Effect of bee venom on some blood and biochemical parameters in formaldehyde induced arthritis male rats in comparison with prednisolone drug

Background Bee venom(BV) has been used to treat and reduce chronic inflammatory diseases such as rheumatoid arthritis(RA), pain, skin diseases and to treat cancerous tumors. The aim of this study was to investigate the effects of bee venom on some blood and biochemical parameters in male albino rats with Arthritis and compare it with prednisolone drug. Materials & methods: (42) mature male albino rats (135-150) gm, divided into (7) groups, (6) male rats for each group. The experiment was continued for (14) days and included: the first group (negative control) was injected by (0.1ml/animal) from physiological normal saline (0.9% Nacl),the second group(Arthritis group) was injected by (0.1ml/animal) formaldehyde, the third group (normal group) was injected by bee venom(i.p) (1mg / kg of B.W), the fourth group (normal group) was treated orally with prednisolone (5mg / kg of B.W), the fifth group (Arthritis group) was injected by bee venom(i.p) (1mg / kg of B.W), the sixth group (Arthritis group) was treated orally by prednisolone (5 mg / kg of B.W) and seventh group (Arthritis group) was injected by bee venom (1 mg/ kg of B.W) and treated orally with prednisolone (5mg / kg of B.W). Results: Our results showed that 2nd group (Arthritis) significantly increased (p≤ 0.05) in the number of WBCs and platelets compared with control group, while the 3rd and 4th groups showed a significant decrease in WBCs and PLT compared with 2nd group. The 5th, 6th and 7th groups showed a significant decrease (P≤0.05) in the number of WBCs and platelets compared with 2nd group. On the other hand, the present study demonstrated a significant decrease (p≤ 0.05) in RBCs, hemoglobin (Hb) and hematocrit (HCT) in the 2nd group (Arthritis group) compared with control group, while the 3rd and 4th groups showed a significant increase in RBCs, Hb and HCT compared with 2nd group. The 5th, 6th and 7th groups showed increase (p≤ 0.05) in RBCs, Hb and HCT compared with 2nd group. The results showed also, a significant increase in levels of cholesterol, triglycerides, LDL and a significant decrease in HDL level in 2nd group compared with control group, while 3rd,4th, 5th, 6th and 7th groups showed a significant decrease (P≤0.05) in cholesterol, triglycerides, LDL and a significant increase in HDL level compared with 2nd group. Conclusion: We conclude from the present study that bee venom attenuates development of Arthritis by improving some Blood and Biochemical parameters.


Introduction
Rheumatoid arthritis is one of the most autoimmune disease prevalence in the present time and it is the most common in the female at ratio 3:1 [1], it causes many adverse effects in the body systems include lymphatic, hematology, respiratory and muscular system as well as, it's effect on heart and kidneys [2]. RA is a disease condition, occurs in any age especially between 35-50 age [3]. There are many factors that play an important role in appearance of RA such as heat shock protein-70, smoking, Rheumatoid factor, stress and cytokines especially tumor necrosis factor [4,5]. Many drugs are used to treat RA such as Non-steroidal anti-inflammatory drugs (NSAIDS), Disease modifying anti rheumatic drugs (DMARDS) and corticosteroids such as prednisolone and prednisone [3].
Glucocorticosteroids are used as a single agent or in combination with other anti-rheumatic drugs. In short-term (duration of use <1 year) regimens, treatment is often aimed at controlling symptoms in periods of high disease activity either with unchanged background anti-rheumatic therapy or while awaiting the effects of newly started other disease-modifying drugs [6], but used of these drugs for long period, cause adverse effects in cardio vascular system, Renal failure, an ulcer in stomach and intestine [3].
In the recent time, researchers have been directed towards used of anti-inflammatory from natural sources because the adverse effects of drugs that used to treat arthritis. One of these natural sources, Bee venom that has both anti-inflammatory and anti-microbial effects involving no side effects in animal model [7]. BV composed of melittin which forms the main component of BV, as well as apamin, adolapin, phospholipase A2, Hyalurornidase, biologically active amines such as (Histamine, Epinephrine), also BV contains lipids, carbohydrates and free amino acids [8,9].
This experiment was designed to study the protective effect of bee venom on some blood and biochemical parameters in male albino rats with Arthritis and compare it with prednisolone drug.

