Pharmacokinetics and Tissue Distribution of Bee Venom-Derived Phospholipase A2 Using a Sandwich ELISA after Subcutaneous Injection of New Composition Bee Venom in Rats.

Bee venom is a traditional drug used to treat the nervous system, musculoskeletal system, and autoimmune diseases. A previous study found that bee venom and one of its components, phospholipase A2, can protect the brain by suppressing neuroinflammation and can also be used to treat Alzheimer's disease. Thus, new composition bee venom (NCBV), which has an increased phospholipase A2 content of up to 76.2%, was developed as a treatment agent for Alzheimer's disease by INISTst (Republic of Korea). The aim of this study was to characterize the pharmacokinetic profiles of phospholipase A2 contained in NCBV in rats. Single subcutaneous administration of NCBV at doses ranging from 0.2 mg/kg to 5 mg/kg was conducted, and pharmacokinetic parameters of bee venom-derived phospholipase A2 (bvPLA2) increased in a dose-dependent manner. Additionally, no accumulation was observed following multiple dosings (0.5 mg/kg/week), and other constituents of NCBV did not affect the pharmacokinetic profile of bvPLA2. After subcutaneous injection of NCBV, the tissue-to-plasma ratios of bvPLA2 for the tested nine tissues were all <1.0, indicating a limited distribution of the bvPLA2 within the tissues. The findings of this study may help understand the pharmacokinetic characteristics of bvPLA2 and provide useful information for the clinical application of NCBV.

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells.

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206+ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206+ DCs could be a predictor of the bvPLA2 response of different individuals.

Bee Venom: From Venom to Drug.

Insects of the order Hymenoptera have a defensive substance that contains many biologically active compounds. Specifically, venom from honeybees (Apis mellifera) contains many enzymes and peptides that are effective against various diseases. Different research papers stated the possibility of using bee venom (a direct bee sting or in an injectable form) in treating several complications; either in vivo or in vitro. Other reports used the active fractions of bee venom clinically or at labratory scale. Many reports and publications have stated that bee venom and its constituents have multiple biological activities including anti-microbial, anti-protozoan, anti-cancer, anti-inflammatory, and anti-arthritic properties. The present review aims to refer to the use of bee venom itself or its fractions in treating several diseases and counteracting drug toxicities as an alternative protocol of therapy. The updated molecular mechanisms of actions of bee venom and its components are discussed in light of the previous updated publications. The review also summarizes the potential of venom loaded on nanoparticles as a drug delivery vehicle and its molecular mechanisms. Finally, the products of bee venom available in markets are also demonstrated.

Targeting of adipose tissue macrophages by bee venom phospholipase A2 attenuates high-fat diet-induced obesity.

BACKGROUND/OBJECTIVES: Adipose tissue macrophages (ATMs) exist in either the M1 or M2 form. The anti-inflammatory M2 ATMs accumulate in lean individuals, whereas the pro-inflammatory M1 ATMs accumulate in obese individuals. Bee venom phospholipase A2 (bvPLA2), a major component in honeybee (Apis mellifera) venom, exerts potent anti-inflammatory effects via interactions with regulatory T cells (Treg) and macrophages. This study investigated the effects of bvPLA2 on a high-fat diet (HFD)-induced obesity in mice. SUBJECTS/METHODS: For in vivo experiments, male C57BL/6, CD206-deficient, and Treg-depleted mice models were fed either a normal diet 41.86 kJ (ND, 10 kcal% fat) or high-fat diet 251.16 kJ (HFD, 60 kcal% fat). Each group was i.p. injected with PBS or bvPLA2 (0.5 mg/kg) every 3 days for 11 weeks. Body weight and food intake were measured weekly. Histological changes in the white adipose tissue (WAT), liver, and kidney as well as the immune phenotypes of the WAT were examined. Immune cells, cytokines, and lipid profiles were also evaluated. The direct effects of bvPLA2 on 3T3-L1 pre-adipocytes and bone marrow-derived macrophages were measured in vitro. RESULTS: bvPLA2 markedly decreased bodyweight in HFD-fed mice. bvPLA2 treatment also decreased lipid accumulation in the liver and reduced kidney inflammation in the mice. It was confirmed that bvPLA2 exerted immunomodulatory effects through the CD206 receptor. In addition, bvPLA2 decreased M1 ATM and alleviated the M1/M2 imbalance in vivo. However, bvPLA2 did not directly inhibit adipogenesis in the 3T3-L1 adipose cells in vitro. CONCLUSIONS: bvPLA2 is a potential therapeutic strategy for the management of obesity by regulating adipose tissue macrophage homeostasis.

