Bee venom was purchased from Sigma (St. Louis, MO, USA) and diluted with saline. In this experimental paradigm, the mice were divided into four groups: non-transgenic mice treated with saline at ST36, Non-Tg, n = 9; hSOD1G93A mice treated with saline at ST36, CON, n = 10; hSOD1G93A mice treated with BVA at ST36, ST36, n = 10; and hSOD1G93A mice injected i.p. with BV, IP, n = 10. The ST36 acupoint is based on the human acupoint landmark and a mouse anatomical reference [31 (link)]; it is anatomically located at 5 mm below and lateral to the anterior tubercle of the tibia. Bee venom was injected into 14-week-old (98-day-old) male hSOD1G93A transgenic mice. The Non-Tg and CON groups were bilaterally injected (subcutaneously) with an equal volume of saline at the ST36 acupoint. BV treatment at 0.1 µg/g was performed three times per week for two weeks. The total amount of BV treatment at ST36-mice or IP-mice was 0.6 µg/g for two weeks.
Bee venom
Manufactured by Merck Group
Sourced in United States
Bee venom is a natural product derived from the venom of honeybees. It contains a complex mixture of peptides, enzymes, and other compounds. The core function of bee venom is to serve as a defense mechanism for the honeybee colony.
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Lab products found in correlation
Isopropyl myristate, by Merck Group (2 mentions)
Melittin, by Enzo Life Sciences (1 mentions)
Penicillin, by Cytion (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Hacat cell line, by Cytion (1 mentions)
Recombinant human tnf α, by R&D Systems (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Corn oil, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Nigericin, by Merck Group (1 mentions)
Anti asc n 15 r, by Santa Cruz Biotechnology (1 mentions)
Lps escherichia coli o55 b5, by Merck Group (1 mentions)
Prolong diamond antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
3 n morpholino propanesulfonic acid mops, by Merck Group (1 mentions)
5 protocols using bee venom
1
Bee Venom Therapy in ALS Mice
2
Bee Venom and Melittin Effects on HaCaT Cells
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The human keratinocyte HaCaT cell line (Cell Lines Service GmbH, Eppelheim, Germany) was cultured in high glucose DMEM supplemented with 10% (v/v) FBS and antibiotics (100 U·mL−1 penicillin and 100 μg·mL−1 streptomycin) at 37°C in a humidified 5% CO2 incubator. HaCaT (5.0 × 105 cells·mL−1) cells were seeded in the complete medium. Twenty‐four hours later, the medium was changed to serum‐free medium with bee venom (1, 10 and 100 ng·mL−1; Sigma) and melittin (0.1, 0.5 and 1 μg·mL−1; Enzo, Plymouth, PA, USA). After 30 min, the cells were stimulated with 10 ng·mL−1 TNF‐α/IFN‐γ for 9 h. PBS (pH 7.4), FBS, DMEM, penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Recombinant human TNF‐α and IFN‐γ were purchased from R&D Systems (Minneapolis, MN, USA).
3
Topical Transdermal Delivery Optimization
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All chemicals including TMA (98 %), bee venom, prednisolone, isopropyl myristate (98 %), dimethylsulfoxide, ethanol and corn oil were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). TMA was dissolved in a mixed solvent of acetone (Merck, Darmstadt, Germany) and isopropyl myristate (4:1, v/v) immediately before use. bee venom was dissolved in saline, and prednisolone was dissolved in a mixed solvent of dimethylsulfoxide, ethanol and corn oil (5:3:92, v/v/v).
4
Inflammasome Activation Mechanisms
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The following reagents were used in this study: Escherichia coli LPS O55:B5, nigericin and bee venom (Sigma-Aldrich, Madrid, Spain); caspase-8 inhibitor II X-IETD-FMK (Merck-Millipore, Darmstadt, Germany); fluorogenic substrates for caspase-1 YVAD-AFC, caspase-3 DEVD-AFC and caspase-8 IETD-AFC (PromoKine, Heidelberg, Germany); crosslinking reagent SDA (Thermo Scientific, Waltham, MA, USA); rabbit polyclonal antibody against IL-1β, caspase-1 p10 (M-20) and anti-ASC (N-15)-R (Santa Cruz Biotechnology, Dallas, TX, USA); ECL horseradish peroxidase-conjugated secondary antibody for immunoblot analysis (GE Healthcare, Uppsala, Sweden); and Alexa Fluor 647-conjugated donkey anti rabbit IgG secondary antibody and ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Paisley, UK).
5
Synthesis and Purification of Citrullinated Melittin
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Melittin was purified from bee venom (Sigma, St. Louis, MO, USA) by ion exchange chromatography on SP-Sephadex C-25 54 . Solid phase synthesis of citrullinated melittin (Cit-Mel) was carried out on methylbenzhydrylamine resin, using commercial Boc-amino acid residues (Chem-Impex, Wood Dale, IL, USA) and BOP as the coupling reagent (Matrix Innovation, Quebec City, QC, CAN). Acidolytic Boc removal was obtained by treating the protected peptide-resin with trifluoroacetic acid (TFA)/methylene chloride (45%). After a final Boc deprotection step, following a cleavage with hydrofluoric acid (Matheson, Edmonton, AB, CAN) containing m-cresol (10%) as a scavenger, a crude peptide preparation was isolated and washed with ethylether. The crude material was purified by reverse-phase HPLC using an acetonitrile (ACN) gradient in aqueous TFA (0.1%). Pure fractions corresponding to the expected mass of Cit-Mel, as established by MALDI-TOF mass spectrometry, were pooled, evaporated to remove ACN, and lyophilized. DPPC and DPPS were purchased from Avanti Polar Lipids (Alabaster, AL, USA).
Ethylenediaminetetraacetic acid (EDTA), NaCl, and 3-[N-morpholino]propanesulfonic acid (MOPS) were obtained from Sigma (St. Louis, MO, USA). All chemicals were used as received.
Ethylenediaminetetraacetic acid (EDTA), NaCl, and 3-[N-morpholino]propanesulfonic acid (MOPS) were obtained from Sigma (St. Louis, MO, USA). All chemicals were used as received.
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