Experiment animals
42 male albino rats (135-150) gm. Were obtained from animal house of vet. med. College, University of AL-Qadisiyah. The animals were placed in plastic cages in an air conditioned room with temperature maintained at 25 ± 2 Cº. the experiment was performed under controlled conditions (temperature, humidity and 12 h light -dark cycle Rats were given sterile food pellets and tap water. All rats were divided randomly in to (7) groups, (6) rats for each one and were treated as below for (14)

Chemicals:
Bee venom ( BV) was obtained from (Sigma-Aldrich, USA) in packages contain (1gm) of BV powder, prepare at a dose (1mg/ kg of B.W) according to(10).Prednisolone was obtained from market and prepare at a dose (5mg/ kg of B.W) according to [11].

Induction of arthritis:
Arthritis was induced in rats by administration of (0.1 ml) formaldehyde (2%) in to the right hind paw in the first and third day of the experiment [12].

Blood collection:
Blood collection was done at 14 days of the experiment via abdominal vein. Blood samples were collected after 14 days placed (2ml) of blood in test tubes with anti-coagulated to hold blood parameters and (3ml) of blood placed in test tubes without anti-coagulated that allowed coagulating for 15 min. Serum was separated from coagulated blood samples by centrifugation at 3000 rpm for 15 min and then kept in the frozen at -20Cº until using to estimate lipid profile.

Estimation of blood parameters:
Blood parameters was measured by Hematology analyzer apparatus which include: WBCs, RBCs, Hb, HCT and platelets (PLT).

Statistical analysis:
Grouped date was statistically read by spss program, methods of testing include one way ANOVA for comparisons among groups followed by least significant differences (LSD) test to compare among groups, P values of P ≤ 0.05 were considered to record statistical significance, all data were expressed as mean ± standard error (SE) (16).

Blood parameters
Our results showed that 2 nd group significantly increased (P≤ 0.05) in the value of WBCs (14.18 ± 1.42) compared with control group. The 3 rd and 4 th groups showed a significant decrease in WBCs compared with 2 nd group, the 3 rd and 4 th groups showed non significant changes in WBCs compared with control group. Also the results showed a significant decrease in the value of WBCs in 5 th (6.50 ± 0.81), 6 th (5.63 ± 0.44) and 7 th (3.13 ± 0.67) compared with 2nd group. The 5 th group showed non significant increase in WBCs compared with 6 th group as in table (1).
The results showed a significant decrease (P≤ 0.05) in RBCs value (5.21 ± 0.17), Hemoglobin (Hb) (10.30 ± 0.42) and Hematocrit value (33.12 ± 1.84) in the 2 nd group compared with control group. The 3 rd and 4 th groups showed a significant increase in RBC, Hb and HCT compared with 2 nd group, while these groups showed non significant changes in RBCs, Hb and HCT compared with control group. Also the results showed an increase in RBCs, Hb and HCT values in the 5 th , 6 th and 7 th groups compared with 2 nd group, while 5 th group showed non significant increase in RBCs, HCT and a significant increase in Hb compared with 6 th group as in table (1).
Platelets values showed significantly increased (P≤ 0.05) in the 2 nd group (739.75± 38.66) compared with control group, The 3 rd and 4 th groups showed a significant decrease in PLT value compared with 2 nd group, while the 4 th group showed non significant changes in PLT value compared with control group. Also the results showed a significant decrease (P ≤ 0.05) in PLT values in the 5 th group (390.25± 43.21), 6 th group (462.25± 29.36) and 7 th group (552.50 ± 8.64) compared with 2nd group, while the 5 th group showed non significant decrease in PLT value compared with 6 th group as in table (1) Also results showed a significant decrease (P ≤ 0.05) in the values of cholesterol, triglycerides , LDL and a significant increase in value of HDL in the 5 th , 6 th and 7 th groups compared with 2 nd group, while the 5 th group showed a significant decrease in the levels of cholesterol, triglycerides, LDL and non significant increase in the HDL level compared with 6 th group, as in the