Lipolytic Activity of a Carboxylesterase from Bumblebee (Bombus ignitus) Venom.

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme's role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

Phospholipase A2 way to hydrolysis: Dint formation, hydrophobic mismatch, and lipid exclusion.

Phospholipase A2 (PLA2) exerts a wide range of biological effects and attracts a lot of attention of researchers. Two sites are involved in manifestation of PLA2 enzymatic activity: catalytic site responsible for substrate binding and fatty acid cleavage from the sn-2 position of a glycerophospholipid, and interface binding site (IBS) responsible for the protein binding to lipid membrane. IBS is formed by positively charged and hydrophobic amino acids on the outer surface of the protein molecule. Understanding the mechanism of PLA2 interaction with the lipid membrane is the most challenging step in biochemistry of this enzyme. We used a combination of experimental and computer simulation techniques to clarify molecular details of bee venom PLA2 interaction with lipid bilayers formed by palmitoyloleoylphosphatidylcholine or dipalmitoylphosphatidylcholine. We found that after initial enzyme contact with the membrane, a network of hydrogen bonds was formed. This led to deformation of the interacting leaflet and dint formation. The bilayer response to the deformation depended on its phase state. In a gel-phase bilayer, diffusion of lipids is restricted therefore chain melting occurred in both leaflets of the bilayer. In the case of a fluid-phase bilayer, lateral diffusion is possible, and lipid polar head groups were excluded from the contact area. As a result, the bilayer became thinner and a large hydrophobic area was formed. We assume that relative ability of a bilayer to come through lipid redistribution process defines the rate of initial stages of the catalysis.

Bee Venom Phospholipase A2 Ameliorates Atherosclerosis by Modulating Regulatory T Cells.

Atherosclerosis is a chronic inflammatory disease caused by lipids and calcareous accumulations in the vascular wall due to an inflammatory reaction. Recent reports have demonstrated that regulatory T (Treg) cells have an important role as a new treatment for atherosclerosis. This study suggests that bee venom phospholipase A2 (bvPLA2) may be a potential therapeutic agent in atherosclerosis by inducing Treg cells. We examined the effects of bvPLA2 on atherosclerosis using ApoE-/- and ApoE-/-/Foxp3DTR mice. In this study, bvPLA2 increased Treg cells, followed by a decrease in lipid accumulation in the aorta and aortic valve and the formation of foam cells. Importantly, the effect of bvPLA2 was found to depend on Treg cells. This study suggests that bvPLA2 can be a potential therapeutic agent for atherosclerosis.

Bee Venom Phospholipase A2 Induces Regulatory T Cell Populations by Suppressing Apoptotic Signaling Pathway.

Bee venom phospholipase A2 is a lipolytic enzyme in bee venom that catalyzes hydrolysis of the sn-2 ester bond of membrane phospholipids to produce free fatty acid and lysophospholipids. Current evidence suggests that bee venom phospholipase A2 (bvPLA2) induces regulatory T cell expansion and attenuates several immune system-related diseases, including Alzheimer's disease. The induction of Treg cells is directly mediated by binding to mannose receptors on dendritic cells. This interaction induces the PGE2-EP2 signaling pathway, which promotes Treg induction in CD4+ T cells. In this study, we investigated the effects of bvPLA2 treatment on the apoptotic signaling pathway in Treg populations. Flow cytometry was performed to identify early apoptotic cells. As a result, early apoptotic cells were dramatically decreased in bvPLA2-treated splenocytes, whereas rapamycin-treated cells showed levels of apoptotic cells similar to those of PBS-treated cells. Furthermore, bvPLA2 treatment increased expression of anti-apoptotic molecules including CTLA-4 and PD-1. The survival rate increased in bvPLA2-treated Tregs. Our findings indicate that bvPLA2-mediated modulation of apoptotic signaling is strongly associated with the Treg induction, which exhibits protective effects against various immune-related diseases. To our knowledge, this study is the first to demonstrate that bvPLA2 is the major bee venom (BV) compound capable of inducing Treg expansion through altering apoptotic signal.

