Experimental study of antigenicity test of Sweet Bee Venom in Guinea Pigs

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(1)ISSN 2093-6966. Journal of Pharmacopuncture. 23 http://dx.doi.org/10.3831/KPI.2011.14.4.023. Original Article. 기니픽을 이용한 Sweet Bee Venom의 항원성 평가. Experimental study of antigenicity test of Sweet Bee Venom in Guinea Pigs. Received : Aug 31, 2011 Revised : Dec 8, 2011 Accepted : Dec 14, 2011. Byung Jun Cho1), Ki Rok Kwon2). KEY WORDS: Melittin; Sweet Bee Venom; antigenicity; ASA; PCA. 2). 1) Sangji University Graduate School of Oriental Medicine, Wonju, Korea Research Center of Pharmacopuncture Medicine, Korean Pharmacopuncture Institute, Seoul, Korea. Abstract Objectives: This study was performed to examine the antigenic potential of pure melittin (Sweet Bee Venom - SBV) extracted from the bee venom by utilizing protein isolation method of gel filtration. Methods: All experiments were conducted at Biotoxtech (Chungwon, Korea), authorized a non-clinical studies institution, under the regulations of Good Laboratory Practice (GLP). Antigenic potential of SBV was examined by active systemic anaphylaxis (ASA) and passive cutaneous anaphylaxis (PCA) in guinea pigs. SBV was subcutaneously administered at 0.07 and 0.28 and also as a suspension with adjuvant (Freund's complete adjuvant: FCA). Ovalbumin (OVA) as a suspension with adjuvant was used to induce positive control response (5 - FCA). Results: 1. In the ASA test, experimental groups showed some symptoms of anaphylaxis like piloerection, hyperpnea and staggering gait. 2. In the PCA test, low dosage group did not show any antibody responses, whereas high dosage group showed positive responses. 3. In the weight measurement and clinical observation, experimental groups didn't show any significant changes compared with control group. 4. In the autopsy of body, the abnormalities of lung were detected in the corpse. This means that the cause of death may induced anaphylactic shock. Conclusions: Above findings suggested that SBV had antigenic potential in guinea pig. Further studies on the subject should be conducted to yield more concrete evidences.. I. 서론 Sweet Bee Venom(. SBV. (Bee Venom). cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.. 2011 Korean Pharmacopuncture institute. melittin Corresponding Author Ki Rok Kwon. Research Center of Pharmacopuncture Medicine, Korean Pharmacopuncture Institute, Seoul, Korea Tel: +82-2-2658-9051 E-mail: [email protected] http://pharmacopuncture.co.kr.

(2) 24. Journal of Pharmacopuncture 제 14권 제 4호( 2011년 12월). SBV Melittin. rat. 40-50% 26. 2840. 4 (Fig. 1) SBV anaphylaxis(active systemic anaphylaxis-ASA) anaphylaxis(passive cutaneous anaphylaxis-PCA). allergy 10,000 lipase A2, hyaluronidase ) histamine. (phospho-. melittin SBV. II. 재료 및 방법 1. SBV (LA230S, CP323S, Sartorius, Germany) vortex mixer. 2. 2.1 Fig. 1. Molecular modeling of oligomeric melittin. The packing of melittin chains in the tetramer. Chain A is the chain built into the electron density map. Chain B is associated with chain A through an 2-fold axis approximately perpendicular to this page. Chain C and D are associated with chains A and B through a precise 2-fold axis almost in the plane of the page, as indicated.. SBV. SBV. SBV (anaphylaxis). 5. Hartley 250-300g. Guinea Pig. Sartorius, Germany) 1. (BP4100S, 7. 21.6~23.7 C. 38.7~59.3% (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C) 2005-79 Laboratory Practice (GLP). Good.

(3) 25. 2.2. 2.4 SBV. 75 5 (Table 1, 2). 10. Table 1. Experimental design and treating groups for active systemic anaphylaxis (ASA) test.. 0.1 ~ 0.4 60kg 0.007 / 10 0.07 / 4 0.28 / / + FCA 0.28 / OVA 10 / FCA FCA 10 /. /human/. 0.28. 5 / 5 / ,. Number of animal. Dose Group. Pathway (Code No.). G1. (sc),. Negative 0. 1.0. 5 (1101~1105). iv. G2 Low dose g.. 0.07. 1.0. 5 (1201~1205). iv. G3 High dose g.. 0.28. 1.0. 5 (1301~1305). iv. G4 High dose+FCA 0.28. 1.0. 5 (1401~1405). iv. 1.0. 5 (1501~1505). iv. control group. 2.5 2.5.1 0. 3. 6, 9, 12 FCA. G5 Positive control 5 (OVA-FCA). 15 0, 7. 14. Table 2. Experimental design and treating groups for passive cutaneous anaphylaxis (PCA) test. Number of animal. Dose Group. (Code No.) G1. (iv). 2.5.2 (PCA) ASA PCA. (ASA) 1 4. 1. Negative 0. 1.0. 10 (1601~1610). iv. G2 Low dose g.. 0.07. 1.0. 10 (1701~1710). iv. G3 High dose g.. 0.28. 1.0. 10 (1801~1810). iv. G4 High dose+FCA 0.28. 1.0. 10 (1901~1910). iv. 1.0. 10 (11001~11010). iv. control group. 2.5.3. G5 Positive control 10 (OVA-FCA). 2.3 SBV Ovalbumin (OVA) evans blue Sigma (St. Louis, MO, USA) Freund's complete adjuvant (FCA) Difco (Grand Island, NY, USA). 12. 1. 2~3. 2. (3,000rpm, 10min, 4 ). ; UNION 32R Plus. 2.5.4 14 3.4.1.

