Mini-αA-Crystallin Stifled Melittin-Induced Haemolysis and Lymphocyte Lysis

Melittin, the most potent pharmacological ingredient of honey bee venom, induces haemolysis, lymphocyte lysis, long-term pain, localised inflammation, and hyperalgesia. In this study, efforts were made to subdue the melittin’s ill effects using a chaperone peptide called ‘mini-αA-crystallin’ (MAC) derived from eye lens αA-crystallin. Haemolytic test on human red blood cells, percentage viability, and DNA diffusion assay on Human peripheral blood lymphocytes (HPBLs) were performed with melittin in the presence or absence of MAC. Propidium iodide and Annexin V-FITC dual staining were performed to analyse quantitative levels of necrotic and apoptotic induction by melittin in the presence or absence of MAC on HPBLs using a flow cytometer. A computational study to find out the interactions between MAC and melittin was undertaken by modelling the structure of MAC using a PEP-FOLD server. The result showed that MAC inhibited melittin-induced lysis in nucleated (lymphocytes) and enucleated (RBC) cells. Flow cytometric analysis revealed a substantial increase in the necrotic and late apoptotic cells after treating HPBLs with melittin (4 µg/ml) for 24 h. Treatment with MAC at a 2:1 molar ratio prevented HPBLs from developing melittin-induced necrosis and late apoptosis. In the docking study, hydrogen, van der Waals, π-π stacking, and salt bridges were observed between the MAC and melittin complex, confirming a strong interaction between them. The MAC-melittin complex was stable during molecular dynamics simulation. These findings may be beneficial in developing a medication for treating severe cases of honeybee stings.


Introduction
The Apis genus of the bee clade includes honey bees, which are eusocial flying insects. They are Eurasia natives that carry important pollination functions of a wide range of farm crops, and hence are commercially valuable (Hoover and Ovinge 2018;Suran et al. 2021). Only eight honey bee species and 43 subspecies have been identified, accounting for a small portion of the over 20,000 bee species that have been documented (Wilson 2014). The three species of bees most commonly known for causing human envenoming are Apis mellifera (A.m. mellifera), A.m. ligustica in Europe, and A.m. scutellate in Africa (Schmidt et al. 2018). Between 2001 and 2012, there were 1,192,667 envenomation and 2,664 deaths reported in Brazil, with 66,283 cases (5.6%) of bee envenomation and the second-highest rate of case fatalities (0.33%; 216 fatalities) after snakebites (0.43%; 3,394 deaths) (Chippaux 2015).
Melittin is a small cell membrane lytic peptide (26 amino acids, 2.846 KDa, and 2 α-helical segments) that can damage membrane phospholipids, including those found in erythrocytes and lymphocytes, causing haemolysis and lymphocyte lysis ( Van et al. 2008;Gajski and Garaj-Vrhovac 2013;Kim 2021;Jin et al. 2023). Under physiological conditions, it has been demonstrated that melittin exists as a random coil, but when it comes in contact with the cell membrane, it forms a monomeric α-helix and interacts with the membrane, leading to membrane lysis (Buck 1998;Goto and Hagihara 1992;Raghuraman and Chattopadhyay 2007;Saini et al. 1999). Melittin quickly attaches to the erythrocyte membrane's outer layer and forms brief pore through which around 40 haemoglobin molecules can escape, causing haemolysis followed by rhabdomyolysis and severe renal failure (Jamasbi et al. 2016). Renal failure or heart hurdles could lead to the victim's death (Vetter et al. 1999). However, many researchers have probed the therapeutic potential of melittin and found it to be effective as an anti-cancer (Gajski and Garaj 2013), antiviral (Memariani et al. 2020), and anti-microbial (Asthana et al. 2004;Hanulova et al. 2009) agent. The use of melittin has been restricted due to its side effects, particularly its haemolysis (DeGrado et al. 1982;Tender et al. 2021;Jin et al. 2023).
Over 90% of the lens proteins in the human eye are comprised of three types of crystallins, known as α, β, and γ-crystallin. In mammalian lenses, α-crystallin can account for up to 50% of the total structural protein mass (Hoenders and Bloemendal 1981) and possesses chaperone-like effects (Horwitz 1992). It is a member of the small heat shock protein (sHsp) family and is composed of two subunits-A and B. In most vertebrate eyes, the subunits are grouped in a 3:1 ratio, however the ratio changes depending on the species and the individual animal's age (de Jong et al. 1998). Because of their sequence, composition and domain topology similarities, β-and γ-crystallins are often lumped together. α-crystallin is believed to have many physiological roles, including the ability to inhibit unfolding protein precipitation and enhance cellular immunity to stress (Augusteyn 2004). Additionally, in various stress situations, α-crystallin subunits can prevent protein inactivation and neuronal aggregation (Klopstein et al. 2012;Derham and Harding 1999;Horwitz et al. 1999). The peptide (KFVIFLDVKHFSPEDLTVK,MAC) inhibited the precipitation and aggregation of unfolding proteins, just like full-length native α-crystallin subunits (Raju et al. 2016;Posner et al. 2022). According to several researchers, MAC has the same chaperone function as their parent proteins and may be used therapeutically to treat a range of disorders (Sreekumar et al. 2013;Zhang et al. 2015;Nahomi et al. 2013). A previous study has shown that melittin interacts with MAC parent protein αA-crystallin (Sharma et al. 2000). This research looked at how MAC impacts melittin toxicity on nucleated (lymphocytes) and enucleated (RBC) cells. This study might aid in the development of drugs to treat severe cases of honeybee stings. Unlike previous studies with synthetic peptides (Blondelle et al. 1996;Hewish et al. 1996;Lam et al. 2002), this research involves testing melittin inhibitory activity in the natural peptide.

