INTRODUCTION:

MTX is often considered to be a "toxic" drug. Methotrexate (MTX), formerly known as methopterin, is a chemotherapeutic medication sold under the trade names Rheumatrex and Trexall¹. Low dosages are used to treat autoimmune disorders such as psoriasis and rheumatoid arthritis^2,3, while large doses are used to treat malignancies such as solid organ tumors and leukemia^4-8.

Inhibition of dihydrofolate reductase, a key enzyme in the folic acid metabolism, is at the basis of MTX's ability to treat a wide range of conditions.

The depletion of nucleotide precursors such as thymidylates and purines caused by a malfunction in the folic acid metabolism hinders the synthesis of DNA, RNA, and proteins. As an added effect, MTX blocks the entry of low folates into cells and the activity of thymidylate synthase. Cells that proliferate fast and those that remain intact, notably those found in the mucous membranes of the mouth, colon, and bone marrow, are directly affected by Methotrexate's direct action^9-12. Because MTX's effects on rapidly proliferating cells are not limited to malignant cells, other cells, such as those in the bone marrow's hematopoietic system, are adversely impacted^4,13,14.

Monounsaturated fatty acids are found mainly in Vegetable oils, nuts, and seeds. showed that MUFA reduced food intake and short-term hunger in overweight subjects¹⁵. palmitoleate is an omega7 monounsaturated fatty acid (16:1n-7). With a total of 16 carbon atoms and a double bond in the seventh carbon atom counting from the methyl group¹⁶. It is mostly present in blood and adipose tissue^17,18. Stearoyl-CoA desaturase 1 (SCD1)is an enzyme that allows mammals to manufacture the cis form of palmitoleic acid, which is created from the saturated fatty acid palmitate19. Plant sources of palmitoleate are macadamia nuts (Macadamia integrifolia), seabuckthorn (Hippophaer hamnoides), Durian fruits (Durio graveolens), and blue-green algae^20,21. Only macadamia nuts contain 15–22% of the total amount of palmitoleate that is present in its cis form^22-24. Numerous studies have shown that omega-7 fatty acids are helpful for your health, including their anti-inflammatory, antioxidant, and lowering effects on blood cholesterol levels²⁵.

Aim:

Evaluation of possible protective effects of omega 7 against MTX-induced blood toxicity.

MATERIAL AND METHODS:

Chemical and drugs:

Omega 7 purchasedfrom Source Naturals (USA), Methotrexate purchased from Ebewe(Austria) 50mg/2 ml and Liquid paraffin had been obtained from Riedel-de Haen AG, Germany.

Animals and Treatment Protocols:

Experiments were conducted using 30 albino Swiss mice (Mus musculus). They were taken from the College of Pharmacy/University of Baghdad's Animal House. at a weight Between 23 and 27 grams each, they were in all, they were separated into five independent groups, each of which was housed in a separate plastic cage. Temperatures ranged from 23 to 25°C, and the animals had access to ad libitum water and free excess food. Five groups of six mice each were formed, as follows

· Group I: Liquid paraffin was administered to six mice. The dosage was administered orally for seven consecutive days. This group represents negative control.

· Group II: mice were fed liquid paraffin For seven consecutive days, and methotrexate (20mg/kg) was administered intraperitoneally on day eight. this group represents a positive control

· Group III: Omega 7 was given orally to mice for seven days at a dose of 100mg/kg.

· Group IV: Omega 7 was given orally to mice for seven days at a dose of 50mg/kg and a methotrexate (20mg/kg) intraperitoneal injection was administered on day eight.

· GroupV: Omega 7 was given orally to mice for seven days at a dose of 100mg/kg and a methotrexate (20mg/kg) intraperitoneal injection was administered on day eight.

The animals had been weighed and starved overnight, Under Diethyl ether anesthesia, blood samples were taken from the retro-orbital venous plexus and hematological tests were performed. the animal was then killed by cervical dislocation.The study included measuring of, Concentration of platelet,Hemoglobin estimation, Packed Cells Volume (PCV), Mean corpuscular volume (MCV), Mean Corpuscular Hemoglobin (MCH). Total WBCs count. and Differential count of leukocytes. The hematological tests were performedautomatically by auto hematology analyzer BC5150/BC 5000

Statistical analysis:

As a consequence of the research, all findings were presented in the form of Mean and Standard Deviation (SD). Data entry and analysis were carried out using the Statistical Package for Social Sciences software version 25 (SPSS V 25) and a T-test was used to compare the test groups²⁶.

RESULTS:

Effect of Omega7 in parameters related to RBCs The results shown in table (1) it is found that each of the MCV and MCH decreasesignificantly in methotrexate compared with the negative control group (p<0.05), The other blood parameter and platelet showed a non-significant decreasewhen compared MTX group with the negative control group (p>0.05). The results showed a significant increase at (P <0.05) between the means of the MTX alone and the means of omega7(100mg/kg)in the MCV parameter(p<0.05) all other blood parameters and platelet showed a non-significant increase between these groups. Omega 7 (50mg/kg with MTX 20mg/kg) showed a non-significant increase in all blood and platelet parameters (p>0.05) when compared with the MTX group,while omega7 (100mg/kg with MTX 20mg/kg)showed a significant increase in Hb and PCV(p<0.05) when compared with MTXgroup, while the other blood and platelet parameter showed non-significant increase Hb and PCV showed a significant difference indifferent omega7 doses treatment (p<0.05) when compared with each other,Concerning the other parameters, which include all of the MCV, MCH, and plateletit was observed that the differences in the means of these parameters did not live up to the level of significance in different omega7 groups doses (p>0.05).