4.Discussion
The object of the present study was to identify the pharmacological effects of bee venom on arthritis

Blood changes
The results of the present study showed a significant increase in the values of WBCs (P≤ 0.05) in the 2 nd group compared with control group, the cause of these results belong to inflammation that accompanying with the onset of arthritis induced [17] or these result may be due to the inflammatory processes that occur in the body that lead to produce some inflammatory cytokines which in turn causing increased proliferation of WBCs [18]. Also an increase in the total number of WBCs in the first treatment group may be due to exposure the animals to oxidative stress which causes an increase in the secretion of ACTH from pituitary gland which in turn causing increased in the production of corticosterone hormone from cortex of adrenal gland, therefore the number of WBCs increase [19]. The significant decrease in the total number of WBCs (P≤ 0.05) in the 5 th group compared with 2 nd group may be due to Immunosuppressive effect of bee venom that cause decrease in the total number of WBCs [20], While the significant decrease in the total number of WBCs (P≤ 0.05) in the 6th group may be due to the ability of prednisolone to prevent the infiltration of WBCs to the inflamed tissues [21,22].Also [23] showed that prednisolone and Atorvastatin have anti-inflammatory effect and contribute in the reduction of WBCs count, through preventing recruitment and adhesion of WBCs to the vascular endothelium and the antiinflammatory effect of prednisolone and Atorvastatin may be due to inhibition of the production of isoprenoids which contribute in the biosynthetic pathway of cholesterol, as well as isoprenoids have effects on G-protein, adhesion molecules and cell proliferation and blocking the production of isoprenoids has many effects on the functional systems which related with inflammation such as WBCs [24,25].
The present study showed that the 7 th group demonstrated a significant decrease in the total number of WBCs and this because of the combination effect of BV and prednisolone.
Our present results showed a significant decrease in the values of RBCs, Hemoglobin(Hb) and hematocrit(HCT) in the 2 nd group (first treatment group) compared with control group, these due to the disease activity in bone marrow suppression or erythropoiesis may be inactive [26] and these decreased may be due to defect in the function of thyroid gland because of toxins secretion that generate in the body of animal as a result of arthritis induction by formaldehyde, hormones of thyroid gland affect on metabolism processes in the body as well as their in direct effects on production of RBCs from bone marrow [27].
The results showed a significant increase (P≤ 0.05) in the values of RBCs, hemoglobin (Hb) and non significant increase in the value of hematocrit(HCT) in the 5 th group compared with 2 nd group, these results because of BV increases coronary and peripheral circulation and improves circulation of blood in the micro blood vessels, as well as its role in stimulation of building erythrocytes [28]. [29] showed that BV has preventive role in the protection of the body from radiation and contribute in the improvement renewal of RBCs in the rats that exposure to gamma radiation, While the significant increased in the values of RBCs, Hb and HCT(P≤ 0.05) in the 6 th group (fifth treatment group) due to role of prednisolone in the improvement of blood parameters such as RBCs, Hb and HCT, where it works on the regulation of hematopoiesis and stimulation of erythropoiesis in direct form through increasing the production of erythropoietin in kidney [30,31].The significant increase in the values of RBCs, Hb and HCT(P≤ 0.05) in the 7 th group (sixth treatment group) due to the combination effect of BV and prednisolone.
The platelets results showed that the 2nd group (first treatment group) significantly increased (P≤ 0.05) in the PLT value compared with control group, due to present of Rheumatoid arthritis inflammation in the early stage [32], Other studies showed these increasing in the PLT value due to the great effect of cytokines especially IL-6, where it works on the maturation of generated cells of PLT and causes increasing production of PLT [33].
The results showed a significant decrease in the value of PLT (P≤ 0.05) in the 5 th group compared with 2 nd group because of BV has antioxidant effect (29), where the antioxidants act to reduce the damages that result from oxidative stress [28], oxidative stress occurs as a result of inflammatory cytokines especially IL-6 that causes an increase in the number of PLT and BV works on inhibition the activity of IL-6 and thus less number of PLT [34], While the 6 th group demonstrated a significant decrease in the value of PLT (P≤ 0.05) compared with 2 nd group, these result due to the role of prednisolone to control on the deterioration in the blood parameters as a result of arthritis, as well as prednisolone has anti rheumatoid and antioxidant effects [31].Finally, the significant decrease in the number of PLT (P≤ 0.05) in the 7 th group due to the combination effect of BV and prednisolone.

