Chun-Hsiung Huang, Department of Urology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung City 807, Taiwan. e-mail: email@example.com
What's known on the subject? and What does the study add?
Ovary hormone deficiency and the age-related changes in post-menopausal women are subjected to a number of urological dysfunctions, including overactive bladder syndrome. Green tea is a popular healthy drink worldwide and its extract catechin has strong anti-inflammatory and antioxidant properties.
EGCG, the major type of catechin, is an antioxidant polyphenol flavonoid isolated from green tea. EGCG supplement could prevent ovariectomy-induced bladder dysfunction in a dose-related manner through its anti-oxidant, anti-fibrosis and anti-apoptosis effects.
• To evaluate whether green tea extract, epigallocatechin gallate (EGCG), could prevent ovariectomy-induced overactive bladder (OAB) and to investigate its antioxidant, anti-apoptotic and anti-fibrosis effects.
MATERIALS AND METHODS
• In all, 48 female Sprague–Dawley rats were divided into four groups. After bilateral ovariectomy, the first group served as the ovariectomy control, the second group received EGCG 1 µM/kg daily i.p. injection after ovariectomy surgery, and the third group received EGCG 10 µM/kg daily i.p. injection. The fourth group was taken as the sham without ovariectomy surgery. The rats were killed after 6 months after ovariectomy surgery.
• Cystometrograms were performed for the measure of bladder overactivity.
• Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay was used to evaluate apoptotic cells.
• Western immunoblots were performed to determine the expressions of inflammatory markers, apoptosis-associated proteins and oxidative stress markers.
• Long-term ovariectomy significantly increased non-voiding contractions and decreased bladder compliance. Treatment with EGCG significantly increased bladder compliance and diminished non-voiding contractions.
• Ovariectomy significantly increased apoptotic cells and enhanced interstitial fibrosis in bladders. The expression of caspase-3 significantly increased, while that of Bcl-2 notably decreased after ovariectomy.
• Inflammatory and fibrosis markers, TGF-β, fibronectin and type I collagen expressions were significantly increased after 6 months of ovariectomy surgery. Treatment with EGCG significantly decreased TGF-β and type I collagen expressions.
• Oxidative stress markers, nitrotyrosine and protein carbonylation levels were significantly increased in the ovariectomy group. EGCG could attenuate this oxidative damage in dose-dependent fashion.
• Ovariectomy increased oxidative damage, enhanced voiding frequency and decreased bladder compliance.
• EGCG could restore ovariectomy-induced bladder dysfunction in a dose-dependent fashion through antioxidant, anti-fibrosis and anti-apoptosis effects.
Postmenopausal women, as a result of ovary hormone deficiency and age-related changes, are subject to a number of urological dysfunctions, including overactive bladder (OAB) symptoms, stress incontinence and recurrent urinary tract infections [1,2]. Ovariectomized animals have been used as a potential model to mimic post-menopause in humans in previous studies . Ovariectomized rat studies have reported that oestrogen deficiency can cause atrophy of bladder smooth muscle (SM), decrease SM contractility, enhance mucosa atrophy, diminish bladder blood flow and eventually result in tissue hypoxia [4,5]. Up to 40% of postmenopausal women were estimated to have symptomatic urogenital atrophy . Additionally, ovariectomized rats voided more frequently than the controls. Changes in voiding patterns occurred in ovariectomized rats, which could be attributed to ovary hormone deficiency . Oestrogen replacement decreased the frequency of voids, but did not return the animals to the pre-ovariectomized pattern .
