Research Article

Effect of Aloe Vera Extract on Some Parameter Complete Blood Count and Liver Functions Induced by Azathioprine in Changes Male Rats

Ahmed Al-Hasnawi*, Wafaa Kadhim Jasim

Physiology, Pharmacology and Biochemistry Department, Veterinary Medicine College, University of Kerbala, Karbala, Iraq.

Received | October 04, 2023; Accepted | October 18, 2023; Published | November 22, 2023

*Correspondence | Ahmed Al-Hasnawi, Physiology, Pharmacology and Biochemistry Department, Veterinary Medicine College, University of Kerbala, Karbala, Iraq; Email: ahmdalhsnawy817@gmail.com

Citation | Al-Hasnawi A, Jasim WK (2023). Effect of aloe vera extract on some parameter complete blood count and liver functions induced by azathioprine in changes male rats. Adv. Anim. Vet. Sci., 11(12):1911-1917.

DOI | https://dx.doi.org/10.17582/journal.aavs/2023/11.12.1911.1017

ISSN (Online) | 2307-8316

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The liver is crucial for a number of physiological processes, including the intrahepatic detoxification of xenobiotics and hormones like angiotensinogen and insulin-like growth factors (Al-Mutairi et al., 2022). It is also a digestive organ with key mechanisms for producing energy and metabolizing carbohydrates, lipids, and proteins (Judge and Dodd, 2020). Instead of regulating blood clot components, the liver produces vital proteins such albumin includes bile acid, cholesterol, glycogen, and lipids like triglycerides (TG), in addition to other vitamins and minerals. It also contains albumin and very low-density lipoprotein (VLDL) (Bizzaro et al., 2019; Ishikawa et al., 2021; Huang et al., 2022). In addition to performing these tasks, the liver is recognized as an immunological organ since it has immune cells that are prepared to remove pathogens from the gastrointestinal (GI) tract (Hastings et al., 2020). Azathioprine (aza) is an immunosuppressive drug that is used to treat leukemia, acute lymphoblastic leukemia, inflammatory bowel disease, and rheumatoid arthritis, Azathioprine in combination with corticosteroids is the best treatment for preventing organ rejection, despite its extensive use, aza has been linked to a number of adverse effects, including the suppression of the patient’s lymphocytes and toxicity in the bone marrow, gastrointestinal system, and liver (Adam et al., 2018). The drug’s toxicity causes free radical generation in organs and tissues, as well as oxidative damage, it works by selectively suppressing purine nucleotide (adenine) synthesis and decreasing DNA synthesis in a range of immune and other specialized cells, including hepatocytes, its influence on the creation of reactive oxygen radicals Tissue alterations, necrosis, increased mitochondria and the rough endoplasmic reticulum are all caused by this drug (Hashim et al., 2022). Since ancient times, medicinal powers have been utilized, Plants and the things they produce have been used as a significant source of medicine for thousands of years, almost 80% of people in the under developed globe still predominantly rely on traditional medications for primary healthcare, aleo Vera, one of these herbs, has a more than 2000-year history of use in traditional medicine, It has been utilized all throughout the world because of its medicinal qualities (Golmohammadi, 2022). To the family Liliaceae belongs aloe Vera (aloes), animals and people have both been shown to benefit from the antioxidant properties of aloe vera extract (Rozani and Kusbaryanto, 2019). It has been used as a herbal medicine for thousands of years in different cultures for a number of purposes, antioxidants, vitamins that are both fat solubles and water solubles, minerals, enzymes, phenolic compounds, and polysaccharides, organic acids are all present in aloe vera extract. additionally, it engages in radical scavenging that is more efficient than its capacity (Singh et al., 2021). It has a variety of biological effects, such as gout reduction, anti-inflammatory, cancer prevention, arthritis, UV protection, dermatitis, cancer prevention, antioxidant, anti-diabetes (hypoglycemic agent), reduction of macrophage activation, antifungal, antiprotozoal, gastro-protective (peptic ulcer), burn treatment, and cancer prevention. promoting the development of epithelial and fibrous tissue (Kusbaryanto and Rozani, 2019). The goal of this study was to Investigate the hepatoprotective effects of Aloe vera extract in rats with Azathioprin-induced liver toxicity. Assessment the activity of Aloe vera extract to improve the toxic effects of treatment of azathioprin on hemotology indices and Histological changes on liver.

MATERIALS AND METHODS

Animals experimental

Forty (40) male white rats weighing (200-220 g) were used in the current study. Taken from the College of Veterinary Medicine, Tikrit University, Iraq and their ages are between (12-15) weeks.