Materials and Methods
Blood sample: A healthy male donor provided human blood for the procedure (26 years of age). Before having their blood sampled, the donor had not been exposed to known genotoxic substances or ionising radiation for diagnostic or therapeutic purposes in a year. Venous blood was taken while maintaining sterility using heparinized vacutainer tubes and lithium heparin as an anticoagulant at the Kasturba Medical College in Manipal, Karnataka, India (Registration No. ECR/146/Inst/KA/2013/RR-19; IEC: 522/2020). Blood was separated into several samples after collection.

Anti-haemolysis Activity Assay
Heparinized human blood was centrifuged using Hettich, MIKRO 220R centrifuge for 10 min at 3500 rpm before being washed three times with phosphate buffer saline (PBS, pH 7.2). The supernatant was discarded, and a 2% suspension of RBC was made in PBS. Serial dilutions of MAC in PBS were used to achieve concentrations ranging from 6.4 to 1.6 µg/ml. Melittin (4 µg/ml) was prepared in PBS and the concentration was selected according to the published paper (Zarrinnahad et al. 2018). The assay was performed in a 96-well microtiter plate where100 µl of 2% RBCs was added to each well of the plate and treated with various molar ratios of MAC with melittin (0.5:1, 1:1, 2:1). Positive (Pos.) and negative (Neg.) controls were used, with Triton X-100 (1%) and PBS, respectively. The plate was incubated in NuAire, NU-5800 incubator for 2 h at 37° C before being centrifuged (Hettich, MIKRO 220R) for 10 min at a speed of 3500 rpm. At 540 nm, the optical density of the supernatant was estimated after the supernatant was transferred to a fresh microtiter tray (BioTek ELX 800 microplate reader) (Zarrinnahad et al. 2018).
The percentage of hemolysis was determined as follows:
The distinct staining of the nucleus was used to differentiate live cells from dead cells. Live cells with a functioning membrane had nuclei that were uniformly stained green, while the nuclei of dead cells were uniformly coloured red. Differential staining was done using acridine orange and ethidium bromide to measure cell viability (Duke 1992). HPBLs were given melittin treatment both with and without of MAC across various time periods. Fifty µl of HPBLs and 2 µl of dye were used to make the slides. An Olympus DP74 upfront microscope was used to examine 100 cells in each repeat with a 40 × objective and fluorescent filters of 515-560 nm (Singh 2000).

