Introduction:

Multiple Sclerosis (MS) is a common disease in many parts of the world (1).  Based on the report published by the World Health Organization (WHO), more than 2.5 million people are affected worldwide. The prevalence of this disease in Iran was 8 to 30 people per thousand people. However in recent years the incidence of MS has increased in the country and cities such as Tabriz, Tehran, Isfahan and Mashhad which have the highest incidence of MS. (2)

MS is a central nervous system disease in which the immune system is disrupted. Axons may also be damaged by the lack of nourishment by surrounding cells. (3) . The most prominent features of this disease are myelin destruction, axonal damage and gliosis (scar formation)(4), which are the basic mechanisms which may be involved in pathogenesis of MS is immune cell trafficking and production of cytokines. The current hypothesis points to the role of cells such as T CD4 +, T CD8 +, B, microglia / macrophages (dendritic, astrocytes) in the pathogenesis of MS(5) . Astrocytes are the major antigen-presenting cells in the nervous system, which Have occupied almost 25 to 50 percent of brain volume.(6) Astrocytes are able to maintain the internal environment of the nervous system by removing excess K+ ions  ,(7) and produce chemokine such as RANTES, IL-8, MCP-1 and 10 IP- and IL-27, IL-17(8) . Interleukin-17 secretion by astrocytes affects Th17 cells via activating signaling pathways which leads to increased astrocyte activity, producing an inflammatory environment that can induce tissue damage in the CNS (9-11). The drugs available for MS are to correct the disease process. Over the past 20 years, various drugs, including interferon beta was used for treatment(9). Venom therapy is one of the most recent methods of treatment in this field (13). It is suggested that animal venom such as snake venom (10), bee venom(11) and scorpion venom are useful in the treatment of autoimmune diseases such as multiple sclerosis (MS)(12). Scorpion venom contains toxins which are effective on cellular channels including potassium (K⁺), calcium (Ca⁺⁺), Chloride (Cl^-) and sodium (Na⁾ channels. Kaliotoxin peptide, with 37 amino acid residues,can reported to inhibit voltage-dependent potassium channels Kv1.3 that selectively blocks T cell activities and hence decreases T cell sensitivity(13). Another toxin   from the scorpion venom that targets the potassium channel (KTx) consists of about 31-39 amino acid residues(14). Venom toxins inhibit the flow of ions through the biological membrane. (17-20) A Kv1.3 channel-based therapeutic approach seems to have an advantage over agents that cause generalized immunomodulation because native and T_CM cells would escape inhibition through upregulation of IKCa1 channels, leaving the bulk of the immune response intact. (11)

Hence the present study was undertaken to investigate the immunomodulatory effect of a toxin present in scorpion Mesobuthus eupeus venom, based on suppression of IL-17 secretion by activated astrocyte cell line. 

  Materials and Methods

Preparation of venom
lyophilized M. eupeus scorpion venom (150mg) provided by venomous animal department of Razi Vaccine and Serum Research Institute was dissolved in distilled water and centrifuged at 14,000 rpm for 15 minutes. The supernatant was collected and protein content determined by Bradford method  (15)  

 Polyacrylamide Gel electrophoresis

Glycine and tricine SDS-PAGEwasperformed for determination of protein pattern in the scorpion venom. The advantage of using tricine instead of glycine in electrophoresis is its greater negative charge than glycine and its higher ionic strength, which results in faster mobility of the ion in the protein and better

  Separation of small proteins (especially in the range of 1 to 10 KDa).

The concentration of the gel separator was 15%, the density of the condensing gel was 4%, and the molecular marker 6.5 to 200 and 3.4 to 26.6KDa which were used for SDS page and tricine SDS- PAGE respectively. Staining of protein bands was done by Coomassie Brilliant Blue. The molecular weight of the proteins was calculated using the Laemmli method  (16).

Size exclusion chromatography

 Solubilized crude venom was applied to a 1.6 × 150 cm column containing sephadex G-50 equilibrated with a 0.1 M ammonium acetate buffer (pH 8.6). The flow rate was adjusted at 60 ml/hr. 5 ml of solubilized venom (120 mg) was loaded to the column and the eluted material was collected in 10 ml fractions. The optical absorbance of the eluent was measured at 280 nm. Fractions were separated and active fraction was selected by molecular weight. The fractions were lyophilized and protein content was determined as described above (17).

