IA and PD₂ of BRL37344 was observed in the model group. These decreases were significantly reversed by administration of SQW.

As shown in Table VII, following the administration of SR59230A (10⁻⁶ M), the inhibitory effect of the model group was significantly higher than that of the young control. Compared with the model group, the PA₂ of the middle- and high-dose SQW groups was significantly decreased.

As shown in Fig. 1A-D, prior to administration of SQW, the BP, ALPP EV and MVP values of the model group were significantly lower than those of the young controls, and no significant difference was observed between the model and SQW groups. Following administration for 6 weeks, the BP, ALPP, EV and MVP of rats in the three SQW groups significantly increased compared with the model group. In addition, the three dosages of SQW were able to significantly raise BP, ALPP, EV and MVP levels compared with levels prior to SQW administration.

As shown in Fig. 1E and F, prior to SQW administration, MBC and VV in the model group were significantly higher than those in the young control group, and no difference was shown in MBC and VV between the model and SQW groups. Following administration for 6 weeks, the three dosages of SQW were able to significantly increase the MBC and VV of rats.

As shown in Fig. 1G and H, BC and PVR were significantly higher in aging rats than in young controls prior to SQW administration, and no significant difference was observed between the medicine and the model groups. Following administration for 6 weeks, the three dosages of SQW exhibited no significant effect on BC. However, SQW at a high dose was able to significantly degrade PVR.

As shown in Fig. 2A and B, prior to SQW administration, the MUCP and MUP in the model group were significantly lower than those in the young control group, and no significant difference was observed in the three groups of SQW. Following administration for 6 weeks, SQW at all three dosages was able to significantly increase MUCP and MUP. In addition, compared with the levels prior to administration, low-, middle- and high-dosage SQW could significantly increase the rats' MUCP and MUP.

As shown in Fig. 2C, prior to SQW administration, the FUL of the model group was significantly lower than that of young controls, and no significant difference was found from that in the SQW groups. Following SQW administration for 6 weeks, high-dosage SQW significantly increased the rats' FUL in comparison with the model group. In addition, the FUL of rats increased significantly following administration of SQW at middle and high dosages compared with levels prior to SQW administration.

As shown in Fig. 3, following SQW administration the expression of β₃-AR mRNA in the model group was significantly lower than that in the young control group. Furthermore, SQW at a high dosage was able to significantly increase the β₃-AR mRNA expression.