DNA Diffusion Assay
This assay was used to detect the type of cell death (Singh 2000;Gajski and Garaj-Vrhovac 2011). In brief, HPBLs were subjected to various molar ratio treatments of MAC with melittin (0.5:1, 1:1, 2:1). Glass slides coated with 0.7% naturally melting agarose were dried at room temperature. On agarose-coated slides, microgels were formed by mixing 20 µl of HPBLs with 50 µl of 0.7% high-resolution agarose and pipetting the resulting mixture onto the slide. Cover glasses were quickly placed over the gel. The slides were labelled and placed on ice to cool for one minute. Cover glasses were taken off, 200 µl of agarose solution at 2% was applied, and they were then replaced and kept on ice for one minute. After removing the cover glasses, slides were placed in a freshly made lysing solution (1.25 M NaCl, 1 mM tetrasodium EDTA, 5 M Tris-HCl pH 10, 0.01% sodium lauroyl sarcosine, 0.2% DMSO, 300 mM NaOH) for 10 min at room temperature. After being lysed, the slides were immersed two times for 30 min each in a neutralising solution (50% ethanol, 1 mg/ml spermine, 20 mM Tris-HCl pH 7.4), after which they underwent air drying and were kept at room temperature. A coverslip was then put on top after 10 min of ethidium bromide (20 g/ml) staining. A fluorescent microscope (Olympus DP74 upfront microscope) was used to examine 300 lymphocytes per slide using a 40 × objective and fluorescent filters of 515-560 nm. According to Singh (2005) figures and instructions, normal cells were distinguished from lymphocytes that were going through apoptosis or necrosis (Singh 2005). The shrinking, hazy, or fuzzy look of the nuclei of apoptotic cells was caused by nucleosomal-sized DNA diffusing into the agarose, which obscured their borders. Necrotic cells had nuclei that were bigger and less clearly defined than those of healthy cells.

Flow Cytometric Analysis
To determine quantitative apoptosis and necrosis in HPBLs, flow cytometry was conducted using an Annexin V-FITC Detection Kit in accordance with the directions from the manufacturer. HPBLs without melittin treatment or MAC were considered as a negative control. The HPBLs were treated for 24 h in triplicate with melittin alone (4 g/ml) and melittin plus MAC at a 2:1 molar concentration ratio. After the treatment, 1 mL of RBC lysing solution was added and incubated for 10-12 min in the dark. The pellet was then washed with phosphate buffer saline after the supernatant from the centrifugation at 3000 rpm (Hettich, MIKRO 220R) for 7 min was discarded. The samples were mixed with 5 µl of PI and 5 µl Annexin-V-FITC and then examined using a flow cytometer (CyFlow Space, Partec-Germany) equipped with a 488 nm laser and Cell Quest software (Becton Dickinson).The populations of viable (both propidium iodide and Annexin V negative), early apoptotic (propidium negative, Annexin V positive), necrotic (propidium iodide positive, Annexin V negative), and late apoptotic (both propidium iodide and Annexin V positive) cells are represented in the lower left, lower right, upper left and upper right quadrants, respectively (Cossarizza et al. 2017;Nykky et al. 2010).

Modelling of MAC Structure and its Docking Studies with Melittin
Peptides identified from protein-protein interaction areas provide great leads for antagonist development (Sillerud and Larson 2005). The 70-88 amino acid residue region of αA-crystallin was reported to interact with melittin (Sharma et al. 2000). Hence, we intended to study the structure that the MAC might assume and whether it will form a stable complex with melittin. The 70-88 amino acid peptide structure can directly be retrieved from the αA-crystallin protein structure available in the Protein Data Bank (PDB) (PDB ID: 3L1F). However, the 70-88 peptide fragment need not adopt a structure similar to the peptide present in the complete protein because the sequence will not be under the restraints imposed by the preceding of the following regions. Hence, the three-dimensional (3D) structure of MAC (residues 70-88) was modelled using the PEP-FOLD server (Maupetit et al. 2009). Using amino acid sequences as its foundation, PEP-FOLD predicts peptide structures. Initially, 200 independent simulations were performed, and the predicted structures were sorted using their energies. For further examination, the best model with the lowest energy was chosen. The structural limitations were eliminated by energy minimization using the protein preparation wizard in the Schrodinger program, and the structure was prepared for missing atoms (Schrodinger LLC.).
Since the MAC is known to have chaperone activity of the parent protein (Horwitz 1992;Sharma et al. 2000), its interaction and stability analysis with melittin were performed. Melittin may spontaneously assemble into cell membranes as well as form a tetramer in water (Terwilliger and Eisenberg 1982). α-Crystallin was reported to interact with the melittin monomer (Ramirez et al. 2020). For the interaction studies, the melittin X-ray diffraction structure with a 2 Å resolution was used (PDB Id: 2MLT). A peptide with two alpha helices separated at proline 14 makes up the 26 amino acid length melittin monomer. Using the protein preparation wizard from the Schrodinger software suite (Schrödinger LLC.), protein structures were created. The heteroatoms were deleted and the structures were altered by assigning the right bond orders, removing water molecules, and introducing missing hydrogen atoms. The ionization and tautomeric states of amino acid residues were corrected by adding hydrogen atoms. The structures were minimized to 0.30 Å root mean square deviation (RMSD) using an OPLS3e forcefield. Peptide-peptide docking of melittin with MAC was carried out using the ZDOCK and HawkDock servers. Maestro (Schrödinger LLC.) was used to analyse the docked structures and assess interactions like π -stacking, van der Waals interactions, and hydrogen bonds.