 High performance liquid chromatography (HPLC)

The active fraction from size exclusion chromatography was further purified by    applying on a C18 RP-HPLC (Amersham Biosciences,UV-900.P-900) column which was equilibrated with solvent A (H2O, 0.1%trifluoroacetic acid), and eluted with a concentration gradient of solvent B (acetonitrile, 0.1% trifluoroacetic acid) from 0 to 100%, at a flow rate of 0.5 mL/min during 80 min. The peaks were detected through the A280. Each RP-HPLC peak was collected individually and lyophilized and each individual fraction was tested for activity. The active fraction was further purified by repeating the purification until a single peak was obtained (18).

50% lethal toxicity of venom (LD50)

Balb/c male mice weighing (18± 20 g) were used in this research. The ethical code for this study is RVSRI. REC. 9800004. The venom as well as the fractions’ lethal toxicity was assayed.  Crude venom and fractions were subjected to 4-5 serial dilutions by 1.25 factor using isotonic sodium chloride solution. (From 20 to 100 µg/ml). Aliquot of 0.3 ml of each dilution was intravenously injected into 4 mice. Dilutions ranges covering the entire Mortality range from 0% as non-lethal dose and - 100% as a dose which kills all the animals. The mice dying in each group during 24h following injection were considered as toxicity. The LD50 was calculated using the spearman karber  method (19).

Cytotoxicity assay

 For cytotoxicity evaluation of crude venom and fractions on 1321N1 cell line growth a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was used. The assay is based on the cellular conversion of a tetrazolium salt (MTT) into a formazan product that is easily detected.  

The prepared astrocyte cells (0.7 × 10⁵ cells well) were added into  each well  and exposed to various concentrations of crude venom, F₃ fraction and sub fractions at concentrations (40, 20, 10 and 5 μg/mL respectively),  for 24 h,  then medium in each well was replaced with 100 μL of medium containing 0.5 mg/mL MTT, followed by incubation at 37°C for 3 h. DMSO was added into each well and read at optical density 580 nm  using micro plate reader (Bio-Rad 550). In each experiment, 6 wells were used, and experiments were repeated 3 times.  As a positive control 50 ug/mL of cisplatin was used. (20)

 Determination of IC50
 This indicates a level of venom that reduces 50% of the biological activity of the astrocyte cell compared to the control cell.

% viability = 100 - Cell Inhibition Percentage

Cell Inhibition Percentage   = (1 – (OD sample wells - OD control wells)   × 100  

 Release of Lactate Dehydrogenase (LDH) enzyme  

The necrotic effect of venom, fractions and subtractions on cells were examined by lactate dehydrogenase (LDH) assay. The increase in lactate dehydrogenase activity is proportional to the number of lysed cells in each well(21)

 Measurement of cytokine IL 17: Astrocyte cell line grown in wells for 24 hours, was activated by exposure of cells to 50 pg lipopolysaccharide for 8 hours. The activated cells were then exposed to various concentrations (40, 20, 10 and 5 μg/mL) of crude venom, fraction F₃ and sub fractions respectively for 24 hours. The Supernatant of cell culture (control and activated) were used for specific cytokine IL-17 Immunoassay: ELISA kits (ebioscience Company).  (22)

Statistical analysis

 All the tests were repeated 3 times.  Obtained data were analyzed with statistical software Sigma plot 12 using one-way ANOVA. The results expressed as mean ±SD and P < 0.05 was considered as significant  (23).

 Results:

Venoms lethal toxicity

The toxicity of venom was tested by determination of LD₅₀ of crude venom in mice during 24 hours. The average LD₅₀ of crude venom was found to be 78 ±   3.2   μg/mice. The LD50 for fraction MFs3 was almost half of crude venom with 38 ± 4.6 μg/mice. Total protein of the crude venom was found to be 80.4 mg after removal of mucoproteins.  

Cytotoxic effect of scorpion venom  

 The astrocyte 1321N1 cell line cultured on medium containing DMEM high glucose + FBS (10 %) showed spindle-shaped rods when observed under the inverted phase microscope (Figure No 1A). Following the administration of venom on these cells, no significant changes were observed in the shape, size and morphology of the cells (Figure No 1B). However with the increase in venom concentration some changes in appearance like deformed rods sticking together was observed (Figure No. 1C).

