Anti-Inflammatory Applications of Melittin, a Major Component of Bee Venom: Detailed Mechanism of Action and Adverse Effects

Inflammation is a pervasive phenomenon triggered by the innate and adaptive immune systems to maintain homeostasis. The phenomenon normally leads to recovery from infection and healing, but when not properly phased, inflammation may cause immune disorders. Bee venom is a toxin that bees use for their protection from enemies. However, for centuries it has been used in the Orient as an anti-inflammatory medicine for the treatment of chronic inflammatory diseases. Bee venom and its major component, melittin, are potential means of reducing excessive immune responses and provide new alternatives for the control of inflammatory diseases. Recent experimental studies show that the biological functions of melittin could be applied for therapeutic use in vitro and in vivo. Reports verifying the therapeutic effects of melittin are accumulating in the literature, but the cellular mechanism(s) of the anti-inflammatory effects of melittin are not fully elucidated. In the present study, we review the current knowledge on the therapeutic effects of melittin and its detailed mechanisms of action against several inflammatory diseases including skin inflammation, neuroinflammation, atherosclerosis, arthritis and liver inflammation, its adverse effects as well as future prospects regarding the use of melittin.


Introduction
Inflammation is a primary process of the immune response that is triggered by any stimulus such as infection, injury, and exposure to contaminants that poses a real or perceived threat to homeostasis [1]. It is a protective process for the body, however chronic inflammation can cause the development of different diseases like rheumatoid arthritis, cardiovascular disease, diabetes, obesity, inflammatory bowel disease, asthma, and CNS related diseases such as Parkinson's disease and Amyotrophic Lateral Sclerosis (ALS) [2].

Application for Skin Inflammation
Acne vulgaris is a long term skin disorder of the hair follicle in the face and upper trunk, and Propionibacterium acnes (P. acnes) is the main cause of the inflammation of acne [33]. Toll-like receptor 2 signaling activated by P. acnes causes keratinocytes and monocytes to secrete pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-8 [34,35]. A wide range of agents, including antibiotics, is used to suppress inflammation in acne vulgaris, but antibiotics can cause side effects [36].
The protective effects of melittin on P. acnes-induced inflammatory responses in vitro and in vivo were reported by Lee et al. [37]. They investigated the anti-inflammatory effects of melittin treatment in heat-killed P. acnes-treated HaCaT cells. Melittin treatment attenuated the increased phosphorylation of IKK, IκB, NF-κB as well as p38 by heat-killed P. acnes in HaCaT cells. These data show that melittin treatment abrogates P. acnes-induced inflammatory cytokine production through blocking NF-κB signaling as well as p38 MAPK signaling in HaCaT cells. In addition, the anti-inflammatory effect of melittin was examined in a live P. acnes-induced inflammatory skin disease animal model. In the animal model, melittin-treated ears show markedly reduced P. acnes-injected swelling and granulomatous responses, as compared with ears injected with live P. acnes alone.
Another study showed the inhibitory action of melittin against heat-killed P. acnes-induced apoptosis and inflammation in human THP-1 monocytic cells [38]. Melittin treatment suppressed the cleavage of the caspase-3, -8, and PARP in heat-killed P. acnes-treated THP-1 monocytic cells; in addition, administration of melittin significantly decreased the expression of TNF-α and IL-1β. This inhibition results in reduced inflammation in skin, arota, joint, liver, and neuronal tissue. TLR, toll-like receptor; CD, cluster of differentiation; NEMO, nuclear factor kappa-B essential modulator; PDGFRβ, Platelet-derived growth factor receptor beta.

Application for Skin Inflammation
Acne vulgaris is a long term skin disorder of the hair follicle in the face and upper trunk, and Propionibacterium acnes (P. acnes) is the main cause of the inflammation of acne [33]. Toll-like receptor 2 signaling activated by P. acnes causes keratinocytes and monocytes to secrete pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-8 [34,35]. A wide range of agents, including antibiotics, is used to suppress inflammation in acne vulgaris, but antibiotics can cause side effects [36].
The protective effects of melittin on P. acnes-induced inflammatory responses in vitro and in vivo were reported by Lee et al. [37]. They investigated the anti-inflammatory effects of melittin treatment in heat-killed P. acnes-treated HaCaT cells. Melittin treatment attenuated the increased phosphorylation of IKK, IκB, NF-κB as well as p38 by heat-killed P. acnes in HaCaT cells. These data show that melittin treatment abrogates P. acnes-induced inflammatory cytokine production through blocking NF-κB signaling as well as p38 MAPK signaling in HaCaT cells. In addition, the anti-inflammatory effect of melittin was examined in a live P. acnes-induced inflammatory skin disease animal model. In the animal model, melittin-treated ears show markedly reduced P. acnes-injected swelling and granulomatous responses, as compared with ears injected with live P. acnes alone.
Another study showed the inhibitory action of melittin against heat-killed P. acnes-induced apoptosis and inflammation in human THP-1 monocytic cells [38]. Melittin treatment suppressed the cleavage of the caspase-3, -8, and PARP in heat-killed P. acnes-treated THP-1 monocytic cells; in addition, administration of melittin significantly decreased the expression of TNF-α and IL-1β.