Table 1: The effect of omega7 in parameters related to RBCs and platelet

Treatment	Mean±SD
Treatment	Platelet 10³/ml	Hb (gm/dL)	PCV (%)	MCV fl	MCH pg
Liquid paraffin (Negative control)	952.8±87.02	13.4±0.78	0.41±0.06	48.62±2.29	15.48±0.24
Methotrexate (MTX) (Positive control) 20mg/kg	912.6±16.01	12.8±0.64	0.37±0.04	43.74±1.85*	14.28±0.89*
Omega-7 100 mg/kg	944.8±92.89	13.08±0.86	0.41±0.03	47.22±1.90a	15.12±0.41
Omega-7 50 mg/kg with MTX with 20 mg /kg	935.2±186.00	12.84±0.70A	0.41±0.02A	46.12±2.84	14.54±0.93
Omega-7 100 mg/kg with MTX with 20 mg /kg	938.4±51.45	14.38±0.63Aa	0.45±0.02Aa	45.56±2.41	12.54±4.49

Data are expressed as mean±SD.

*Denote Different from liquid paraffin in a significant way (P<0.05).

• Small letters superscripts (a) denote significantly different to methotrexate (P<0.05).

• Capital letters superscripts (A) denote significantly different when a comparison between group III and Group IV and GroupV (P<0.05).

DISCUSSION:

In the current investigation, The change in hematological parameters in Group II over the experimental period was seen.According to Table 1, in the MTX group, compared to the negative group, the MCV and MCH levels have decreased significantly, with a decrease in the hemoglobin level,A possible link to methotrexate's inhibition of dihydrofolate reductase, which causes a drop in folate levels, might be found here²⁷.Other investigations showed that MTX significantly decreased the amounts of antioxidant enzymes, making cells more susceptible to reactive oxygen species (ROS)^28,29.Because of the high concentration of oxygen and hemoglobin in erythrocytes' membranes, as well as the latter's potential role as an effective oxidative process activator, erythrocytes are particularly vulnerable to oxidative damage³⁰.The release of free iron ions from ROS interactions with hemoglobin leads to the destabilization of the heme and globin structure and is a key factor in the production of ROS. Any alteration to the cellular antioxidant reserves or endogenous ROS production might modify the associated oxidative DNA changes, which can disturb cellular functions³¹. The deleterious consequences of the above-mentioned actions have stimulated studies on the mechanisms of action of biologically relevant natural antioxidants such as mono and polyunsaturated fatty acids³². One such potentially health-promoting substance is omega7³³.The result in the current study showed omega 7 at a high dose try to normalize the effect of MTX back to normal result in the negative control group. When compared to the negative control group (Group I), the group that received just omega7 (Group III) showed no significant change, demonstrating that omega7 had no harmful impact. Table 2 showed the results of the complete blood count. There is a significant decrease in the number of WBCs (p < 0.05) Among the WBCs, there was a decrease in the levels of neutrophils,and a significant increase in the number of monocytes count in the MTX group compared to the negative control. It is well known that myelosuppression and pancytopenia are characteristic features of chemotherapy.Methotrexate has a systemic impact that causes high lymphocytes and low neutrophils ratio³⁴, or an increase owing to spleen tissue damage, thus lymphocytes grow to eliminate damaged cells, additionally, monocytes rise in reaction to liver tissue damage from hyperthyroidism, causing infiltration of these cells from the liver³⁵,or due to the affected bone marrow by methotrexate³⁶.

In the present study, omega7 normalizes monocytes count to a significant value and decrease the effect of MTX on total WBC count and neutrophils count. This effect returns to the role of omega7 antioxidant and anti-inflammation,animal studies showed Administration of omega7 resulted in considerably lower levels of fat-related inflammatory cytokines (signaling molecules). Omega7's capacity to inhibit NF-kappa B, the master inflammatory regulatory complex, may play a role in many of these positive anti-inflammatory actions³⁷.

Knowing that the NF-B pathway modulates the response to oxidative stress³⁸Supplementation with omega7 fatty acid may have resulted in a decrease in NF-B gene expression, which might be linked to the improvement in the oxidative stress state³⁹.

CONCLUSION:

Data concluded that omega7 hasa significant protective role as an anti-inflammatory and antioxidant by increasing Hb and PCV and decreasing monocytes values that were affected by MTX toxicities.

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Received on 27.08.2022 Modified on 10.10.2022

Research J. Pharm. and Tech 2023; 16(4):1709-1713.

DOI: 10.52711/0974-360X.2023.00281

Treatment	Mean±SD
Treatment	Total WBC	Neutrophil (%)	Lymphocytes (%)	Monocytes (%)
Liquid paraffin (negative control)	4920±1221.47	0.12±0.04	0.83±0.03	0.11±0.04
Methotrexate (MTX) (Positive control) 20mg/kg	3000±1113.55*	0.08±0.02*	1.16±0.41	0.21±0.05*
Omega-7 100 mg/kg	4000±1640.12	0.10±0.02	0.81±0.03	0.10±0.02 a
Omega-7 50 mg/kg with MTX with 20 mg /kg	3840±763.54	0.08±0.01	0.86±0.04	0.06±0.01a
Omega-7 100 mg/kg with MTX with 20 mg /kg	4120±782.3	0.09±0.01	0.86±0.02	0.07±0.02 a