Molecular Dynamics (MD) Simulation Studies
To evaluate the stability of peptide-peptide interactions, MD simulation was used. The MD simulation was performed using the Gromos53a6 force field and Gromacs v4.5.5 software. The system underwent 20,000 steepest descent minimization steps in a vacuum before explicit solvation took place in the middle of a cubical system containing water and specific point charges (SPC/E). Na + , Cl − , and 0.1 M NaCl salt were added to the solution to neutralise the ionizable residues. The linear constraint (LINCS) method was used to determine the lengths of the H-bonds, and the particle mesh Ewald (PME) method was used to deal with longrange electrostatic interactions. In order to limit the length of the H-bond, the SHAKE algorithm and 20,000 iterations of solvated system steepest descent energy minimization were applied. A 20 ns MD simulation of a fixed number of particles at a predetermined pressure and temperature was carried out after the system had undergone a 100 ps NVT and NPT equilibration. The outputs were examined using the Gromacs distribution package's tools and the visual molecular dynamics (VMD) analysis program (Fayaz and Rajanikant 2015).

Statistical Analysis
In GraphPad Prism version 8.4.0 software, the Bonferroni multiple comparison test tests were used for all statistical analyses. Data were presented as mean with ± SEM (standard error of the mean) and two-way ANOVA was used to analyse them. ***p < 0.001, **p < 0.01, *p < 0.05 was considered statistically significant.

Determination of Lymphocyte Morphological Changes and Viability
Cells were given melittin treatment both with and without MAC for varied times to investigate the effects of melittin on the viability of HPBLs. The addition of melittin to HPBLs resulted in decreased cell viability. Live cells and dead cells were distinguished from one another using the nucleus' distinctive staining (Fig. 2). At 4 µg/ml of melittin the viability of cells at 1, 6, and 24 h was 64%, 49%, and 23% respectively. The percentage cell viability in presence of MAC with melittin at 0.5:1, 1:1, and 2:1 molar concentration ratios were 73%, 77%, 80% for 1 h, 61%, 68%, 74% for 6 h, 51%, 53%, 57% for 24 h, respectively (Fig. 3) suggesting the inhibition of melittin-induced cell death by MAC.

Flow Cytometric Analyses
PI/Annexin V-FITC dual staining was carried out and assessed using a flow cytometer to analyse the melittininduced cell death quantitatively in HPBLs. The quadrants depict necrosis, early and late apoptosis, and normal cells.

Melittin and MAC Docking Studies
Docking of αA-crystallin with melittin revealed that the amino acid residues 67 to 92 of αA-crystallin bind to melittin, as reported experimentally (Sharma et al. 2000). The modelled MAC exhibited two antiparallel betasheets, similar to the corresponding sequence present in the full-length native structure (Fig. 7). Docking studies showed that the MAC was interacting with melittin. The shapes MAC were complementary to the melittin and fit perfectly to form a stable complex (Fig. 8) with the docking score ranging from − 2783.70 to − 1572.49 and binding free energy ranging from − 37.69 (Kcal/mol) to − 18.06 (kcal/ mol) for the top 10 models. Residues 74 (Phe) and 82 (Pro) of MAC interacted with Pro 14, Lys 23, Arg 24, and Gln 25 of melittin by forming van der Waals bonds and, π-π stacking. Moreover, interactions such as hydrogen bonds, and salt bridges were found between MAC and melittin (Table 1) suggesting that the MAC had a strong affinity towards melittin and these interactions may be the reason for the inhibition of the haemolytic and lymphocyte lysis action of melittin.

Molecular Dynamics Simulation Studies
Visual inspection of molecular dynamics simulation revealed that MAC is stably bound to melittin at the αA-crystallin binding region. The melittin and MAC complex was stable throughout the MD trajectories thereby confirming the docking results. The RMSD of the MAC and melittin complex was within 2 Å during the entire 20 ns, and it was stable in the last 5 ns illustrating the stability of the complex (Fig. 9). This indicates that the MAC might form a stable complex with melittin at physiological conditions.