Green tea is a popular healthy drink worldwide. Previous studies have identified strong anti-inflammatory/antioxidant properties of green tea and its associated polyphenols. Many investigations have suggested that the biological activity of green tea is mediated by its major polyphenolic components . Epigallocatechin gallate (EGCG), the major type of catechin, is an antioxidant polyphenol flavonoid isolated from green tea. EGCG has been found to prevent and/or reduce the deleterious effects of oxygen-derived free radicals associated with various chronic diseases, such as neurodegenerative disorders [7,8]. Both cell culture and in vivo studies have elucidated the molecular mechanisms and cell signalling pathways of EGCG, including inhibition of growth factor receptor phosphorylation, activation of protein kinase C , stimulation of extracellular signal-regulated kinase 1 and 2 expression, and inhibition of nuclear factor-kappa B involving neuroprotective action [9–12]. Although studies of the effect of green tea on bladder tissue are limited, it has been shown to exhibit bladder tumour inhibition activity in different animal models [13–15]. EGCG has also been found to inhibit bladder cancer cell growth and suppress in vitro migration capacity of cancer cells via down-regulation of N-cadherin and inactivation of Akt signalling . In substance P or cyclophosphamide-induced cystitis, catechins, major components of green tea, can ameliorate bladder neurogenic inflammatory damage via down-regulation of reactive oxygen species (ROS) production and decreasing intercellular adhesion molecular expression [17,18].
A previous investigation (Y-S Juan et al., unpublished data) showed that EGCG could ameliorate ovariectomy-induced OAB symptoms through decreasing bladder intramural nerve damage and diminishing M2, M3 muscarinic receptors overexpression. In extension of that work, the aim of the present study was to evaluate whether EGCG could prevent ovariectomy-induced OAB through reducing oxidative stress and apoptotic processes in a surgical menopause rat model.
MATERIALS AND METHODS
Experiments were performed on adult female Spraque–Dawley  rats purchased from the animal centre (BioLASCO, Taipei, Taiwan) and weighing between 200 and 250 g. Forty-eight female rats were divided into four groups. Three groups of rats underwent bilateral ovariectomy under halothane anaesthesia. Both ovaries were excised through bilateral abdominal incisions and then closed with 2-0 silk. After bilateral ovariectomy, 12 rats received an i.p. saline injection (the ovariectomy control group), 12 received a daily injection of EGCG 1µM/kg i.p and the other 12 received a daily injection of EGCG 10 µM/kg i.p. over a period of 6 months. EGCG was initially dissolved in dimethyl sulphoxide (DMSO) at a concentration of 10 mmol/L and kept at −20 °C for the remaining experiment. The EGCG stock was diluted with saline to 1 µM or 10 µM before treatment. The dilution multiple of saline/DMSO was either 104 or 103. Accordingly, the concentration of DMSO was less than 0.1% in the experiment. The remaining 12 rats comprised the sham group, which received anaesthesia, surgery and manipulation of the ovaries without ovariectomy. The rats were killed 6 months after ovariectomy surgery. The present study was approved by the Animal Care and Treatment Committee of Kaohsiung Medical University. All experiments were conducted in accordance with the guidelines for laboratory animal care.
The isovolumetric cystometrograms (CMGs) were performed according to a previously described method . In brief, for each experiment, the rats were anaesthetized with Zoletil50 (1 mg/kg i.p.). The bladder catheter (PE50 tube) was connected to both a syringe pump (KD Scientific 100, KD Scientific, Holliston, MA, USA) and a pressure transducer (MLT 0380, ADI Instruments, Colorado Springs, CO, USA). This single catheter was used to fill the bladder and to measure bladder pressure. Before the beginning of each CMG, the bladder was emptied and saline was then infused at a steady rate (0.08 mL/min), during which pressure was measured via a small-volume pressure transducer in line with the catheter. A voiding contraction was defined as an increase in bladder pressure that resulted in urine loss. The CMG was recorded until the bladder pressure was stable and at least five filling/voiding cycles were measured in each rat. Pressure and force signals were amplified (ML866 PowerLab, ADI Instruments), recorded on a chart recorder and digitized for computer data collection at 1000 samples/s (Labchart 7, ADI Instruments, Windows 7 operating system). CMG variables recorded for each animal included filling pressure (pressure at the beginning of the bladder filling), threshold pressure (bladder pressure immediately before micturition), micturition pressure (the maximal bladder pressure during micturition), micturition interval (time between micturition events), void volume and the presence or absence of non-voiding contractions.
