J Arthropod-Borne Dis, December 2017, 11(4): 497–503 M Khosravi et al.: The Effects of … 497 http://jad.tums.ac.ir Published Online: December 30, 2017 Original Article The Effects of Isolated Fractions of Mesobuthus eupeus Scorpion Venom on Humoral Immune Response *Mohammad Khosravi 1, Mansour Mayahi 2, Farnoosh Kaviani 3, Mohammad Nemati 4 1Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 3Student of Veterinary Medicine, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4Razi Reference Laboratory of Scorpion Research, Razi Vaccine and Serum Research Institute, Karaj, Iran (Received 19 Dec 2015; accepted 20 Dec 2017) Abstract Background: Many elements such as immunosuppressive, chemotactic and anti-inflammatory peptide that could effect on human and animals physiologic system were determined in venom. This study evaluated the use of Meso- buthus eupeus scorpion venom fractions as an immunomodulator. Methods: The venom fractions collected from Khuzestan Province in South West of Iran were purified by ion ex- change chromatography. Elution of the bounded elements was done by using a linear gradient of sodium chloride (0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 2 molar). The fractions were analyzed by Bradford spectrophotometric and SDS-PAGE method. After treatments of chicken with venom fractions and sheep red blood cell (SRBC), direct hae- magglutination test in microtiter plate was used for the determination of the chicken SRBC antibody titer. Results: The fraction released by NaCl 1.25M had the highest protein concentration. The highest and lowest anti- body titer was determined at the fifth (NaCl 0.75 molar) and seventh fraction (NaCl 1.25 molar), respectively. Conclusion: Different protein profile of isolated fractions, were associated with various effect on immune response. Both enhancing and suppressing of the chicken humoral immune response to SRBC were observed after M. eupeus faction’s venom treatment. It is due to biological functions of venom components. Purification of these elements would provide the new agents for immune responses manipulation. Keywords: Venom, Mesobuthus eupeus, Immune response Introduction Scorpion venom contains biological com- pounds as short-chain peptides, bioactive sub- stances such as enzymes, nucleotides, lipids, mucoprotein, mucopolysaccharides, biogenic amines, neuroactive peptides, protease inhib- itors, phospholipase, hyaluronidase (1) and other unknown compounds, which could af- fect on the physiologic system of vertebrate and invertebrate organisms (2). Only 400 pep- tides out of the expected 100000 peptides of venom have toxic effects on human and an- imals (3). Envenomated organism produces various mediators, which contains both pro- and anti-inflammatory cytokines (4). Scorpi- on venom could induce local and systemic inflammatory responses. The local effects can lead to the activation of vascular endothelium, increase the vascular permeability and leuko- cytes migration to the affected tissues. The sys- temic inflammation persuades the acute phase *Corresponding author: Dr Mohammad Khosravi, E-mail: dr.khosravim@gmail.com J Arthropod-Borne Dis, December 2017, 11(4): 497–503 M Khosravi et al.: The Effects of … 498 http://jad.tums.ac.ir Published Online: December 30, 2017 response (5). The consequence of this inflam- matory reaction is undergoing by a variety of factors such as duration of the stimulus and the balance between the inflammatory and an- ti-inflammatory mediators (4). Different methods, used for fractionation, purification, analysis of the structure and char- acterization of toxins have made it possible to clarify the components of venoms. Identi- fication of toxins or others biologically im- portant peptide were the outcome of these efforts (6). Many elements such as immuno- suppressive (7) chemotactic (8, 9) and anti-in- flammatory peptides were determined in ven- om and are as potential therapeutic agents (10). Scorpion’s venom has been used in med- icine for relation of pain, osteoporosis, neu- rological diseases, homeostasis and rheology, cancer and autoimmune disease (11, 12). Scorpions were divided into 13 families and about 1400 species and subspecies (13). The medical importance scorpion species, belong- ing to the family Buthidae mainly, Androcto- nus, Buthus, Mesobuthus, Buthotus, Parabu- thus, and Leirus (4). The M. eupeus venom