Biochemical changes:
Our results showed a significant increase (P≤ 0.05) in the values of cholesterol, triglycerides, LDL and significantly decrease in the value of HDL in the 2 nd group compared with control group, these results due to systemic inflammation occurs as a result of elevation C-reactive protein and ESR concentration which consider independent risk factor and play an important role in atherosclerosis, as well as inflammatory mediators such as cytokines, chemokines and immune cells cause many metabolic effects in arthritis and these inflammatory mediators play an important role in atherosclerosis and dyslipidemia such as increase LDL, cholesterol, triglycerides levels and decrease HDL level [35,36,37]. [38] showed that increasing in the level of cholesterol and TG due to injection the animals with formaldehyde which causing release of toxins in their bodies, these toxins affect on the function of thyroid gland and cause hypothyroidism, reduction the concentration of T3 and T4 hormones affect on metabolism of cholesterol in the body and increased its concentration in the blood. Also reduction the concentration of T3 and T4 hormones adversely affect on lipoprotein lipase that works on analyze of TG, therefor the concentration of TG increase in the blood [39]. [40], showed that elevation of cholesterol level in the animal blood infected by arthritis related directly with level of LDL and adversely with level of HDL and this explain the significant increase in the level of LDL and significant decrease in the level of HDL.
The results showed that the 5 th group (fourth treatment group) significantly decreased (P≤ 0.05) in the levels of cholesterol, TG,LDL and significantly increased in the level of HDL because of Bee venom has hypolipidemic activity in the mice induced by diabetes [41] and this hypolipidemic effect of BV attributes to melittin, phospholipase A2 and poly peptide that are together make up more than 62% of BV [42]. There is another mechanism explains reason of dyslipidemia in animals with diabetes, this mechanism is link metabolism of glucose and fatty acid by improving insulin action in fat cells which lead to lower LDL, TG and increased HDL levels [43], also the enzymatic activity of phospholipase A2 in BV plays an important role in reducing the level of cholesterol, TG, LDL and increasing HDL level as well as regulating of lipid profile in the blood [44].
The significant decrease in the levels of cholesterol, TG, LDL and increasing in HDL level (P≤ 0.05) in the 6 th group (fifth treatment group) compared with 2 nd group due to use of prednisolone in low dose causing an elevation in the levels of cholesterol and HDL [45], as well as used prednisolone in high dose has appropriate effect on lipid profile. Also [46], showed that glucocorticoids cause an increasing in the level of HDL and total cholesterol, while [40], showed that the level of cholesterol link inversely with the level of HDL, therefore the reason of this result that the most acceptable is the glucocorticoids more affect on HDL  [46] and cause increasing the level of HDL, therefore the elevation of HDL causes decreasing level of cholesterol in the blood.
Finally, the significant decrease in the levels of cholesterol, TG, LDL and increasing in HDL level (P≤ 0.05) in the 7 th group(sixth treatment group) because of the combination effect of BV and prednisolone.