Application for Neurodegenerative Diseases
In vitro assays have revealed the potential of melittin as an agent for the prevention of neurodegenerative diseases. Moon et al. showed that melittin has a potent suppressive effect on pro-inflammatory responses of BV2 microglia and suggested that melittin may have potential to treat neurodegenerative diseases accompanied with microglial activation [39]. Melittin suppresses expression of NO and iNOS by blocking LPS-induced activation of NF-κB in BV2 microglial cell line. These results indicate that melittin suppresses COX-2/PGE2 expression resulting in anti-inflammatory properties. Meanwhile, Han et al. investigated the anti-apoptotic effects of melittin in a H 2 O 2 -induced cytotoxicity model using the SH-SY5Y human neuroblastoma cell line [40]. Melittin treatment increased cell viability and decreased apoptotic DNA fragmentation. Melittin inhibited the H 2 O 2 -induced decrease of anti-apoptotic factor Bcl-2 expression and increase of pro-apoptotic factor Bax expression.
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects motor neurons in the brain and the spinal cord, resulting in weakness and atrophy of muscles [53]. Yang et al. showed that melittin treatment (0.1 µg/g twice a week s.c. acupuncture on bilateral point ST36) improves anti-neuroinflammatory ability of proteasome in the CNS of ALS model mice [41]. In this model, administration of melittin reduces phosphorylation of p38 and the microglial cell number in the brainstem and spinal cord; in addition, melittin-treated mice exhibit declined neuronal death in the spinal cord and improved motor control that improves motor function. Furthermore, melittin alleviates the misfolding of proteins by activation of chaperones and reducing post-transcriptional modification of α-synuclein, a major process of ALS pathogenesis. The results demonstrate the anti-neuroinflammatory effects of melittin. Although the main problem of ALS occurs in CNS, ALS also affects other organs, including the liver, spleen, and lung. Melittin treatment attenuates inflammation and stimulates the signaling for cell survival in the spleen and lung in an animal model of ALS [42]. Administration of melittin suppresses the expression of CD14 and Iba-1 (inflammatory proteins) in the lung of symptomatic ALS transgenic mice. In addition, melittin increases the expression of pERK and Bcl-2 (cell survival factors) and suppresses the expression of C14 and COX-2 in the spleen of symptomatic ALS mice.
Dantas et al. examined the pharmacological effects of melittin in mice, with particular emphasis on dopaminergic related behaviors [54]. The animals were submitted to behavioral tests, such as the apomorphine rotation test, catalepsy test, and open field test. The results showed that melittin-treated mice displayed reduced effects caused by apomorphine, though there is no alteration in motor activity or cataleptic effects with melittin treatment. The authors reported that melittin exhibits anti-psychotic properties and could be an alternative to treat psychotic disorders, cutting down the side effects of neuroleptic medicines. Melittin also decreases apomorphine-induced stereotypes. The data indicate the anti-psychotic activity of melittin in a mice model.