After cystometric studies, experimental rats were perfused with saline solution through the left ventricle, and the bladders were removed, cut open in a sagittal direction and further fixed overnight. Owing to the small size of the bladder, not all rats could be included in every analysis. All animals were randomly assigned to the various analytical groups to avoid subjectivity. A quantity of 1 mL of blood was obtained at the termination of the experiment for oestrogen analyses. Blood was separated by centrifugation at 4 °C. The microtitre wells of the 17-β oestradiol ELISA kit (Cayman Chemical Co., Ann Arbor, MI, USA) were coated with an antibody directed towards a unique antigenic site on the oestradiol molecule. After addition of the substrate solution, the intensity of colour developed was inversely proportional to the concentration of oestradiol in the rat sample as measured by ELISA (Bio-Tek ELX 800, BioTek, Bad Friedrichshall, Germany). The mean absorbance values for each set of standards, controls and samples were calculated. A standard curve was obtained by plotting the mean absorbance obtained from each standard against its concentration with absorbance value. Use of the mean absorbance value for each sample allowed the concentration of the samples to be read directly from this standard curve.
The bladder tissue samples in the same area from different groups were embedded in paraffin blocks, and serial sections of 5 µm thickness were obtained. Deparaffinized sections were stained with Masson's trichrome stain (Masson's Trichrome Stain Kit, DAKO, Glostrup, Denmark). The standard Masson's trichrome staining procedure was followed to stain connective tissue in blue and SM in red. The transverse section of each specimen was captured by digital camera in five random, non-overlapping frames at 100 × magnification and the entire bladder wall thickness was included in each region analysed. The colour setting and image-associated quantification were determined by image analysis software (Image-Pro Plus, Media Cybernetics, MD, USA). The blue-stained collagen and red-counterstained SM were highlighted for each image. The total areas occupied by SM and collagen were determined and the ratio of collagen: SM was calculated.
To detect the cells undergoing programmed cell death, tissue sections were processed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche, Pleasanton, CA, USA). The DNA strand breaks can be identified by labelling free 3′-OH termini with fluorescence in dUTP plus terminal transferase. Permeabilized tissues with 0.25% trypsin (Invitrogen, Grand Island, NY, USA) were incubated at 37 °C in an oven for 25 min. Tissue sections were incubated with the TUNEL reaction mixture at 37 °C for 1 h. Samples were then captured and analysed under a fluorescein isothiocyanate fluorescence microscope. For each sample, the apoptotic cells in each image were highlighted by manually selecting the pixel value within the stained nucleus using image analysis software (Image-Pro plus, version 5.0). The number of TUNEL-positive cells in 10 randomly selected non-overlapping fields of 200 magnifications in the bladder was calculated and compared between groups. In each experiment, negative controls with label solution (without terminal transferase) instead of TUNEL reaction mixture were done to elucidate non-specific immunostainings.
For protein isolation and Western blot analysis, frozen tissue samples of bladder were homogenized on ice in the buffer (50 mM Tris, pH 7.5, 5% Tiron-X100) containing the Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA) at 100 mg/mL. After the addition of 1% concentration of sodium dodecyl sulphate, the sample was boiled and centrifuged. Protein concentration in the supernatant was determined using the bicinchoninic acid protein assay (Pierce, Rokford, IL, USA) against a BSA protein standard in a SpectraMAX Plus microplate reader (Molecular Devices, Sunnyvale, CA, USA). Equal amounts of total protein (20 µg) from the bladders were loaded on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (Immobilon-P, Millipore, MA, USA) membranes with Towbin buffer. Immobilon-P membranes were then blocked with 5% non-fat milk in 0.05% Tween 20 in tris-buffered saline (TTBS) at room temperature for 1 h and were then incubated with the primary antibody.