Figure No. 1(A, B, C):A.  Astrocyte cell (control), B: Activated Astrocyte cell (with LPS) and C: Exposed Aastrocyte .Cells in Proximity to LD₅₀ Levels of   Scorpion venom (Zoom × 10X). The morphology of the astrocyte cells (control and activated) is similar and only the number of astrocyte cells in the activated state shows an increase, but due to the increase in scorpion venom  the cells are more deformed and  stuck together.

Determination of IC₅₀  

The IC₅₀ (Half maximal inhibitory concentration) was calculated and plotted after drawing the curve and obtaining the line equation using different concentrations of venom and percentage of live cells, the IC₅₀ in the  1321N1 astrocyte cell  against the  M.eupeus scorpion crude venom is equal to 82± 3.3 µg/ml

 Size exclusion chromatography

 After elution from gel filtration column 5 peaks were obtained (Figure No.2) out of which the third peak (F ₃) was found to be toxic on mice with low molecular weight when checked with SDS-PAGE. The content of protein in F₃ was found to be 19.26 mg when estimated by Bradford method.

Electrophoretic Profile  

The crude venom of M.eupeus scorpion contained proteins raging from less than 6 KDa. up to 116 KDa. Fraction F₁ contains molecular weight proteins ranging from 31 to 116.5 KDa and fraction F₂ contains proteins of molecular weight 14.5 to 31 KDa. As it is shown in Electrophoresis (Figure 3.1)    Fraction F₃, has small molecular weight proteins ranging from 14.5 to less than 5 KDa. Sub fractions of F3 has molecular weight proteins ranging from3.4 to 6.5 KDa. (Figure3.2A). the molecular weight of F331 is almost 4.5 KDa. (Figure: 3.2B)

 Purification of active peptide by RP-HPLC Method:

After  loading of F₃ fraction  onto  RP-HPLC , 7 sub-fractions, including F_3.I, F ₃₂ , F_33., F_34., F_35., F_36., F₃₇  were obtained.(Figure No.4.A). Fractions were injected into 18- to 20-g mice after being lyophilized to remove toxic acetonitrile content of each fraction. Each fraction was tested on astrocyte (control and activated) cell line. From F33 sub Fraction, 5 sub fractions F₃₃₁, F₃₃₂, F₃₃₃, F₃₃₄ and F₃₃₅ were obtained (Figure No.4.B). F₃₃₁ was a peptide purified from M.eupeus scorpion venom (Figure No4.C).   

Determination of cell viability by MTT assay:

 The astrocyte cell viability in all groups including control and activated cells exposed to crude venom, fractions, subtractions and   purified peptide were determined. Concentration of 80 µg/ ml of crude venom are shown in (Table:2) 57.7±  2.35 % survival rate  of astrocyte cell (control)  and lower doses  did not show  significant toxicity on cells ( 86.4 ± 1.9 %viability). (Table3)  However fraction F3 at concentration of 20 μg / ml showed significant effect (P<0.05) on activated astrocyte cells. Although fraction F35 reduced survival rate to 52±1.8 %, but F33 of HPLC did not affect the viability of activated cells by endotoxin (Table3). On the other hand the purified peptide (F331) at 10 µg/ml   did not show significant toxicity (viability 89±4.2) on activated astrocyte cells

 LDH release rate:

Release of mitochondrial enzyme lactate dehydrogenase (LDH), which indicates the cytotoxic effects of scorpion venom on the cell, was determined. The fraction F₃ showed a significant effect on cells to release lactate dehydrogenase with 95% confidence. However the purified peptide did not show significant necrotic effect of LDH release on activated astrocyte cell line (1321N1) (Table: 4, 5)

   Release rate of lactate dehydrogenase enzyme from astrocyte cells (control and active cell). Table 4: Enzyme release following scorpion venom fractions. The effect of F₃ fraction on activated astrocyte cells was significant at P< 0.05 and it was not significant at the P < 0.05 on astrocyte control cells, Table 5: Sub-fraction F₃₅ causes lactate dehydrogenase (LDH) release in the cell and is significant on both activated and control cells. 