Application for Atherosclerosis
Atherosclerosis is an inflammatory disease in which plaque builds up inside arteries. This chronic inflammatory disorder of the arteries is a one of major causes of death in adults. Plaque is made up of cholesterol, triglycerides, remnants of dead cells, and immune cells. In the plaque, inflammatory immune cells including macrophage and helper T cells produce inflammatory cytokines, a main trigger of plaque growth [55]. Modulation of NF-κB signaling, which plays a critical role in apoptosis and cell proliferation, is regarded as a potential therapeutic target for the treatment of atherosclerosis [56,57].
In vitro studies show an effect of melittin on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The NF-κB signal pathway was investigated as a potent mechanism for apoptosis of cultured rat aortic VSMCa. Melittin suppresses not only platelet-derived growth factor receptor (PDGFR) β-tyrosine phosphorylation, but also downstream intracellular signal transduction in rat aortic VSMCa [43] and blocks phosphorylation of AKT induced by the PDGRF signal [44]. In addition, phosphorylation of extracellular signal-regulated kinase 1/2, an upstream signal of NF-κB, is slightly suppressed by melittin treatment. Melittin treatment, besides, induces expression of pro-apoptotic proteins, including p53, Bax, and caspase-3, but decreases expression of anti-apoptotic protein Bcl-2 [44]. In addition, in vivo experiments demonstrate that melittin treatment suppresses atherosclerosis via athero-protective functions in High-Fat/LPS treated mice [45]. Melittin treatment decreases total cholesterol and triglyceride levels and recovers heart and descending aorta. In addition, melittin decreases the expression levels of TNF-α, IL-1β, VCAM-1, ICAM-1, and TGFβ-1 in atherosclerotic mice.
Cho et al. used mass spectrometry and gel electrophoresis to elucidate the anti-atherosclerotic mechanisms of melittin. They identified differentially expressed proteins by melittin treatment in human VSMCs stimulated by TNF-α [46]. In their experiments, a proteomics analysis identify 33 proteins with consistently different expression patterns after melittin treatment. The identified proteins include anti-apoptotic proteins like as stress-70 protein and annexin A1, Several target molecules of inflammation and proliferation such as prohibitin, annexin-1, and calreticulin were identified. Furthermore, intracellular signal pathway analysis shows that EGFR and NF-κB are the main signaling molecules of inflammation in TNF-α treated human VSMCs.

Application for Arthritis
The Hong group proposed target inactivation of NF-κB by directly binding to the p50 subunit as anti-arthritic mechanism of bee venom and melittin. They demonstrated that melittin inhibits LPS-induced p50 translocation into nucleus resulting in reduced transcription of inflammatory genes [20]. Melittin directly binds to p50 (affinity [Kd] = 1.2ˆ10´8 M). They also investigated the anti-inflammatory effect of melittin via interaction with IKKs. In a mouse macrophage cell line and synoviocytes acquired from rheumatoid patients, melittin suppresses the TNF-α/LPS-induced production of NO and PGE2 [47]. In addition, they demonstrated that the JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-κB activation by melittin [48].

Application for Liver Inflammation
Park et al. reported that melittin attenuates hepatic injury, inflammation and hepatic fibrosis. Melittin inhibits TNF-α secretion and expression of IL-1β and IL-6 in the TNF-α-treated hepatic stellate cells (HSCs). Melittin attenuates inflammation and fibrosis by inhibiting the NF-κB signaling pathway in thioacetamide-induced liver fibrosis. In addition, they suggested that the regulated inflammatory response may affect anti-fibrotic effect of melittin in the activated HSCs [49]. Subsequently, they demonstrate that melittin inhibits liver failure via blocking NF-κB signaling and apoptotic pathways in the D-galactosamine/LPS-induced mouse liver failure model [50].
Melittin decreases the high rate of lethality, alleviates hepatic pathological injury, attenuates hepatic inflammatory responses and inhibits hepatocyte apoptosis. They also elucidated the inhibitory mechanism of melittin for NF-κB transcription in TNF-α-induced hepatic damage [51]. In TNF-α-treated hepatocytes, melittin inhibits DNA binding of NF-κB as well as promoter activity of NF-κB, suggesting that melittin inhibits apoptosis of hepatocytes through suppression of NF-κB activation. Recently, they showed that administration of melittin attenuated inflammation and fibrosis of the bile duct. Their experimental results suggest that melittin may have therapeutic applications in chronic liver injury [52]. Figure 1 summarizes the major mechanisms of the anti-inflammatory action of melittin.

Adverse Effects of Melittin
Melittin is the major constituent of apitoxin and is known as an allergenic peptide. It is also responsible for cell lysis and death. Accumulated melittin peptides disrupt phospholipid packing in the cell membrane, resulting in cell lysis [23]. Melittin provokes the lysis of plasma membranes and intracellular membranes. Melittin and bee venom phospholipase A 2 (PLA 2 ) show synergistic action with lipid membranes, leading to cell damage [58].