Monoclonal antibody to nitrotyrosine, 2,4-dinitrophenol (DNP), TGF-β (R&D, Minneapolis, MN, USA, 1:1000), fibronection (BD, San Jose CA, USA, 1:1000) Type 1 Collagen (Abcam, Cambridge, MA, USA, 1:1000), Caspase-3 (Cell Signaling, Danvers, MA, USA, 1:1000), Bd-2 (Cell Signaling, Danvers, MA, USA, 1:1000), and actin (Upstate Signaling, Billerina, MA, USA) were used to determine the changes in oxidative stress markers, interstitial fibrosis, and pro-apoptotic and anti-apoptotic protein expressions. In each experiment, negative controls without the primary antibody and protein molecular weight markers were carried out to exclude non-specific bands.
After treatment with the primary antibody, the membranes were washed with TTBS and incubated with secondary antibody (goat anti-mouse immunoglobulin G). The unbound antibodies were removed by washing with TTBS at room temperature. The expression of protein bands was visualized by adding ECL-Plus (Amersham Pharmacia Biotech, Little Chalfont, UK) for 2 min. Immobilon-P membrane was sealed in a hybridization bag, scanned and analysed with a Kodak Image Station 440CF and Kodak ID image analysis software.
All data are expressed as the mean ± SDs and assessed statistically using a one-way anova followed by Bonferroni's test for individual comparisons. In all tests, a P < 0.05 was considered to indicate statistical significance.
Cystometric variables are shown in Table 1 to examine the bladder function after ovariectomy with or without EGCG treatment. The cystometric bladder capacity significantly decreased while maximum voiding pressure and non-voiding contractions significantly increased in the ovariectomized group. These observations imply bladder hyperactivity with an increase in micturition frequency in ovariectomized rats. By contrast, the micturition intervals, bladder compliance and voiding volumes were significantly lower than in the controls.
The bladder compliance at different bladder capacity is shown in Figure 1. As noted, a higher bladder pressure indicates lower bladder compliance. Ovariectomy significantly decreased bladder compliance. Treatment with EGCG, at both 1 and 10 µM, significantly increased bladder compliance. At a higher concentration of EGCG, the voiding pattern was similar to that in the control.
The serum oestradiol concentration was 22.5 ± 4.5 pg/mL for the sham group, 14.7 ± 3.9 pg/mL for ovariectomy group, 13.6 ± 2.3 pg/mL for ovariectomy group with 1 µM EGCG treatment, and 16.1 ± 3.7 pg/mL for ovariectomy with 10 µM EGCG treatment group. Ovariectomy, with or without EGCG treatment, significantly decreased serum oestradiol concentration in comparison with the sham-operated controls (P < 0.01). There was no statistical significant difference between then ovariectomy-alone and EGCG treatment (either 1 or 10 µM) groups.
To investigate the fibrosis process after ovariectomy, with or without EGCG treatment, the expressions of TGF-β, fibronectin and type I collagen at protein level were examined by Western blotting. The results show that TGF-β, fibronectin and type I collagen expressions were significantly increased 6 months after ovariectomy (Fig. 2). In addition, treatment with EGCG significantly decreased TGF-β and type I collagen expression. These findings show that administration of a higher concentration (10 µM) of EGCG after ovariectomy reduced the fibrotic biosynthesis and protected the bladder from ovariectomy-induced inflammatory damage.
Masson's trichrome stain also showed that there was significant interstitial fibrosis and collagen accumulation between SM bundles in ovariectomized bladders (Fig. 3). For the EGCG treatment and sham groups, there was only sparse collagen accumulation between SM bundles. Morphometric analysis of trichrome stain showed a notable increase in collagen:SM ratio in the ovariectomy group. Nevertheless, treatment with EGCG significantly decreased bladder interstitial fibrosis (Fig. 3).
The TUNEL and DAPI stainings for the detection of degenerating apoptotic cells in the bladder after ovariectomy surgery and EGCG injection are shown in Figure 4. There were significant differences between the control and ovariectomy groups. The number of apoptotic cells in the urothelium and SM layers was about five times higher in the ovariectomy group than in the control group. At the lower concentration of EGCG, the numbers of apoptotic cells in the urothelium and SM layers were three and two times higher, respectively. Notwithstanding this, there was essentially no difference in the number of apoptotic cells between the sham group and the group treated with the higher concentration of EGCG.