could affect on cytokine releases with both pro and anti-inflammatory properties (14, 15). Venom components that affect on immune re- sponse could be used as immunomodulatory agents. We investigated the possibility of using different M. eupeus scorpion venom fractions as immunomodulator like elements for ele- vation or suppression of immune response. Materials and Methods Venom Mesobuthus eupeus scorpions were col- lected from Khuzestan Province in South West of Iran (31°19′–32°73′N, 48°41′–49°4′E) milked by electric stimulation. The total protein concentration was measured using the usual Bradford spectrophotometric method with bo- vine serum albumin (BSA) as standard. Chickens Forty adult Rass 308 chickens were se- lected and kept in the isolation facility. Feed and water were provided during the experi- ment. Experimental procedures were accord- ing to the guidelines of the animal care. Purification of venom fractions The venom fractions were purified by ion exchange chromatography. Anion-exchange chromatography was performed using diethyl- aminoethyl cellulose (DEAE-C) column (Sig- ma, Product Number: D3764) at a flow rate of 1ml/min. The column-stabilizing buffer was 0.05 molar Tris-HCl, pH 8.6. Tow milliliter of M. eupeus venom were dissolved in stabi- lizing buffer and loaded on the column (12 mg/mL). After washing away of the unbound components, elution of the bound elements was done by using a linear gradient of sodi- um chloride (0.1, 0.25, 0.5, 1, 1.25, 1.5, 1.75 and 2 molar) in 3ml of stabilizing buffer. All the fractions were dialyzed overnight against distilled water, pH 7.2 at 4 °C. The protein concentrations of the collected fractions were measured by Bradford spectrophotometric method (Accu Reader, Serial No: 96501575) with BSA as standard. SDS-PAGE The protein profiles of crude venom, as well as the purified fractions, were analyzed by SDS-PAGE (16), the stacking and resolving gel concentration was 4% and 11%, respec- tively. Samples were denatured by boiling in loading buffer containing SDS and β-mer- captoethanol prior to loading onto the gel. Pro- teins were stained with 1% Coomassie blue R 250. Molecular mass standard (Vivantis, prod- uct No: PR0602) was run in parallel in order to calculate molecular weights of the proteins. J Arthropod-Borne Dis, December 2017, 11(4): 497–503 M Khosravi et al.: The Effects of … 499 http://jad.tums.ac.ir Published Online: December 30, 2017 Immunomodulatory analysis of the purified fractions The purified fractions were diluted to 200 μg/ml with pyrogen-free water. The 0.5ml of each fraction injected subcutaneously at breast region of four adult chickens. The sheep red blood cells (SRBC) were prepared by cen- trifugation of Ethylenediaminetetraacetic acid (EDTA) anticoagulated whole blood, followed by 3 times washing with normal saline. The suspension of fresh 2% SRBC was prepared and the 0.5ml injected in intra-muscularly route. The clinical symptoms were recorded during the test periods. Two ml of the ve- nous blood were collected from the injected chickens 5 and 10 d after the injections. The serum was separated and kept at minus 20 °C until use. Hemagglutination test Direct hemagglutination test in microtiter plate used for the determination of the SRBC antibody titer. Serum samples were serially diluted in a microtiter plate and 1% SRBCs were added. After mixing, the plates were in- cubated at 37 oC for 45min and examined for hemagglutination. The test repeated 3 times for each samples and antibody titer was de- termined as the equal of the maximum dilu- tion that exhibiting hemagglutination. Results Ion exchange chromatography The total protein concentration of the eluted fractions has been measured and as shown in Table 1, the eluted fraction by NaCl 1.25M had the highest percentage of protein. SDS-PAGE Venom fractions had a different protein profile (Fig. 1). Proteins of the venom had 15 detectable bands, which were between ≤ 5 and ≥ 175 kDa on 12% polyacrylamide gel electrophoresis. Hemagglutination test The fifth fraction (NaCl 0.75) had the lowest hemagglutination titer, this was less than the control titer. The seventh fraction (NaCl 1.25) had the highest anti SRBC titer (Table 2). Table 1. The total protein concentration of the venom fractions which eluted by a linear gradient of sodium chloride Elution (Molar) Tris 0.05 NaCl 0.1 NaCl 0.25 NaCl 0.5 NaCl 0.75 NaCl 1 NaCl 1.25 NaCl 1.5 NaCl 2 Protein Concentration (mg/ml) 4.2 1.27 1.20 1.61 3.95 3.8 4.3 2.05 0.82 Table 2. The hemagglutination titer against sheep red blood cells (SRBC) in chickens which treated with venom fractions or phosphate buffered saline (PBS) Fraction (Molar) PBS SRBC 1 2 3 4 5 6 7 8 9 5 days titer 0 10 10 20 30 20 5 30 60 20 10 10 days titer 0 20 20 40 40 30 10 40 160 40 20 J Arthropod-Borne Dis, December 2017, 11(4): 497–503 M Khosravi et al.: The Effects of … 500 http://jad.tums.ac.ir Published Online: December 30, 2017 Fig. 1. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Mesobuthus eupeus scorpion venom. The Lane M: Marker proteins (175, 130, 95, 70, 62, 51, 42, 29, 22, 14 and 10, respective- ly). The lanes 1 to 9 are eluted fractions by 0.05 M Tris- HCl and 2, 1.5, 1.25, 1, 0.75, 0.5, 0.25 and 0.1 M of NaCl, respectively Discussion The isolated fractions of M. eupeus ven- om have immunomodulatory effects. The hu- moral immune response to the SRBC was a sensitive endpoint to evaluate drug modifi- cation of the humoral immunity (17). The cooperation and interaction of antigen pre- senting cells, T helper and B cells are in- volved in the production of the anti-SRBC (17). Both inductions and suppression of the humoral immune response to SRBC were ob- served in treated chickens with venom frac- tions. The six fractions out of nine fractions increase the anti SRBC titer, from 1.5 to 4 fold. Several proinflammatory mediators, such as leukotrienes, phospholipases A2, prostaglan- dins, kinins, H2O2, NO production and ac- tivation of complement system, were increased in scorpion envenomated peoples (18–20). The fractionated extracts of venom may improve the phagocytic efficacy of the PMN cells and stimulate innate immune response. In addi- tion, venom induces the release of cytokine and activation of endogenous immunological and inflammatory mediators (18). Moreover, scorpion venoms could enhance the release of different inflammatory mediators which cause leukocytosis and raise the cytokines levels such as IL1b, IL6, IL8, IL10, TNFa and NO (21, 22). A mixture of peptide with diverse potential for induction of pro-in- flammatory mediator can exist in M. eupeus venom. Production of IFN-𝛾 and IL-4 en- hance the effect of venom on innate and hu- moral immune responses (23). Improved hu- moral response to SRBC was reported for the administration of NNAV (23). The ele- vation of Th1 and higher proinflammatory re- sponse were induced by Bothrops venom (24). The venom of Hemiscorpius lepturus and An- droctonus crassicauda scorpion stimulate the monocytes immune response by IL12 pro- duction (25, 26). An inhibitory effect on humoral immune response of chickens to SRBC was observed on one fraction of M. eupeus venom. The M. eupeus venom was contained effective anti- inflammatory mediators (14). In addition, the other venom has suppressive effects on im- mune system. Significant inhibition of the im- mune responses and interfering with the syn- thesis of immunoglobulin G were observed after viper snakes venom treatment (27, 28). Some of venom derived peptides have im- munosuppressant properties and used for the treatment of autoimmune diseases and the or- gan transplantation (29). In addition, muco- polysaccharides of venom inhibit nitrous ox- ide and interleukin human chondrocytes (1), hence have anti-inflammatory properties. The Naja naja atra venom had anti-inflammatory effects (30). The cobratoxin had anti-inflam- matory and inhibitory effect on activation of NF-𝜅B (31). Both pro- and anti-inflammatory cytokines were produced after scorpion envenomation (22, 32). The IFN-γ, TNF-α, GM-CSF IL-1α, IL-6 and IL-10 levels were increased in en- venomed peoples (22, 33). The B. erythro- melas and C. d. cascavella venom have sig- nificant immunomodulatory effects. Howev- er, B. erythromelas enhance a proinflamma- tory profile and that C. d. cascavella venom J Arthropod-Borne Dis, December 2017, 11(4): 497–503 M Khosravi et al.: The Effects of … 501 http://jad.tums.ac.ir Published Online: December 30, 2017 has anti-inflammatory effects (34). The ven- om of T. serrulatus contains substances with immunomodulatory effects (35). Conclusion Different protein profiles of isolated frac- tions of M. eupeus venom are associated with various effects on the immune response. 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