Twenty-four SD rats of each gender were obtained from a specific pathogen-free facility (ORIENTBIO Inc., Gyeong- gi, Korea) at 5-weeks of age and were used after a week of quarantine and acclimatization. The animals were housed in a room maintained at 20.0 ─ 23.0℃ under a relative humidity of 42.8% ─ 68.9%. The room was illuminated with artificial lighting from 07:00 to 19:00 hours and 10 ─ 15 air changes per hour. Three animals were housed in suspended stainless-steel wire-mesh cages and were allowed sterilized tap water and commercial rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Harlan Laboratories, Inc., U.S.A.). The protocol of this study was approved by the institutional Animal Care Committee of Biotoxtech, Co. (Oh Chang, Korea).
SBV was prepared using a freeze dryer (FD 8508, Ilshin Lab Co. Ltd., Korea) by Ginseng & Venom (G & V) Co. (Wonju, Korea) and was stored at ─ 20℃. A high-performance liquid chromatography (HPLC, Agilent 1100 series, Agilent Technologies Ltd., U.S.A.) analysis was performed to confirm that SBV was pure melittin.
Twenty healthy male and 20 healthy female rats were selected by average weights, and 5 rats of each gender were assigned to 1 of 4 groups: control (normal saline), low dosage (0.1 mL/animal), medium dosage (0.5 mL/animal), and high dosage (1.0 mL/animal). In a pilot study, no mortalities had been observed at a dosage of 1.0 mL/animal of SBV in male and female SD rats; based on that results, we set the high dose at 1.0 mL/animal. SBV was administered to the rats by intravenous injection into the caudal vein. The control group was administered 1.0 mL/animal of normal saline (Lot No. 12115, Choongwae Pharma Corp., Korea).
All animals were observed for clinical signs at 30 minutes, 1 hour, 2 hours, 4 hours and 6 hours after the injection of SBV. Clinical signs were observed daily from the injection day to 14 day after the first injection. The body weight of each rat was measured at the initiation of treatment and at 3 days, 7 days and 14 days after the injection.
The animals were fasted for 18 hours prior to necropsy and blood collection. Blood samples were drawn from the abdominal aorta by using a syringe needle under ether anesthesia. Blood samples were collected into tubes containing ethylenediaminetetraacetic acid (EDTA) and were analyzed by using a blood counting analyzer (ADVIA 120, SIEMENS, Germany) to determine the red blood cell count (RBC), hemoglobin concentration (HGB), hematocrits (HCT), mean corpuscular cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular cell hemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC), WBC differential count, reticulocyte (Reti) count, prothrombin time (PT) and active partial thromboplastin time (APTT).
For the serum biochemistry analysis, blood samples were centrifuged at 3,000 rpm for 10 minutes and analyzed using an auto-analyzer (7180, HITACHI, Japan). Serum biochemistry parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio (A/G ratio), total cholesterol, triglycerides (TG), phosphorus, glucose, calcium, chloride, sodium and potassium, were examined.
Tissue was obtained from the injection site on all animals and was then fixed with 10% neutral buffered formalin solution. The tissue was routinely processed, embedded in paraffin, and sectioned. The sections were stained with hematoxylin & eosin (H&E) stain, and bone tissue was decalcified with Calci-Clear-RapidTM (National diagnostics, U.S.A.) for microscopic examination.
Data on the weights and from the hematology and serum biochemistry results were tested by using a statistical analysis system (SAS, version 9.3, SAS Institute Inc., U.S.A.). The variance was checked by using the Bartlett test (P < 0.05). If the variance was homogeneous, the data were subjected to a one-way analysis of variance (ANOVA, P < 0.05). If a significant difference was observed between the groups, the data were analyzed by using the multiple comparison procedure of the Dunnett’s test (P < 0.05, P < 0.01). If the variance was not homogeneous, the data were analyzed by using the Kruskal-Wallis test (P < 0.05).