Discussion
Research into the venomous systems provides essential insights into their biological actions and the information can be used to overcome the problems caused by them effectively. Melittin, which makes up about 40-60% of the dry weight of honeybee venom, is the main biologically active ingredient (Peiren et al. 2005;El Adham et al. 2022). The phospholipid bilayer identifies melittin as a normal pore-forming peptide (Williams and Bell 1972) and often inhibits or enhances phospholipase A2 (PLA2) function (Nishiya 1991;Mollay and Kreil 1974). Melittin produces prolonged painful stimulation by acting on primary nociceptor cells through direct and indirect mechanisms, whereas other polypeptides of honeybee venom contribute to transient and much shorter pain (Chen et al. 2016). The direct action occurs when the PLA2 cascade pathway activates the potential vanilloid 1 (TRPV1) thermal nociceptor transient receptor, resulting in primary nociceptor sensitization (Wall et al. 2006). For indirect activity, melittin's pore-forming abilities are necessary because they allow mast cells to release chemicals that induce pain, including H + , adenosine triphosphate (ATP), and 5-hydroxytryptamine (5-HT) (Lu et al. 2008). The majority of the patients will have a local response that includes redness, tenderness, swelling, and discomfort at the sting spot, and about 21% will have Type I hypersensitivity (Nittner-Marszalska et al. 2004). Melittin, however, has been the subject of many studies for its therapeutic potential, and it has been discovered to be effective as an HPBLs + MAC + melittin (2:1) (D). The quadrants represent the cells at normal state, Q 4 early and Q 2 late apoptosis, and Q 1 necrosis anti-cancer (Gajski and Garaj 2013), antiviral (Memariani et al. 2020), and anti-microbial (Asthana et al. 2004;Hanulova et al. 2009) agent. Due to its side effects, especially its hemolysis, melittin's use has been restricted (DeGrado et al. 1982;Tender et al. 2021). Previous research has demonstrated that the binding of synthetic peptide Ac-IVIFDC-NH2 to melittin blocks the interaction with lipid layers (Hewish et al.1996;Houghten et al. 1991). Using a combinatorial library (Blondelle et al. 1996), over 20 hexapeptides were identified as melittin activity inhibitors. When the melittin inhibitory activity of the synthetic peptides was measured by RBC haemolytic assay, Ac-IIIYFE-NH2 (14 mg/ml) peptide was able to suppress the haemolysis to the tune of 70% by melittin (7.5 mg/ml) after 30 min of preincubation (Blondelle et al. 1996).
The current study using human eye lens crystallinderived peptide to subdue melittin-induced haemolysis and lymphocyte lysis brings out a possible newer way of treating such cases. The 70 KFVIFLDVKHFSPEDLTVK 88 region of the αA-crystallin also known as MAC can suppress aggregation of unfolded proteins and protects cells from apoptosis (Sharma et al. 2000;Sreekumar et al. 2013;Raju et al. 2014). Additionally, it was discovered to have anti-inflammatory properties at low concentrations by scavenging oxygen free radicals on HaCaT cell lines in a lipopolysaccharide-induced free radical scavenging experiment and significantly reducing the volume of the rats' paws in a carrageenan-induced paw edema test (Muralidharan et al. 2021), making it more suitable for treating honey bee stings known to be accompanied with inflammation.
In this study, we found that MAC inhibits melittin-induced lysis in nucleated (lymphocytes) and anucleated (RBCs) cells. Four μg/ml of melittin has induced 82 ± 0.882% haemolysis, in 2% RBCs suspension. The equimolar concentration of MAC suppressed melittin-induced haemolysis in 2% RBCs suspension. MAC reduced hemolysis most effectively with a 34 ± 0.917% hemolysis rate when combined with melittin at a 2:1 molar ratio. Melittin enhanced the proportion of apoptotic and necrotic in HPBLs throughout time and dose. MAC reduced the proportion of apoptotic and necrotic cells produced by melittin over time (1, 6, and 24 h). When HPBLs were treated with 4 µg/ml of melittin for 24 h, the flow cytometric analysis revealed a substantial increase in the necrotic (13.21%) and late apoptotic (4.84%) cell percentage. With MAC plus melittin at a 2:1 molar ratio for 24 h, the proportion of necrotic and late apoptotic HPBLs was 3.13% and 3.76%, respectively. The percentage of viable cells in HPBLs treated with MAC-melittin group was highest with 82.20% followed by untreated HPBLs group (76.27%) and melittin group (72.66%). The chaperone peptide has delayed the natural cell death and inbitted melittin induced cell lysis which may be the reason for higher viable cell population in MAC-melittin group than untreated group. The MAC treatment completely inhibited the ill effects of melittin in HPBLs.