Illustrating Interfacial Interaction between Honey Bee Venom Phospholipase A2 and Supported Negatively Charged Lipids with Sum Frequency Generation and Laser Scanning Confocal Microscopy.

Phospholipase A2 is an important enzyme species which can widely be found in animals, plants, bacteria, and so on. A large number of studies have shown that phospholipase A2 is highly catalytic toward the lipids. Here, sum frequency generation (SFG) vibrational spectroscopy and laser scanning confocal microscopy (LSCM) were applied to study the interaction between honey bee venom phospholipase A2 (bvPLA2) and the negatively charged DPPG bilayer. In both cases without and with the calcium ions (Ca2+), the bvPLA2 molecules were adsorbed onto the outer leaflet surface with the orientational order, and the adsorbed bvPLA2 molecules damaged the order of the packed outer leaflet. In comparison to the case without Ca2+, the addition of Ca2+ can accelerate the attaching process of bvPLA2 to the outer leaflet surface and decelerate the process of damaging the outer leaflet order. The experimental result also confirmed, with the help of the Ca2+, the DPPG molecules in the outer leaflet were hydrolyzed, with both hydrolysates, that is, lysophospholipids and fatty acids, remaining at the interface, showing a distinct difference from previous published literatures regarding neutral lipids [Phys. Chem. Chem. Phys. 2018, 20, 63-67] and PLA1 [Langmuir 2019, 35, 12831-12838].

Phospholipase A2 from bee venom increases poly(I:C)-induced activation in human keratinocytes.

Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly(I:C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly(I:C)-mediated interleukin 8 production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly(I:C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly(I:C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.

Polyunsaturated Phospholipid Modified Membrane Degradation Catalyzed by a Secreted Phospholipase A2.

To optimize the compositions of the lipid-based nanomedicine and to advance understanding of the roles of polyunsaturated phospholipids in biological membranes, this study examined the effects of polyunsaturated phospholipids on the degradation of giant unilamellar vesicles catalyzed by a secreted phospholipase A2 (sPLA2) using fluorescence microscopy. Molecular interfacial packing, interaction, and degradation of the films containing various mixing ratios of saturated and polyunsaturated phospholipids were quantified using a Langmuir trough integrated with synchrotron X-ray surface scattering techniques. It was found that a high molar fraction (0.63 and above) of polyunsaturated phospholipids not only enhanced the rate of sPLA2-catalyzed vesicle degradation but also changed the vesicle deformation process and degradation product morphology. Hydrolysis of the saturated phospholipids generated highly ordered liquid crystal domains, which was reduced or prohibited by the presence of the polyunsaturated phospholipids in the reactant film.

Prophylactic Effects of Bee Venom Phospholipase A2 in Lipopolysaccharide-Induced Pregnancy Loss.

Spontaneous abortion represents a common form of embryonic loss caused by early pregnancy failure. In the present study, we investigated the prophylactic effects of bee venom phospholipase A2 (bvPLA2), a regulatory T cell (Treg) inducer, on a lipopolysaccharide (LPS)-induced abortion mouse model. Fetal loss, including viable implants, the fetal resorption rate, and the fetal weight, were measured after LPS and bvPLA2 treatment. The levels of serum and tissue inflammatory cytokines were determined. To investigate the involvement of the Treg population in bvPLA2-mediated protection against fetal loss, the effect of Treg depletion was evaluated following bvPLA2 and LPS treatment. The results clearly revealed that bvPLA2 can prevent fetal loss accompanied by growth restriction in the remaining viable fetus. When the LPS-induced abortion mice were treated with bvPLA2, Treg cells were significantly increased compared with those in the non-pregnant, PBS, and LPS groups. After LPS injection, the levels of proinflammatory cytokines were markedly increased compared with those in the PBS mouse group, while bvPLA2 treatment showed significantly decreased TNF-α and IFN-γ expression compared with that in the LPS group. The protective effects of bvPLA2 treatment were not detected in Treg-depleted abortion-prone mice. These findings suggest that bvPLA2 has protective effects in the LPS-induced abortion mouse model by regulating Treg populations.

