Preparation of KH-204
The major ingredients of KH-204 are fruits obtained from five plants: Cornus officinalis Sieb. et Zucc. (CO; 32%), Lycium chinense Miller (LC; 32%), Rubus coreanus Miquel (RC; 16%), Cuscuta chinensis Lam (CC; 16%), and Schisandra chinensis Baillon (SC; 4%). These herbs were purchased from Andong Excellent Medicinal Herbs Distribution Center Co., Ltd. (Andong, Korea), and identified by one of the authors (S.Y. Hwang). Voucher specimens (KH204-CO, KH204-LC, KH204-RC, KH204-CC, and KH204-SC) of each plant were deposited at the R&D centre of KEMIMEDI (KMD) Co. Ltd. (Andong, Korea). Each herb (20 kg) was extracted in 200 L of distilled 30% ethanol and refluxed at 98 ± 2 °C for 3 h. The extract was filtered, and the liquid from the filtrates was removed by a rotary evaporator and a spray dryer. KEMIMEDI (KMD) Co. Ltd. (Seoul, Korea), a venture company that develops Oriental herbal medicines, developed this product as a health supplement.
Marker Compounds for Each Plant
A marker compound for each plant was selected, and their chemical structures are shown in Table 1.
The presence of the marker compound for each plant was confirmed by high-performance liquid chromatography (HPLC). Each peak in the HPLC profile was identified by comparison with the retention times and UV spectra of standard compounds (Fig. 1).
Animal Groups and Treatment Protocol
Thirty-six 6-week-old male Sprague-Dawley rats, supplied by Orient Bio Inc. (Gyeonggi-do, Korea), were treated under a protocol approved by the Institutional Animal Care and Use Committee at the School of Medicine, The Catholic University of Korea (Approval Number: CUMC-2016-0111-01) and handled according to the guidelines of the National Institutes of Health (NIH). Rats were divided equally into three groups (n = 12 each): Control, the normal control group; HFC, the HC group induced by high fat and cholesterol diet; and HFC + KH, HFC rats administered 400 mg/kg of KH-204. Animals were housed three per plastic cage and identified by the presence or absence of ear punches. The lighting of the animal rooms was controlled by artificial light for 12 h from 7 o’clock in the morning to 7 o’clock in the evening, and 18–23 °C room temperature and 40–60% humidity were maintained. All rats in the HFC and HFC + KH groups received a high-fat and cholesterol diet, supplied by Feedlab, (Gyionggi-do, Korea). Rats in the Control group received a normal chow diet for 12 weeks. The formulation of the HFC diet is shown in Table 2. Rats in the HFC + KH group received KH-204 (400 mg/kg) for 12 weeks. KH-204 was dissolved in distilled water and administered orally through an 8 F red Rob-Nel catheter once a day. After 12 weeks, all rats were weighed and underwent intracavernosal pressure (ICP) measurement. After ICP measurement, blood from the internal carotid artery and corporal tissue were sampled.
Serum Level of Total Cholesterol (TC), Low Density Lipoprotein (LDL)/Very Low Density Lipoprotein (VLDL) Cholesterol, High Density Lipoprotein (HDL) Cholesterol, and Triglyceride
To measure serum TC, LDL/VLDL-C, HDL-C, and TG, commercial assay kits [HDL and LDL/VLDL Cholesterol Assay Kit, STA-391 (Cell Biolabs Inc., San Diego, CA, USA); Triglyceride Quantification Colorimetric/Fluorometric Kit, K622-100, Biovision Inc. (Milpitas, CA, USA)] were used according to manufacturer instructions. Absorbance at 570 nm was read with a PowerWave HT Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Sample cholesterol concentrations were determined by interpolation from a standard curve.