The percentage death of lymphocytes induced by melittin was inhibited by MAC as confirmed in this study from cell viability assay, DNA diffusion assay and flow cytometry analysis. To our knowledge, no natural peptide has been reported to suppress melittin-induced haemolysis and lymphocyte lysis. αA-crystallin's amino residues 70-88 have been identified as the protein's major chaperone site and as a molecular chaperone, it plays an important role. This region is believed to be highly conserved among several small heat shock proteins (Sharma et al. 2000;de JONG et al. 1984).
Melittin molecules are thought to attach perpendicularly to membranes, generating a pore and leading to cell lysis ( Van et al. 2008). With addition of MAC, there was decreased in haemolysis and lymphocyte lysis when compared to melittin alone, suggesting that the perpendicular binding action of melittin on RBCs and lymphocyte cells might have been prevented by MAC (Fig. 10). To confirm this, peptide-peptide docking and stability of MAC melittin complex were performed to identify their binding regions/ amino acid residues involved in stable complex formation. It has been demonstrated that the lysis activity of melittin needs proline 14 and the polar residues 23 to 26 at the C-terminus (Otoda et al. 1992;Rivett et al. 1996). In our study, we found that Phe (corresponding to residue 74 in αA-crystallin) and Pro (corresponding to reside 82 in parent protein) of MAC interacted with Pro 14, Lys 23, Arg 24, and Gln 25 of melittin by forming van der Waals bonds and π-π stacking (Table 1), suggesting that this interaction may be the reason for the haemolytic and lymphocyte lysis prevention action of the peptide chaperone. Further, MAC also showed interaction with other amino residues of melittin by forming hydrogen, van der Waals, π-π stacking, and salt bridges. The molecular dynamics simulation studies of the melittin-MAC complex revealed that the RMSD and potential energy did not fluctuate much and stayed almost constant indicating it to be a stable complex. Hence, it can be said that one molecule of MAC perfectly binds with one molecule of melittin by interacting with amino acids viz., Pro 14, Lys 23, Arg 24, and Gln 25 that are critical for its cell lysis properties.
After honey bee sting, melittin induces haemolysis and lymphocyte lysis leading to wide range of health complications, ranging from localized oedema to life-threatening systemic anaphylactic shock. Majority of patients will have a local response that includes redness, tenderness, swelling, discomfort and pain at the sting spot, and about 21% will have Type I hypersensitivity reactions (Peiren et al. 2005). These complications can be minimized by administering appropriate dose of MAC. However, more studies are necessary to confirm these effects using preclinical and clinical studies.
Findings from this study may aid in the development of treatment for honeybee stings and a number of other diseases for which melittin shows promise as a treatment but whose use is restricted due to side effects. At  the appropriate dose, it is feasible that this combination may be successful in treating a range of disorders for which melittin has promised therapeutic advantages.

Conclusion
MAC which represents the amino acid residues 70-88 of αA-crystallin has inhibited haemolysis and lymphocyte lysis induced by melittin. The interaction between MAC and melittin, specifically with amino acid residue 14 and the polar residues 23-26 at the C-terminus of melittin, may be the cause of the inhibition of haemolysis and lymphocyte lysis activity of MAC. This finding may be helpful in developing medication to treat honey bee stings as well as a variety of other diseases. Melittin, despite having good therapeutic value (Gajski and Garaj 2013, Memariani et al. 2020, Asthana et al. 2004Hanulova et al. 2009), its use is restricted due to its toxicity. Given that MAC has been shown to be able to prevent melittininduced haemolysis and lymphocyte lysis, it is conceivable that, at the right dosages, this combination may be effective in treating a variety of illnesses for which melittin has promising therapeutic benefits. Therefore, the therapeutic effects of melittin administered with MAC need to be assessed in light of the current study's findings to see if the venomous impact of melittin mitigated by the chaperone