(4) Journal of Pharmacopuncture 제 14권 제 4호( 2011년 12월). 26. [-] [. 3.4. Asymptomatic. ]. Mild. 1~4. [+]. Moderate. 1~10. [++]. Severe. 1~19. [+++]. Death. 3.4.1. 2.5.5 12 20, 40, 80, 2560, 5120,. 160,. 320,. 640,. ( 10, 1280,. 13 1 0.05 2. 1 5. 10 4. 2% Evans blue 30. CO2. 3.4.2. 3. shock. 1. (Restlessness) 2. (Piloerection) 3. (Tremor) 4. (Rubbing or licking nose) 5. (Sneezing) 6. (Coughing) 7. (Hyperpnea) 8. (Urination) 9. (Evacuation) 10. (Lacrimation) 11. (Dyspnea) 12. (Rhonchus) 13. (Cyanosis) 14. (Staggering gait) 15. (Jumping) 16. (Gasping and writhing) 17. (Convulsion) 18. (Side position) 19. Cheyne-Stokes (Cheyne-Stokes respiration) 20. (Death) 3.4.2. 3.1 1. 5. 3.2 3.5. 29. CO2. 3.3 30. 4 shock. CO2.

(5) 27. III. 결과. 3.. 1.. shock 3/5 (Evacuation). (Urination) 1/5 (Table 3) Table 3. Clinical signs and mortality. 2.. 225g 195g. 214g, 205g SBV. 203g. (Table 4).. 2/5 (Restlessness) 4/5 (Piloerection) 1/5 (Rubbing and licking nose) 1/5 (Sneezing), 1/5 (Coughing) 1/5 4/5 3/5 (Rhonchus) 1/5 (Gasping and writhing) 1/5 (Convulsion) 1/5 (Side position) 1/5 Cheyne-Stokes (Cheyne-Stokes respiration) 1/5 (Death) 5/5 3/5 2/5 1/5 2/5 (Hyperpnea) 5/5 2/5 1/5 2/5 (Strggering gait) 5/5 5/5 1/5 (Tremor) 3/5 1/5 3/5 5/5 2/5 1/5 (Dyspnea) 3/5 2/5. Table 4. Change of body weights. 5/5 5/5. 4/5 3/5 5/5. 3/5 5/5 5/5. 4/5 (Jumping) 3/5 5/5 5/5. 4/5 3/5 3/5. 5/5. 5/5 Cheyne-Stokes (Table 5)..

(6) Journal of Pharmacopuncture 제 14권 제 4호( 2011년 12월). 28. Table 5. Active systemic anaphylaxis (ASA) test.. (3) High dose of Sweet Bee (4) High dose + FCA Venom. (5) Positive control (OVA-FCA)) Fig. 2. Skin test for Passive Cutaneous Anaphylaxis (PCA) by Sweet Bee Venom in guinea pigs. (1) Negative control group (NCG) was treated by normal saline, (2) Low dose group of Sweet Bee Venom (SBV: 0.07mg/kg) (3) High dose group of Sweet Bee Venom (SBV: 0.28mg/kg), (4) High dose + FCA (Freund's complete adjuvant) (5) OVA (Ovalbumin) + FCA (Freund's complete adjuvant). 4. 5 5. 1. 2 1/5 20. 1/5 5120. 20 1/5 160. 1/5. Table 6. Passive cutaneous anaphylaxis (PCA) test.. 640 5 (Fig. 2, Table 6).. 5. (1) Negative control. (2) Low dose of Sweet Bee Venom.

(7) 29. FCA 1. shock. 5 Shock FCA (Enlarged) (Dilatation) (Table 7) Table 7. Necropsy finding of active systemic anaphylaxis (ASA) test.. 1 3. Good Laboratory Practice (GLP). IV. 고찰. 1 shock. IgE type. shock. shock crosslink. Melittin. shock allergen (poison, toxin, toxicant). 26 40-50% Melittin phospholipase A2 melittin. peptide 2846.

(8) 30. Journal of Pharmacopuncture 제 14권 제 4호( 2011년 12월). melittin. melittin. nuclear factorB NF- B lipopolysaccharide(LPS), tumor necrosis factor- (TNF- ) sodium nitroprusside(SNP) (Fig. 3). shock 1. 5. SBV shock. 20 640 5,120 SBV Fig. 3. Possible anti-inflammatory mechanism of melittin. NF- B activity is stimulated by many inflammatory stimuli. The IKK complex, which consists of the kinases IKK , IKK and the regulatory subunit NEMO (also known as IKK ), is a point of convergence for all 3 signaling pathways. The IKK complexes phosphorylate I B , which leads to its degradation and allows the NF- B dimers to enter the nucleus, where they bind to the cognate DNA binding sites and activate the expression of the pro-inflammatory gene.. SBV LD50 1/50-100 SBV SBV. 30. V. 결론 gel filtration Sweet BV(melittin). / shock shock. SBV (melittin). 1. shock. shock. shock. shock.

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