Interleukin 17 release:

As shown (in Figure 5.A) the level of interleukin-17 released in cultured media by control astrocyte cells was found to be 78±1.9 pg /ml. When the cells exposed to LPS at a concentration of 50ng/ml, the release of IL-17 increased to 147± 2.7pg/ml which was statistically significant (p<0.05). Crude venom could reduce the release of IL-17 in cells exposed to LPS (103±3.4 pg/ml).  The activated 1321N1 cells when exposed to fraction F3 showed a reduced IL-17 release up 93± 3.3 pg / ml. However partially purified fraction F33 as well as purified peptide reduced the IL-17 of activated cells to 82 ± 2.9   which were statistically significant at P< 0.05 (Figure 5.B)

 Discussion:

 Multiple Sclerosis (MS) is a chronic central nervous system disease (1) and affects about 2.5 million people worldwide (24) . According to available reports, the prevalence of this disease in Iran is about 0.07% (2).The mostly accepted hypotheses in this case is the role of autoimmune mechanisms, including abnormal increase in the number of astrocyte cells  (25). Recently the use of venom as a tool for treatments is considered a hope for new effective drugs (15) and   this study investigated the potency of scorpion venom as well as its peptides for modulating the hyperactivity of activated astrocytes using the cell line 1321N1. Today, the use of activated astrocyte-cells is considered as a prefect tool for determination of drugs efficacy to subside the clinical effects of MS disease (26).   Scorpion venom contain a mixture of peptides, toxins and many other bioactive compounds (17, 18).  The cells were activated by LPS. Cell activation was identified by a rise in Interleukin 17 (18). The assay of MTT and LDH release of astrocyte cells exposed to venom was determined and the highest dose of venom that was not cytotoxic to cells was used as treatment dose. (26, 27). In the present study  sequential gel filtration and RP-HPLC were used  to purify active peptide from M.eupeus scorpion venom  (27). Results of the present study clearly showed that F331 peptide was able to significantly reduce the secretion of interleukin 17 in the activated astrocytoma cells.  Researchers demonstrate that blocking the IL-17 pathways in astrocytes is a  promising therapeutic approach for MS disease, which does not interfere with systemic immune responses which is  major concern in conventional MS therapy(27).Astrocytes can modulate the excitability of neurons by changing the concentration of potassium ions in the extracellular environment, a process called K⁺ clearance  (28).  It is suggested that astrocytes, in addition to their modulation of neuronal excitability at the synaptic level, are strategically located to act as “synaptic managers” that oversee the overall synaptic activity (18). Several studies have confirmed that Kv1.3 channel is highly expressed in macrophages, microglia, and TEM cells, suggesting that Kv1.3 plays a crucial role in immune and inflammatory responses to human diseases such as multiple sclerosis (MS). Over 120 different peptides including agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin were isolated and shown to recognize and block with distinct affinities and varieties  of different K+ channel(17). Several naturally peptides, especially from scorpion, have been reported to be effective blockers of Kv1.3 channels  (29). Most peptides purified from scorpion venom with 33 – 55 residues reported to affect Kv channels have been isolated from species of family Buthidae . The F331, a purified peptide from M. eupeus scorpion venom, may modulate the hyperactivity of activated 1321N1 cells, through blocking the K channel.  Scorpion venom heat resistant peptide (SVHRP) is a toxin purified from scorpion venom decrease in glial fibrillary acidic protein (GFAP) as an indicator of hyperactivity in astrocyte cell (30)  Bin Gao, et al  reported  the purification ,sequencing and functional characterization of a  K+ channel blocker (MeuKTX) with molecular weight of 3.5 KDa from the venom of the scorpion M.eupeus (31). However the molecular weight of peptide purified in the present study was found to be 4.5 KDa.  Previous reports by some  previous research scientists working on toxins blocking Kv1.3 derived from scorpion, sea anemone, snakes, and other animals    are found to be 3 to 6.5 KDa (31).Hence the molecular mass of F331 purified from M.eupeus is in the range and confirmed previous reports 

Conclusion:

Based on the results obtained in the present study, the peptide with molecular weight of about 4.5 KDa in venom of scorpion Mesobouthus eupeus    may act as a modulatory tool for activated astrocyte 1321N1 cells through reduced release of IL17. More studies are require to characterize and confirm the in vivo modulatory action of F331 peptide. 

Acknowledgments:

Authors are thankful to the Department of Venomous Animals and Anti venom Production, Razi Vaccine and Serum Research Institute, Karaj, for providing facilities and Supports.

Conflict of interest: None to be declared.

Funding and support: None.