Analysis of Western blotting indicated that the expression of the anti-apoptotic protein Bcl-2 significantly decreased in ovariectomized bladder tissues (Fig. 5). Treatment with EGCG, especially at a higher concentration, could reverse this change to near the control level. For pro-apoptotic protein, the expression of caspase-3 significantly increased in the ovariectomy bladder tissue (Fig. 5). Treatment with EGCG, at either the lower or higher concentration, could ameliorate this change.
To determine the bladder damage induced by oxidative stress in ovariectomized rats, the protein carbonylation and nitrotyrosine expressions were measured after ovariectomy and EGCG treatment (Figs 6A,B). These figures show that the ratio of DNP to actin six times higher in the ovariectomy group, five times higher in the low-concentration EGCG group and three times higher in the high-concentration EGCG group than in the control (Fig. 6A). Additionally, the expression of nitrotyrosine was four times higher in the ovariectomy group and two times higher in the low-concentration EGCG group than in the control group, but there was essentially no difference between the high-concentration EGCG group and the control (Fig. 6B). These findings reveal that treatment with EGCG significantly decreased bladder oxidative markers, and nitrotyrosine and protein carbonylation expression, implying that EGCG treatment ameliorated ovariectomy-induced oxidative damage.
The present investigation confirmed that surgical menopause can diminish bladder compliance and bladder capacity, increase voiding frequency, expand oxidative damage and interstitial fibrosis, and increase bladder SM or mucosa cell apoptosis. EGCG can prevent ovariectomy-induced bladder dysfunction in a dose-dependent fashion through antioxidant, anti-fibrosis and anti-apoptotic effects.
In the present study, ovary hormone withdrawal for a period of 6 months was related to a significantly greater collagen:SM ratio. This observation was compatible with previous findings . Such histological changes might be associated with a decrease in bladder compliance and bladder capacity as shown in urodynamic studies. Investigations have shown an association of bladder wall mechanical properties with detrusor muscle elasticity, collagen content and collagen distributions . Myelodysplastic bladders had more collagen deposition than normal bladders in correlation with decreased bladder compliance and increased bladder trabeculations . In dysfunctional bladder studies, Landau et al.  showed that dysfunctional bladders were significantly less compliant than normal controls, and these bladders had increased collagen and collagen:SM ratios. These pathological ultrastructural changes were reflected by abnormal low bladder volume and decreased bladder compliance, which could be indicators of a loss of bladder wall elasticity. In pregnant rats or ovariectomized rabbits treated with oestrogen, their bladders were found to have greater bladder compliance and lower collagen:SM ratios than those in ovariectomized animals [22,25]. Bladders with a higher SM expression would be expected to have greater elastic properties and higher compliance than bladders with a higher proportion of collagen.
In ovariectomized bladders, TGF-β expression was significantly higher than in control bladders. TGF-β is a multifunctional growth factor involving the cell differentiation, proliferation and extracellular matrix remodelling after binding to cell surface TGF receptors . In obstructive renal disease, increasing TGF-β expression led to enhanced macrophage infiltration, myofibroblast formation and type I collagen accumulation [27,28]. The present results also show an increase in fibronectin and type I collagen expression in ovariectomized bladders. A previous study revealed that bladder SM and urothelial cells could synthesize type I and III interstitial collagen, while type I collagen was present in the extracellular matrix and had greater deposition in the lamina propria . The altered bladder compliance after ovariectomy probably corresponded to an altered distribution pattern. Treatment with EGCG significantly decreased TGF-β and type I collagen expression, indicating anti-fibrosis properties of EGCG. Another study indicated that EGCG could ameliorate the overproduction of pro-inflammatory cytokines and mediators, reduce the activity of nuclear factor-kappa B, inhibit the expression of inducible nitric oxide synthase and reduce the interstitial fibrosis process .