Bee Venom Phospholipase A2 Alleviate House Dust Mite-Induced Atopic Dermatitis-Like Skin Lesions by the CD206 Mannose Receptor.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by highly pruritic, erythematous, and eczematous skin plaques. We previously reported that phospholipase A2 (PLA2) derived from bee venom alleviates AD-like skin lesions induced by 2,4-dinitrochlorobenzene (DNCB) and house dust mite extract (Dermatophagoides farinae extract, DFE) in a murine model. However, the underlying mechanisms of PLA2 action in actopic dermatitis remain unclear. In this study, we showed that PLA2 treatment inhibited epidermal thickness, serum immunoglobulin E (IgE) and cytokine levels, macrophage and mast cell infiltration in the ear of an AD model induced by DFE and DNCB. In contrast, these effects were abrogated in CD206 mannose receptor-deficient mice exposed to DFE and DNCB in the ear. These data suggest that bvPLA2 alleviates atopic skin inflammation via interaction with CD206.

Phospholipase A2-Responsive Phosphate Micelle-Loaded UCNPs for Bioimaging of Prostate Cancer Cells.

We report the effective synthesis of biocompatible upconversion nanoparticles (UCNP)-loaded phosphate micelles and successful delivery of UCNPs to prostate cancer cells via secreted phospholipase A2 (sPLA-2) enzyme cleavage of the loaded micelles for the first time. The activity of the (sPLA-2) enzyme toward the synthesized micelles was investigated and confirmed by LC-MS. TEM results showed that the micelles have a size distribution of 80 to 150 nm, whereas UCNP-loaded micelles range from 200 to 350 nm, indicating the successful loading of UCNPs. The selective release of UCNPs to prostate cancer cells rather than other cells, specifically cervical cancer cells, was observed and confirmed by a range of bioimaging studies. Moreover, cytotoxicity assays confirmed the biocompatibility of the UCNP-loaded micelles.

Calcium-dependent hydrolysis of supported planar lipids was triggered by honey bee venom phospholipase A2 with the right orientation at the interface.

Hydrolysis of planar phospholipids catalyzed by honey bee venom phospholipase A2 (bvPLA2) was studied. Experiments demonstrated that Ca2+ ions mediated between the lipids and bvPLA2, induced reorientation of bvPLA2, and activated hydrolysis. One of the hydrolysis products, fatty acids, was desorbed, and the other one, lysophospholipids, self-organized at the interface.

Bee Venom Phospholipase A2 Ameliorates House Dust Mite Extract Induced Atopic Dermatitis Like Skin Lesions in Mice.

Atopic dermatitis (AD) is a biphasic inflammatory skin disease that is provoked by epidermal barrier defects, immune dysregulation, and increased skin infections. Previously, we have demonstrated that bvPLA2 evoked immune tolerance by inducing regulatory T cells (Treg), and thus alleviated Th2 dominant allergic asthma in mice. Here, we would like to determine whether treatment with bvPLA2 exacerbates the AD-like allergic inflammations induced by house dust mite extract (DFE) in a murine model. Epidermal thickness, immune cell infiltration, serum immunoglobulin, and cytokines were measured. Ear swelling, skin lesions, and the levels of total serum IgE and Th1/Th2 cytokines were elevated in DFE/DNCB-induced AD mice. Topical application of bvPLA2 elicited significant suppression of the increased AD symptoms, including ear thickness, serum IgE concentration, inflammatory cytokines, and histological changes. Furthermore, bvPLA2 treatment inhibited mast cell infiltration into the ear. On the other hand, Treg cell depletion abolished the anti-atopic effects of bvPLA2, suggesting that the effects of bvPLA2 depend on the existence of Tregs. Taken together, the results revealed that topical exposure to bvPLA2 aggravated atopic skin inflammation, suggesting that bvPLA2 might be a candidate for the treatment of AD.