Preparation of azathioprine (AZA)

Drug (Imuran®) aza in a commercially accessible 50 mg tablet form, obtainable at a neighborhood drugstore then was dissolved using ordinary saline and administered at a dose of 50 mg/kg body weight.

Preparation of aloe vera gel

Fresh water was used to wash mature, fresh aloe vera leaves that were between 76 and 95 cm long. Pieces of the leaves were transversely sliced. a portion of the thickened epidermis has been removed. The outer layer of the aloe Vera leaf was peeled off to collect the gel directly, and the entire gel was removed using a little spoon. Natural aloe Vera gel was visible. The gel was then put in a blender to create a combination and foam that was ready to be dosed to the research animals (Al-Kaabi et al., 2022).

The experimental design

In the current investigation, 40 male white rats were employed They were split into 4 groups (n = 10) as listed in the points below:

(1) Group (G1): 10 rats administrating with sterile water and administered daily as a single oral dose for 28 days.

(2) Group (G2): 10 rats administrating azathioprine (50mg/kg. b. w) was solved in sterile and administered daily as a single oral dose for 0.28 days (Al-Rekabi et al., 2020).

(3) Group (G3): 10 rats administrating Aloevera gel extract (500mg/kg b.w) was solved in sterile water and administered daily as a single oral dose for 28 days (Rashid et al., 2022).

(4) Group (G4): 10 rats administrating azathioprine (50mg/kg. b. w) and Aloevera gel extract (500mg/kg b.w) was solved in sterile water and administered daily as a single oral dose for 0.28 days.

Laboratory analysis

In this experiment, rats were anesthetized by inhalation of the anesthetic chloroform, and a blood sample was collected by cardiac puncture from each male rat. After four weeks of the experiment, the concentration of vital indicators for measuring the complete blood count (CBC) MCH, MCV, MCHC was measured, and the results were obtained automatically using the Soelab Alpha device, and liver function enzymes were measured using Malondialdehyde, and thiobarbituric acid (TBA) was estimated using the assay method (Firdausa et al., 2022), estimation of serum glutathione-S-transferase (GST) concentration (Onuoha et al., 2023) and serum reduced glutathione (GSH) concentration (Abdel Razzaq et al., 2022). At the end of the experiment, the mice were anesthetized and dissected to take a sample of the liver to study the effect of the toxicity that occurred due to the administration of azathioprine and the improvements that occurred in the liver after the administration of aloe vera extract.

Statistical analysis

Statistical analysis of data was performed using SAS (Statistical Analysis System-version 9.1). One-way ANOVA and Least significant differences (LSD) post hoc test were performed to assess significant differences among means. P < 0.05 is considered statistically significant (Rulkiewicz et al., 2023).

RESULTS AND DISCUSSION

Table 1 showed azathioprine causes MCV, MCH, and MCHC levels reduced that imply that erythropoiesis was suppressed, which led to the development of microcytic hypochromic anemia, add on ROS (reactive oxygen species) promotes hemoglobin glycation, erythrocyte fragility, and direct oxidation can harm bone marrow (Bosing et al., 2012; Gao et al., 2013; Kausar and More, 2019). Reactive oxygen species (ROS) lead to enhance glycemia, erythrocyte fragility, and the risk of direct oxidative damage to bone marrow, more precisely, azathioprine may be etiological by increasing oxidative stress (Niforou et al., 2014). While the result of many studies showed the aloe vera leaf extract significant increases in hematological parameters such as MCV, MCH and MCHC This increase may be due to variations in erythrocyte size, shape, and hemoglobin content (Channa et al., 2014; Egong et al., 2018). This result and elevated level are credited to the effectiveness of vital vitamins like thiamine, riboflavin, and folic acid as well as non-essential and essential amino acids in a. vera that are necessary for the production of hemoglobin (Iji et al., 2010).

While in Table 2 MDA has been utilized as a diagnostic for lipid oxidative damage since it is one of the aldehydes produced by the lipid peroxidation process, MDA is related with numerous ailments, especially liver problems, in the sequence of oxidative stress. MDA causes DNA and protein alterations. Oral treatment of aza to rats raised MDA while dramatically decreasing GSH and G-S transferase levels, indicating the role of oxidative stress and lipid peroxidation in aza-induced liver injury. GSH depletion adds to aza toxicity on hepatocytes, resulting in mitochondrial damage, substantial ATP depletion, and cell death by necrosis (Ahmed et al., 2014). The enhanced lipid peroxidation that destroys tissue and undermines the antioxidant defense mechanism is shown by the raised MDA levels in liver tissue. The observed GSH depletion identified following azathioprine poisoning can be used to explain the increased liver MDA in the azathioprine-treated rats, according to reports, the generation of lipid peroxides and GSH depletion are closely related, azathioprine caused hepatotoxicity, which was evident in the livers of rats treated with the drug by significant pathological alterations, Similar histological lesions have been seen in the rats treated with aza in other instances (Ayala et al., 2014) aldehyde and other by products of lipid oxidation have a significant negative impact on the liver, leading to the creation of high molecular mass protein accumulate inside the membrane (Rosenzweig et al., 2019). Therefore, an elevated level of MDA and its related products, such as conjugated dienes, are a real sign of lipid peroxidation that highlights the harmful effects of MDA on the liver, Thiols are believed to be essential for protecting cells from lipid oxidation and the cellular protein surveillance network includes important components, such as ATP-dependent chaperones, that are engaged in a wide

