MO formula
MO formula is currently being developed by the Kemimedi (KMD) Company (Seoul, republic of Korea) for the treatment of infertility. Originally, KMD Company, which develops oriental herbal medicines, developed this product as a health care supplement. It includes Cornus officinalis Sieb. Et Zucc (32%), Lycium chinense Mil (32%), Rubus coreanus Miquel (16%), Rubus coreanus Miquel (16%), and Schisandra chinensis Baillon (4%). MO formula was obtained from Andong Excellent Medicinal Herbs Distribution Center Co., Ltd. (Andong Korea). The formal identification of the plant material was undertook by S.Y. Hwang who was officer of R&D center of KEMIMEDI (KMD). Voucher specimens (KH204-CO, KH204-LC, KH204-RC, KH204-CC, and KH204-SC) of each plant were deposited at the R&D center of KEMIMEDI (KMD) Co. Ltd. (Andong, Korea). In the present study, 400 mg/kg MO formula was orally administered for 2 weeks. The MO formula was prepared as a mixture of the five herbs. 20 Kg of five herb products was extracted with 200 L of 30% distilled ethanol. After that, each products refluxed at 98 ± 2 °C for 3 h. After the herb extract was filtered and the liquid from the filtrate was removed using a rotary evaporator and a spray dryer.
Features of the MO formula
The quality of the MO formula was confirmed using its marker compounds. Each of the components was selected as a marker compound as in another study [7]. The HPLC profile of the MO formula and its marker compounds were the same as that reported by Jang et al. [7].
Animal groups and study design
A total of 24, 8-week-old, Sprague-Dawley female rats weighing 250–300 g were used. The rats were supplied by Orient Bio Inc. (Gyeonggi-do, Korea) and were treated using the protocol approved by the Institutional Animal Care and Use Committee at the School of Medicine, The Catholic University of Korea (Approval Number: CUMC- 2015-0175-01). Rats were also handled according to the guidelines set by the National Institutes of Health (NIH). The rats were fed a standard rat feed and were granted free access to food and water. The rats were also maintained in a 12 h light-dark cycle, a room temperature of 20 ± 2 °C and a relative humidity of 50 ± 10% throughout the experiment. The 24 female rats were randomly divided into four groups: 1) a sham operation only (no partial urethral occlusion, control group), 2) a partial urethral occlusion-induced DO (DO group), 3) a DO group treated with solifenacin, and 4) a DO group treated with 200 mg/kg of the MO formula. We used a rat model showing persistent DO induced by partial urethral obstruction [15]. All 24 rats were subjected to an abdominal incision to expose the urinary bladder and urethra. In the persistent, detrusor overactive group, and the DO groups treated with solifenacin and the MO formula, a 25 G angio-sheath was placed on top of the urethrovesical junction. We then ligated the urethra with 3–0 nylon to create a partial bladder outlet obstruction model followed by sheath removal. Both ends of the nylon were pulled into the vaginal cavity through the previously made incision. After 2 weeks, the partial urethral occlusion was released by cutting the previously placed nylon through the vagina. The control and overactive groups were administered distilled water for 4 weeks, and the solifenacin- and the MO formula-treated groups were treated with the respective medication for 4 weeks. To prevent urinary tract infection, cephazolin was injected subcutaneously at 7 mg/kg.
Cystometrography
After the 4-week treatment period, a cystometrogram (CMG) was obtained of all rats. Subcutaneous 1.2 mg/kg urethane was injected as the anesthetic. Under anesthesia, midline laparotomy was performed to expose the bladder. A 25 G needle was then inserted into the bladder lumen, with a polyurethane tube connected to the needle. This tube was also connected to a pressure transducer to record the intravesical pressure as well as a Harvard syringe pump for saline infusion by a 3-way stopcock. The rate of saline infusion was 0.04 ml/min. The detrusor contraction, its interval and contraction pressure were recorded using a polygraph (Grass 7D, Grass Institute Co., Quincy, MA).