Protective Effects of Intratracheally-Administered Bee Venom Phospholipase A2 on Ovalbumin-Induced Allergic Asthma in Mice.

Asthma is a common chronic disease characterized by bronchial inflammation, reversible airway obstruction, and airway hyperresponsiveness (AHR). Current therapeutic options for the management of asthma include inhaled corticosteroids and ß2 agonists, which elicit harmful side effects. In the present study, we examined the capacity of phospholipase A2 (PLA2), one of the major components of bee venom (BV), to reduce airway inflammation and improve lung function in an experimental model of asthma. Allergic asthma was induced in female BALB/c mice by intraperitoneal administration of ovalbumin (OVA) on days 0 and 14, followed by intratracheal challenge with 1% OVA six times between days 22 and 30. The infiltration of immune cells, such as Th2 cytokines in the lungs, and the lung histology, were assessed in the OVA-challenged mice in the presence and absence of an intratracheal administration of bvPLA2. We showed that the intratracheal administration of bvPLA2 markedly suppressed the OVA-induced allergic airway inflammation by reducing AHR, overall area of inflammation, and goblet cell hyperplasia. Furthermore, the suppression was associated with a significant decrease in the production of Th2 cytokines, such as IL-4, IL-5, and IL-13, and a reduction in the number of total cells, including eosinophils, macrophages, and neutrophils in the airway.

Bee venom phospholipase A2 ameliorates motor dysfunction and modulates microglia activation in Parkinson's disease alpha-synuclein transgenic mice.

α-Synuclein (α-Syn) has a critical role in microglia-mediated neuroinflammation, which leads to the development of Parkinson's disease (PD). Recent studies have shown that bee venom (BV) has beneficial effects on PD symptoms in human patients or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin-induced PD mice. This study investigated whether treatment with BV-derived phospholipase A2 (bvPLA2) would improve the motor dysfunction and pathological features of PD in human A53T α-Syn mutant transgenic (A53T Tg) mice. The motor dysfunction of A53T Tg mice was assessed using the pole test. The levels of α-Syn, microglia and the M1/M2 phenotype in the spinal cord were evaluated by immunofluorescence. bvPLA2 treatment significantly ameliorated motor dysfunction in A53T Tg mice. In addition, bvPLA2 significantly reduced the expression of α-Syn, the activation and numbers of microglia, and the ratio of M1/M2 in A53T Tg mice. These results suggest that bvPLA2 could be a promising treatment option for PD.

A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Regulatory T Cells Contribute to the Inhibition of Radiation-Induced Acute Lung Inflammation via Bee Venom Phospholipase A2 in Mice.

Bee venom has long been used to treat various inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis. Previously, we reported that bee venom phospholipase A2 (bvPLA2) has an anti-inflammatory effect through the induction of regulatory T cells. Radiotherapy is a common anti-cancer method, but often causes adverse effects, such as inflammation. This study was conducted to evaluate the protective effects of bvPLA2 in radiation-induced acute lung inflammation. Mice were focally irradiated with 75 Gy of X-rays in the lung and administered bvPLA2 six times after radiation. To evaluate the level of inflammation, the number of immune cells, mRNA level of inflammatory cytokine, and histological changes in the lung were measured. BvPLA2 treatment reduced the accumulation of immune cells, such as macrophages, neutrophils, lymphocytes, and eosinophils. In addition, bvPLA2 treatment decreased inflammasome-, chemokine-, cytokine- and fibrosis-related genes' mRNA expression. The histological results also demonstrated the attenuating effect of bvPLA2 on radiation-induced lung inflammation. Furthermore, regulatory T cell depletion abolished the therapeutic effects of bvPLA2 in radiation-induced pneumonitis, implicating the anti-inflammatory effects of bvPLA2 are dependent upon regulatory T cells. These results support the therapeutic potential of bvPLA2 in radiation pneumonitis and fibrosis treatments.