2-1 I-Tiao-Gung
I-Tiao-Gung (ITG) belongs to Glycine tomentella Hayata grown from the Xiguo mountain of Kinmen area in Taiwan and is provided by Kinmen County Agriculture Research Institute. We used the biennial dried main root of ITG in the present study.
2-2 Preparation of I-Tiao-Gung extract
The method for extracting ITG was described previously.1 In brief, the dry root of ITG was grounded, added ethanol, heated at 70°C for 2 hours and filtrated. After doing the previous step twice, the combined liquid extracts were lyophilized and obtained ITG extract. The ITG extract was then stored at 4°C until use.1
2-3 HPLC analysis
The ITG extract was analyzed by a high-performance liquid chromatography (HPLC, Hitachi system) consisting of a LaChrom Pump L-2130, a Programmable Autosampler L-2200, an Interface D-7000 and a LaChrom Diode Array Detector L-2455 (Merck, Vienna, Austria). The reagents of genistin, genistein, daidzin, daidzein, glycitin, glycitein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile
(HPLC grade) and DMSO (analytical reagent grade) were obtained from Merck (Darmstadt, Germany).
2-4 Animals
Thirty female Wistar rats (200-250 g body weight) were purchased from BioLASCO (Taiwan Co. Ltd., Taipei) were housed with a consistent light cycle (light from 07:00 to 18:00). Food and water were provided ad libitum. All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan Normal University and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997).
The rats were anesthetized with intraperitoneal urethane (1.2 g/kg body weight). Urethane was chosen for lacking ganglionic blocking properties.13 The maintenance of deep anesthesia was determined by the persistence of miotic pupils as judged from frequent inspection.13 Body temperature was maintained at 37°C with a heat lamp. After experiments, the anesthetized animals were sacrificed by intravenous KCl.
Thirty rats were divided into control rats (Control, n=6), CYP rats (CYP, n=6), CYP treated rats with ITG (CYP+ITG, n=6), CYP treated rats with Diadzin (CYP+Diadzin, n=6), and one week of ITG preconditioning with CYP rats (ITG+CYP, n=6). The detailed protocols were indicated in Figure 1A.
2-5 ITG and Daidzin given dose
Previous studies showed the safety dose of ITG extract was below 2 g/kg/day in rats for 28 days.31 Our preliminary data has evaluated the dosage at intragastrical 0.117 g/kg, 0.585 g/kg, 1.17 g/kg and 3.34 g/kg on CYP-induced voiding dysfunction. We found ITG extract at 1.17 g/kg displayed the most efficient action on the improvement CYP-induced overactive bladder. The intragastric dosage of 3.34 g/kg increased non-voiding contractions in the bladders implicating a possible toxic effect. Therefore, we applied the dosage of ITG extract intragastrically at 1.17 g/kg/day to the animal. Our ITG extract contained 1.07% Daidzin and 0.77% Daidzein. The ITG extract mixed with 1 mL of distilled water was applied to animals intragastrically. ITG 1.17 g contains 12. 5 mg of Daidzin. Daidzin mixed with 1 mL of distilled water (12.5 mg/kg/day) was applied to animals intragastrically. Control rats were given distilled water.
2-6 Antioxidant activity of ITG, Daidzin and Daidzein
A major reactive oxygen species generated from activated leukocytes via the myeloperoxidase system is hydrogen peroxide (H2O2), which can initiate inflammation.32 We evaluated the antioxidant H2O2
activity of ITG extract, Daidzin and Daidzein on the enhanced chemiluminescent signals from the H2O2-luminol mixture as described previously.32
2-7 CYP-induced cystitis and bladder hyperactivity
CYP-induced hemorrhagic cystitis and bladder hyperactivity were performed with intraperitoneal CYP at a dose of 200 mg/kg, while a control group received intraperitoneal saline.
2-8 Transcystometric model
We utilized a transcystometric technique to evaluate voiding response to CYP.13 These parameters of bladder responsiveness were measured: intercontraction interval (ICI, the time interval between two micturition cycles identified with active contractions [> 10 mmHg]), baseline bladder pressure (BP), threshold pressure for triggering micturition (PTH), maximal voiding pressure (MVP) and contractile amplitude (MVP-BP =AM) (Figure 2A).
2-9 Recording of extraurethral sphincter-electromyogram (EUS-EMG) activity
For measurement of EUS-EMG activity, epoxy-coated stainless steel
wire (50 mm; M. T. Giken Co., Tokyo, Japan) EMG electrodes were placed percutaneously in the extraurethral sphincter (EUS) by using a 30-gauge needle with a hooked EMG electrode positioned at the tip. The needle was inserted into EUS approximately 5–10 mm lateral to the urethra and then withdrawn, leaving the EMG wires embedded in the muscle.13
2-10 In vitro and in vivo chemiluminescence recording for ROS activity
The major reactive oxygen species generated from activated leukocytes via the myeloperoxidase system is hydrogen peroxide (H2O2), which can initiate inflammation.33 We evaluated the antioxidant H2O2 activity of ITG extract and Daidzin on the enhanced chemiluminescent (CL) signals from the H2O2-luminol mixture as described previously.33
2-11 Western blot
The expression levels of respective antibody against M2 (#bs-0441R, Biosynthesis Biotechnology, Littleton, CO, USA), M3 (#ab87199, Abcam's RabMb® technology, Cambridge, UK), P2X2 (#ab10266, Abcam's RabMb ® technology, Cambridge, UK), and P2X3 (#RA14139, Neuromics, Edina, MN, USA), and 3-Nitrotyrosine (3-NT, #ab61392,
Abcam's RabMb® technology, Cambridge, UK) and NADPH oxidase 4 (NOX4, #MBS176126, Mybiosource, San Diego, USA) in the whole bladder homogenates were analyzed by Western blot.34 Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (#406401, Biolegend , San Diego, CA, USA) was used as secondary antibody. The band density was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA, USA). Lipid peroxidation indicated by malondialdehyde (MDA) was determined with the MDA Assay Kit (#ab118970, Abcam's RabMb® technology, Cambridge, UK).
2-12 Multiple cytokine antibody arrays
In response to toxicity, several inflammatory mediators such as cytokines and chemokines could be released by activated macrophages/Kupffer cells in the damaged bladder. Therefore, multiple cytokine expression levels were simultaneously detected and identified with the aid of RayBio®rat cytokine protein array (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's instructions.
2-13 Histology
Five-µm sections of formalin-fixed bladders were stained with
hematoxylin and eosin (H&E) for evaluating leukocytes infiltration, hemorrhage and edema, toluidine blue staining for mast cell labeling32, and Masson’s trichrome for fibrosis. The leukocytes, hemorrhage and edema in the bladder tissue were analyzed and scored as described.35 Bladder sections were immunohistochemically stained with the specific antibody as described in Western blot. For speific immunostaining, the tissue sections were incubated overnight at 4°C with specific antibody. A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was applied followed by streptavidin conjugated to HRP. The chromogen used was Dako Liquid diaminobenzene. Twenty fields were randomly selected for each section, and the value of each positive stain was counted.
2-14 Statistical Analysis
All data were expressed as means ± standard error of the mean (SEM). Data were subjected to one way of analysis of variance, followed by Duncan’s multiple-range test for assessment of the difference among groups. p<0.05 was considered to indicate statistical significance.