Rats were anaesthetised with an intraperitoneal injection of 0.2 ml tiletamine (Zoletil®). With the rat in the supine position, the penis was dissected, and the corpus cavernosum and crus of the penis were exposed. A low, midline abdominal incision was made to access the pelvis, and the pelvic ganglion lateral to the right prostate was exposed. For the measurement of ICP, a heparinised 23-gauge butterfly needle was inserted in the corpus cavernosum of penile proximal portion after the penile skin was degloved and the corpus cavernosum identified. Then, a bipolar electrical stimulator was placed on the ganglion to stimulate the cavernosal nerve for 50 s at 10 V and 2.4 mA for 0.5 ms. Cavernosal nerve stimulation was conducted at least three times and the interval between stimulations was maintained for more than 10 min. Both mean arterial pressure (MAP) and ICP were continuously monitored during electrical stimulation. Systemic MAP was continuously monitored via a 24-gauge polyethyl tube placed in the right carotid artery. Each 23-gauge butterfly needle was inserted in the right and left penile crus for recording ICP. Both the polyethyl tube and the butterfly needle were connected to tubing diluted with heparin (250 IU/ml). ICP and MAP were recorded via pressure transducers connected to a recorder (Grass model S48K, Grass Instrument Division, Astro-Med, West Warwick, RI, USA). Comparisons were made for ICP/MAP and area under the curve corresponding to the duration of electrical stimulation . After the stimulation test, the corpus cavernosum was removed and divided in two. The first part was cryopreserved in liquid nitrogen, and the other part was fixed in formalin.
Masson’s Trichrome Staining
Masson Trichrome staining was performed in paraffin-embedded corporal tissue sections. After ICP, the skin-denuded middle portion of the penile shafts were fixed overnight in 10% formalin, washed, and stored in 70% alcohol at 4 °C until they were processed for paraffin-embedded tissue sectioning (4 μm). After staining, the colour distribution of the muscle tissue was approximated using Adobe Photoshop CS 8.0. The entire colour distribution of the image was calculated and the muscle tissue distribution was selected and expressed as red in colour . There was variation in our calculations due to colour overlays and ambiguity of the colour spectrum in the muscle tissues.
eNOS Expression Test: Western Blot Analysis
Corpora tissue was homogenised in ice-cold lysis buffer containing 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 μM leupeptin, 20 μg/ml chymostatin, and 2 mM phenylmethanesulphonyl fluoride (PMSF). Following centrifugation at 12,000 g for 20 min at 4 °C, the supernatant was extracted and quantified using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to membranes. The membranes were blocked in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 and then probed with anti-eNOS antibody (1:1000; ab5589, Abcam, Cambridge, UK), anti-phosphorylated-eNOS (P-eNOS) antibody (1:1000; #9571, Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody (1:10000; SC47778, Santa Cruz Biotechnology, Dallas, TX, USA) as an internal control. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Densitometric analysis of band intensity was detected by Luminescent Image Analysis System (LAS-3000; Fujifilm, Tokyo, Japan) and measured using Multi Gauge 3.0 software (Fujifilm) .
nNOS Expression Test: Immunohistochemistry
The paraffin sections of corpora tissue were immunostained with the following primary antibodies: neuron-specific β-III tubulin diluted 1:200 (Abcam) and nNOS diluted 1:200 (Santa Cruz Biotechnologies). Sections were mounted with 4,6-diamidino-2-phenylindole (DAPI; Vector laboratories, Inc., Burlingame, CA, USA) to stain the nuclei. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using Zen 2009 (Zeiss) .
Measurement of 8-Hydroxy-2-deoxyguanosine (8-OHdG) in Corpora Tissue
Oxidative stress in corpora tissues was evaluated by measuring the levels of 8-OHdG, oxidatively modified DNA. The DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) and the Total DNA oxidation kit (Highly Sensitive 8-OHdG Check enzyme linked immunosorbent assay (ELISA); Japan Institute for the Control of Aging, Fukuroi, Japan) were used according to the manufacturer’s protocol. The 8-OHdG standard (0.5–40 ng/ml) or 15–20 μg of DNA purified from the testis was incubated for 1 h with a monoclonal antibody against 8-OHdG in a microtiter plate precoated with 8-OHdG. After development of the final colour with the addition of 3,3′,5,5′-tetramethylbenzidine, absorbance was measured at 450 nm. Tissue sample concentration was calculated from a standard curve and was corrected for DNA concentration .
All data are presented as the mean ± standard deviation (SD). Data were analysed using SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Data were evaluated using analysis of variance (ANOVA), with group comparisons made by Scheff’s test. A p-value less than 0.05 was considered to be statistically significant.