Allergic Reactions
Melittin is considered as an allergen of bee venom since research in the 1970s showed that it induces an IgE response in around one-third of honeybee venom-sensitive patients [59] and melittin appears to be allergenic in several patients [60]. However, the results are possibly due to contamination with other bee venom components. Bee venom is a complex mixture of melittin, apamine, mast cell degranulating peptide, histamine, adolapin, oligopeptides, phospholipids, saccharides, phospholipase A 2 , hyaluconidase, acid phosphatase, etc. [26]. Purified melittin potentially contains residues of strong allergens such as hyaluronidase, phospholipase A 2 and acid phosphatasen. As the purity of currently available purified melittin is not high (e.g., Sigma-Aldrich M7391: 65%-85% and Sigma-Aldrich M2272: ě85%, (Sigma Aldrich Inc., St. Louis, MO, USA)) synthesized melittin (e.g., Sigma-Aldrich M4171: ě97%) should be used to confirm whether melittin causes allergic reactions or not.

Hemolysis, Cytotoxic Effects
Melittin is also known for its high lytic activity on human erythrocyte cells [61,62]. It directly binds on erythrocytes and releases hemoglobin. It is reported that the maximum capacity and the apparent dissociation constant for melittin binding to human erythrocytes is 1.8ˆ10 7 molecules/cell and 30 nM, respectively [61]. When melittin provokes hemolysis, swelling of the erythrocytes is observed after leakage of cations occurs from membrane of the cells. In its initial phase, melittin increases permeability of ions and the release of hemoglobin is followed [61,62]. Melittin is cytotoxic for human peripheral blood lymphocytes (HPBLs) in a dose-and time-dependent manner. It leads to granulation, morphologic changes, and finally lysis of cells [63].

Genotoxic Effects
Gajski et al. report that low doses (non-cytotoxic concentrations) of melittin can increase DNA damage in HPBLs [63]. In their experiments, comet and micronucleus assays show decreased proliferation of lymphocyte and increased formation of nuclear buds and micronuclei. Moreover, melittin modulates gene expressions related with apoptosis (Bax, Bcl-2, Cas-3, Cas-7), DNA damage response (TP53, CDKN1A, GADD45α, MDM), and oxidative stress (CAT, GCLC, GPX1, GSR, SOD1). The observed genotoxicity coincides with reduction of glutathione level, increased formation of reactive oxygen species, increased phospholipase C activity and lipid peroxidation, showing the induction of oxidative stress.

Attempts to Overcome the Adverse Effect of Melittin
Even though melittin has demonstrated significant therapeutic properties, its toxicity must be neutralized for use as an anti-inflammatory agent. The cytotoxic effect of melittin is beneficial in part for anti-tumor purposes, but it hinders therapeutic application of melittin for other purposes. Since the native form of melittin causes non-specific cell lysis and toxicity, attempts including mutation and fusion proteins to reduce the toxicity of melittin to deliver it to specific targeted lesions are ongoing. Asthana et al. showed that alanine substitution in the leucine zipper motif of melittin results in a significant reduction of its hemolytic, but not anti-bacterial activity [64]. This suggests that lesion-specific mutations can reduce the toxicity of melittin without affecting its therapeutic activity. Rayahin et al. showed that the melittin fusion protein with glutathione S-transferase exhibits anti-inflammatory properties and minimal toxicity [65]. Development of delivery techniques using nanocarriers enable safe delivery of melittin to targeted lesions without affecting non-targetcells [27,66,67].

Conclusions
Studies over the previous decade have advanced our knowledge about bee venom-derived melittin. Herein, we summarize the inflammation-specific effects of melittin and discuss its underlying mechanisms (Table 1, Figure 1). However, parts of the underlying mechanisms still remain unclear and the toxicity of this peptide still prevents its use for anti-inflammatory activity. Melittin could possibly be used for anti-inflammatory purposes if careful provisions are taken to avoid adverse effects. Technical developments will help to modify this toxic peptide into a safe therapeutic agent. We consider that synthesized melittin and its derivatives [68][69][70] should be used to overcome the effects of contaminants from bee venom and for developing novel pharmaceutical agents. The future therapeutic application of melittin on inflammatory disorders will depend on new study protocols to validate the efficiency and safety of melittin. In addition, the anti-inflammatory effect of bee venom PLA 2 was recently established [71]. As melittin and bee venom PLA 2 are synergistic [58,72], it is worth studying the anti-inflammatory effect of melittin and bee venom PLA 2 co-treatment.