Effect on oxidative stress
In the bladder wall, oxidative stress was evaluated based on Superoxide dismutase (SOD) and 8-OHdG levels. The SOD level was measured using a SOD Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan). In addition, the decrease in the rate of superoxide-mediated reduction of nitroblue tetrazolium was monitored at 450 nm using a spectrophotometer. The oxidatively modified DNA, 8-OHdG level, was measured to identify the level of DNA oxidative stress. Total DNA was extracted from the bladder tissue using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). The 8-OHdG level was measured using a DNA oxidation kit (Highly Sensitive 8-OHdG Check ELISA; Japan Institute for the Control of Aging, Fukuroi, Japan). Absorbance was measured at 450 nm after final color development after the addition of 3, 3′, 5, 5′-tetramethylbenzidine. Tissue sample concentration was calculated from a standard curve and corrected for DNA concentration.
Cytokine analysis for anti-inflammatory effect
To identify the anti-inflammatory effect of MO formula, proinflammatory cytokine such as tumor necrosis-α (TNF- α), and interleukin-8 and 4 (IL-8 and IL-4) were analyzed. The enzyme-linked immunosorbent assay (ELISA) was used as the analysis tool. After checking the cystometrogram, blood was obtained from rats following sacrificing. Blood centrifugation was then performed at 3000 rpm, at 4°C for 10 mins, followed by immediate transfer of the supernatant to a tube. The concentration of each cytokine was measured every 5 mins, for a total of 30 mins. The immunoassay ELISA kit (R&D systems, Minneapolis, MN, USA) and the spectrophotometer at 450 nm were used according to the manufacturers’ protocols.
Western blot
After cystometrography, rats were euthanized by exsanguination method and the bladder tissues were obtained and frozen in liquid nitrogen. The frozen bladder tissue was grounded to a fine powder with a mortar and pestle, and cooled in liquid nitrogen. The total protein in the bladder was extracted using a cell lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM NaEDTA, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Protein extracts were quantified using the BCA Protein Assay reagent (Thermo Scientific, Rockford, IL, USA) and the quantified proteins (60 μg) were boiled in a loading buffer (62.6 Mm Tris-HCl pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.01% bromophenol blue, 10% glycerol, and 100 mM DTT). The proteins were then loaded onto a lane of a 4 to 12% SDS-polyacrylamide gel for electrophoretic separation. Proteins were transferred onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences, Freiburg, Germany), and equal protein loading was verified by Ponceau-S staining (Sigma Aldrich Co., St. Louis, MO, USA). The membranes were blocked by treatment with 5% non-fat milk in tris-buffered saline containing 0.1% Tween 20. The membranes were also probed with endothelial NOS (1:1000, BD Pharmingen, San Diego, CA, USA), Rho A (1:1000, BD Pharmingen, San Diego, CA, USA), ROCK-I (1:1000, BD Pharmingen), ROCK-II (1:1000, BD Pharmingen), M2 (1:1000; Abcam, Cambridge, MA, USA), M3 (1:500; Abcam, Cambridge, MA, USA), and β-actin, followed by incubation with the corresponding secondary antibodies (Santa Cruz Biotechnology, CA, USA) conjugated with horseradish peroxidase. The densitometric analysis of band intensity was performed using the Luminescent Image Analysis System (LAS-3000; Fujifilm, Tokyo, Japan).
Histological analysis
After CMG completion, the bladder wall was excised after the rats were euthanized, and divided into two sections. These two individual sections were used to conduct and identify the histological evaluation and oxidative stress, respectively. One bladder section was fixed in 20% buffered formalin for 96 h and the other section was frozen at − 80 °C. The bladder tissue was embedded in paraffin, cut into 4 mm transverse sections and stained with Hematoxylin-Eosin to identify the collagen-to-muscle ratio.
Statistical analysis
Statistical analysis was performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). The data of each group was compared using the one-way ANOVA test. Significance was defined as a P value < 0.05.