The TUNEL results in the present study show that ovariectomy not only caused SM atrophy, but also significantly increased apoptotic cells in both mucosa and muscle layers. The caspase-dependent apoptotic pathway might be responsible for the programmed cell death. In the present study, caspase-3 expression was significantly increased, while Bcl-2 was decreased 6 months after ovariectomy. A higher concentration of EGCG (10 µM) could reduce both urothelial and detrusor SM apoptotic cells. In rats with intestinal ischaemia–reperfusion injury, EGCG pre-treatment inhibited intestinal ischaemia–reperfusion-induced apoptosis by down-regulating the expression of nuclear factor-kappa B, c-Jun and caspase-3 . Ovariectomy resulted in an ≈50% decrease in circulating oestradiol compared with the sham group. There was no difference in oestradiol concentration between the ovariectomy groups receiving EGCG 1 µM and EGCG 10 µM. Pretreatment with EGCG had no effect on blood oestradiol concentration. Hence, this anti-apoptotic effect of EGCG should not have a direct relation with oestrogen or its receptors.
Interestingly, EGCG has been described as affecting mitochondrial signal transduction in a concentration-dependent manner . In a lower concentration range (1–10 µM), EGCG has been reported to inhibit the pro-apoptotic caspase and induce the degradation of the Bax gene via the proteasome and protein kinase C pathways [31–33]. By contrast, at higher concentrationS (10–50 µM), EGCG exhibited pro-oxidant and pro-apoptotic activity, such as the induction of mitochondrial membrane depolarization and caspase-dependent apoptosis, which might be responsible for the enhanced cell death effects [34,35]. In cancer prevention and/or treatment studies, a higher concentration of EGCG was conducted to induce cancer cell apoptosis . The results of those studies were consistent with the findings of the present study, with 10µM EGCG exhibiting a strong anti-apoptotic effect on ovariectomy-induced bladder dysfunction.
In the present study, oxidative stress markers, nitrotyrosine and protein carbonylation expression were significantly increased in the ovariectomy group. By contrast, there were no quantitative changes in these oxidative stress markers as compared with the sham group and the group treated with high-concentration EGCG. EGCG could reverse the oxidative damage and exhibited antioxidant activity in a dose-dependent fashion. EGCG was reported to exhibit iron- and copper-chelating properties, which could neutralize ferric iron to form redox inactive iron, and protect cell from serum-deprived cell culture . In diabetic mice, EGCG significantly increased the immunoreactivity of Ki-67, CD31, and α-SM actin expressions . These results suggest that EGCG could benefit wound healing by accelerating re-epithelialization and angiogenesis . In addition, several reports showed that EGCG was a powerful hydrogen-donating antioxidant and a free radical scavenger of ROS and reactive nitrogen species. EGCG could inhibit iron ascorbate-induced mitochondrial membranes and synaptosome lipid peroxidation [39,40], thus protecting brain tissue from free radical damage.
In the present study, EGCG was given i.p., which was different from daily consumption habit as a healthy drink. Previous reports showed that the intestinal bioavailability of EGCG was very poor . The tissue and blood concentrations of EGCG only corresponded to 0.1–0.5% of the oral ingested dose. The tissues that are involved in excretion of catechin, such as the bladder, oesophagus and large intestine, appeared to have higher concentrations of catechin. Based on a previous study , it is likely that a daily oral consumption of 1500–1600 mg EGCG in humans will achieve concentrations similar to those of mice injected with EGCG 20 mg/kg i.p. Further investigations to examine the effects of EGCG on bladder dysfunction in different bladder tissue concentrations is warranted.
In conclusion, the present study confirms that ovary hormone deficiency can lead to OAB symptoms. Ovariectomy significantly increased the bladder collagen:SM ratio, decreased bladder compliance, enhanced bladder fibrosis markers, expanded bladder apoptotic cells and augmented oxidative stress damage. EGCG supplementation could prevent ovariectomy-induced bladder dysfunction in a dose-dependent manner through its antioxidant, anti-fibrosis and anti-apoptosis effects.
We are grateful to Professor Chang-Hwei Chen of the University at Albany for his valuable comments on this manuscript. This research is supported in part by the Department of Medical Research, Kaohsiung Medical University Hospital grant KMUH-98-8R07; and in part by the Kaohsiung Medical university grant KMU-Q098015.