 

Table 1: Effect of azathioprine, aloe vera extract and combination on MCV, MCH and MCHC.

Groups	MCV	MCH	MCHC
Control group (G1)	59.30±0.27a	18.84±0.27b	35.30±0.20a
Aloe Vera group (G2)	60.38±0.90a	19.71±0.64b	36.80±0.36a
Azathioprine group (G3)	50.09±0.45b	14.42±0.52a	30.78±0.42b
Aloe vera & Azathioprine group (G4)	58.53±0.78a	22.65±0.47c	37.48±0.95c
LSD	1.95	1.49	1.68

 

*Data represented as mean ± SD different letters significant differences at P-value (P<0.05).

 

Table 2: Effect of azathioprine, aloe vera extract on reduced glutathione (GSH), glutathione s-transferase (G-S transferase) and malondialdehyde (MDA).

Groups	MDA	GSH	GST
Control group (G1)	8.62±0.8a	40.78±2.6a	13.30±0.43a
Aloe Vera group (G2)	9.41±1.0ba	38.62±2.0a	12.98±0.47b
Azathioprine group (G3)	16.52±1.4b	22.52±1.7b	9.16±0.83c
Aloe Vera & Azathioprine group (G4)	11.21±1.1c	35.81±2.1c	15.98±0.76d
LSD	2.03	3.21	1.91

 

*Means with a different letter in the same column are significantly different (P<0.05).

 

range of protein functions (Xu et al., 2021). Additionally, the aloe vera reduced the levels of MDA in the liver and increased the amount of GSH and GST, aloe vera treatment dramatically increased metallothionein induction in male rats treated across the body, reduced lipid peroxidation, and activated antioxidant enzymes crucial for managing oxidative stress and boosting immunity (Farid et al., 2022) aloe vera extract treatment in mice boosted hepatic antioxidant enzymes, which in turn lowered hepatic MDA level and elevated GSH and GST level, suggesting that aloe vera extract has a protective action against oxidative stress-induced cell damage and stimulated protein synthesis (Hashim et al., 2022). A study on the benefits of AV extract against liver damage brought on by oxidative stress found that AV decreased the production of lipid peroxidation, when rats administered with AV were compared to rats administered with AV, the effects on enzyme levels were comparable to antioxidants in terms of glutathione and glutathione s-transferase (Iftkhar et al., 2015; Nahar et al., 2013).

Effects of azathioprine, aloe vera extract on liver histopathological

Following the animals had undergone anesthesia, the liver was removed, and following fixation, sections of the liver tissue were obtained and fixed with formalin (10%), Following processing in an alcohol and paraffin, the blocking of the samples was divided into sections, and the samples were then stained with (H & E) histological examination of liver section of control group were staind with haematocylin and eosin, as showed a normal hepatocyte appearance, normal liver lobular structure, and a strikingly normal central vein, as seen in the Figure 1 (Younes et al., 2018).

 

 

Histological examination of liver section of aloe vera group were staind with haematocylin and eosin, as showed a normal hepatocyte appearance There are no changes in the liver cell’s normal central vein and no changes to the hepatic cells as showed in Figures 1, 2 and that the liver sections have a normal pattern in his investigation of employing aloe vera extract as a hepatoprotective (Al-Abbood et al., 2019). Aloe vera extract treatment has been shown to the ability of antioxidant enzymes in the liver of mice addition to plays a crucial function in protecting hepatocytes from oxidation-induced membrane and cellular damage by increasing the activity of antioxidant enzymes by reducing the amount of malondialdehyde (MDA) that occurs in the liver as a result of oxidative stress. aloe vera extract regulates and has the capacity to increase Glutathione (GSH), the primary factor in enhancing liver damage and improving hepatocyte alterations (Guo and Mei, 2016). Histological examination of liver section of azathioprine group were staind with haematocylin and eosin, as showed a sever central vein dilatation, significant hepatic degenerative changes and marked perivascular polymorph nuclear inflammatory cells infiltration, hepatocytes with significant rounded large nuclei and central vein dilation, azathioprine is a popular immunosuppressant used to treat a variety of diseases, However, its negative side effects are extremely severe, and the most prevalent cause of liver damage is likely due to a variety of factors, including liver oxidation that occurs during drug metabolism and the detoxification process, The most hazardous stages of the liver damage, on the other hand, are fibrosis, cirrhosis, and even hepatic cancer (Mannaa et al., 2015; Roselli et al., 2020) (Figure 3). Azathioprine treatment for abnormal organization and non-organized architecture of hepatic tissue resulted in minor hepatocyte destruction, as seen in the liver portion suffered from apoptosis, or programmed cell death, as well as an evident harm to the liver cell (Meijer et al., 2017). Azathioprine can damage the liver and cause hepatotoxicity due to defects in the function of cellular organelles like the mitochondria and rough endoplasmic reticulum, which affect the pathways of the stress activated protein kinase and also result in a reduction in glutathione levels within hepatocytes, these defects can be seen in the histological section (Matsuo et al., 2014). Additionally, about azathioprine reported an increase in the amount of lipid peroxidation, which stimulated both the apoptosis and necrotic processes in the liver cells and the metabolism of azathioprine in the cells of hepatic tissue can result in a number of problems, including the reduction of the antioxidant Glutathione (GSH), mitochondrial damage, a drop in ATP levels, and cell death by apoptosis (Cui et al., 2014). The rise in reactive oxygen species (ROS), which are free radicals, is one of the main factors contributing to the harmful effects of azathioprine on liver cells, The methylation aberrations that create metabolic abnormalities and indicate through methylated metabolites are typically linked to the hepatotoxicity that occurs in hepatocytes affected by azathioprine, where the buildup of methylated toxic metabolites of azathioprine can induce liver damage (Ardeshiri et al., 2012). Histological examination of liver section of azathioprine and aloe vera group were stained with haematocylin and eosin, as showed mild hepatocytes degeneration and normal central vein with mild hepatic swelling and hepatic inflammatory cells infiltration that Use medicinal herbs and plants as a natural alternative treatment that is not harmful and has curative effects because the toxic effects of many drugs that cause numerous disorders from numerous causes in many organs, with the liver being the most effective and considered to be too sensitive to be treated directly with drugs and chemicals (Al-Abdaly et al., 2021) (Figure 4). Aloe vera is a popular medicinal plant whose gel plays a part in both protecting and healing individuals, The ability of aloe vera gel to protect the liver from injury and toxic effects of many drugs and chemicals is known as hepatoprotection. The active ingredients, such as the high concentrations of polysaccharides that have inflammatory effects, as well as the gel’s ability to act as an antioxidant by reducing the oxidation that occurs in hepatocytes by the final metabolite, malondialdehyde (MDA), is decreased by aloe vera extracts, cell damage is lessened during lipid peroxidation events occurring inside of cells (Al-Abbassi et al., 2023).

CONCLUSIONS AND RECOMMENDATION

The current study concluded, based on the findings, that the hepatotoxicity, or liver damage, caused by immunosuppressive medications such as azathioprine varies from the favorable outcomes obtained by providing (aloe vera extract) to male rats and reducing the toxicity, addition to the increasing the activity of antioxidant enzymes and reducing the amount of malondialdehyde (MDA) that develops in the liver as a result of oxidative stress, Aloe vera extract has been shown to play a significant role in protecting hepatocytes from oxidation-induced membrane and cellular damage. In this current study, we recommend that people who are Using additional plant materials from aloe vera, such as the flowers, Purification of the active ingredient, which is utilized to lessen drug adverse effects and act as a hepatoprotective.

 

 

ACKNOWLEDGMENTs

Regards and gratitude First and foremost, praise be to God Almighty, who prompted me to do this research. I’d want to convey my heartfelt appreciation and gratitude to (Assistant Professor Dr. Wafaa Kadhim Jasim). I would like to express my heartfelt gratitude to the Deanship of the College of Veterinary Medicine at the University of Karbala for accepting me for postgraduate studies in the fields of physiology, biochemistry, and medicine.

NOVELTY STATEMENT

The study’s innovation is that it focuses on physiologically active aloe vera extract, which may be used as a novel anti-toxic treatment due to its antioxidant activity with few side effects and efficient drug to reduce the toxicity of azathioprine.

AUTHOR’S CONTRIBUTION

These authors each contributed equally.

Conflict of interest

The authors have declared no conflict of interest.

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