Protective effect hesperidin on methotrexate induced toxicity in Wistar rats: A histopathological study

Introduction

Chemotherapy has been an important component in human and animal cancer patients for the last many years. Now a days in India cancer cases are rapidly increasing in the animals. Cancer is an uncontrolled growth out of control and spread to other areas of your body. Chemotherapy drugs have a cytotoxic effect on cancer cells as well as normal cells. Chemotherapy drug such as Methotrexate (MTX) has antimetabolite, immunomodulatory, anti-inflammatory, immunosuppressive and cytotoxic properties and used in a wide range of clinical practice (Peters et al. 2000) [17] . Methotrexate widely used as a first line of treatment in autoimmune disease, inflammatory disease such as a rheumatoid arthritis, crones' disease and psoriasis. Methotrexate is also used in the treatment of lymphoma, breast tumours, Osteosarcoma, invasive urinary bladder tumours, autoimmune disease and in veterinary oncology. Numerous organs and tissue are affected by MTX treatment including kidney, spleen, liver, small intestines, ovary, testicles and nerve cells (Li ex et al. 2016) [12] . Flavonoids are categorized as flavones (Apigenin, Tangeretin), flavanols (Quercetin, Rutin, Myricetin) flavanones (Hesperidin, Naringin, flavonols), isoflavones (Genistein, Daidzein), chalcone (Phlorectin, Arbutin) and anthocyanin (Cyanidin, Malvidin) according to structural variations (Panche et al. 2016) [16] . Hesperidin is one of the most commonly used and biologically active compounds in the flavonoid family (Turk et al. 2019) [18] . Hesperidin also has antibacterial, antioxidant, lipid-lowering, anti-inflammatory, antiviral, anti-hypertensive, anti-carcinogenic, and antioedema effects. Hesperidin has some pharmacological effects like effects on anti-fertility, vascular system, action on enzyme, platelet and cell aggregation inhibition, ultra violet protecting activity and also some miscellaneous effect like antiallergic, analgesic, antipyretic activity, effect on wound healing and anti-ulcer activity (Garg et al. 2001) [8] .

Materials and Methods

The study was conducted at small animal house and Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand. The study was approved by the Institutional Animal Ethics Committee (IAEC) of the College of Veterinary Science and Animal Husbandry, Kamdhenu University, Anand, Gujarat, India. The animal facility of Cadila Pharmaceuticals Company Ltd. in Dholka, Gujarat, India provided a total of 40 Wistar rats (20 male and 20 female), which were used in experiments. The procedures for animal care and management followed to those given by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India. All of the rats were kept in polypropylene cages at a laboratory animal house facility (College of Veterinary Science and Animal husbandry, Anand) in a climate-controlled room with a constant temperature of 22 ± 3 ºC and a humidity of 30-70%. There was a 12/12-hour cycle of light and dark. The rats were kept stress-free by using corncob as bedding and following all appropriate management protocols. Before experiments, all rats were acclimated for 15 days. Rats were given free access to a regular pellet food and palatable water for the duration of the trial. The feed was provided by keval sales corporation, Vadodara, Gujarat. Rats were identified by having picric acid applied to their body coats to mark them. One cage had five rats, each of which had a ring at the base of its tail to represent the number of the animal (rats 1, 2, 3, 4, and 5 consecutively). Methotrexate and hesperidin were purchased from cadila healthcare Ltd, Ahmedabad and Sigma Aldrich company respectively. A total of 40 Wistar rats (20 males and 20 females) were divided into four groups at random, each including five female and five male rats. The groups were given numbers ranging from I to IV. Group I (control): Rats treated with vehicle 1% carboxymethylcellulose (CMC) dissolved in distilled water through orally and single dose of normal saline via intraperitoneal route. Group II (MTX): Rats were received single dose of MTX at the dose of 20 mg/kg/bw via intraperitoneally. Group III (MTX and hesperidin 100 mg): Rats received MTX at the dose of 20 mg/kg through intraperitoneally at the 21st day and HSP @ 100 mg/kg/bw through orally for 28 days. Group IV (MTX and hesperidin 200 mg): Rats were given single dose of MTX @ 20 mg/kg i.p at 21st day and HSP at the dose of 200 mg/kg/bw for 28 days via oral route. Every Wistar rats in the study was examined twice a day for morbidity and mortality. The clinical observations have been recorded once daily during the adaptation period. Clinical and behavioural observations were carried out at least twice on each day of dosing during the experimental period (before and after therapy). All the surviving rats were sacrificed on by high dose of sodium pentothal 29th day of the experiment for necropsy. All rats were starved overnight before necropsy. The final body weight was recorded prior to necropsy. For histopathology, kidney, liver, heart, lung, spleen, stomach, intestine, and brain tissues were collected and preserved in 10% neutral buffered formalin and testes have been stored in modified Davidson fluid. Postmortem examination was carried out on all the animals and gross lesions were recorded if any.

The tissue from sacrificed animals, including the kidney, liver, heart, brain, spleen, lungs, testes, stomach, and intestine, were collected. Any adherent tissue was subsequently removed if needed, and the tissue organ was preserved in 10% neutral buffered formalin for at least 48 hours. Tissue processing and staining: During necropsy, the tissue of the kidney, liver, heart, brain, spleen, lungs, testes, stomach, and intestine were collected in 10% neutral buffered formalin. The collected tissues were processed in an automatic tissue processor, and then paraffin blocks were prepared in an automatic tissue embanding station. 5-6 µm sections were cut with an automatic microtome. Sections were stained with H&E (Haematoxylin & Eosin) stains with standard protocol. Under a light microscope, the Haematoxylin and Eosin-stained slides were examined and microscopic lesions were recorded.

Results

Pathomorphology Kidney

In male rats, kidney section showed normal histoarchitecture details in control group (I) rats (Fig. 1.1). The kidney section of methotrexate group (II) revealed severe degeneration of tubules, decreased lumen size of tubules (Fig. 1.2), infiltration of inflammatory cells, congestion in interstitial spaces along with necrosis of glomeruli (Fig. 1.3, Fig. 1.4). The kidney section of hesperidin 100 mg + methotrexate group (III) showed mild degeneration of tubular epithelial cells and presence of red blood cell cast (Fig. 1.5). The kidney section of hesperidin 200 mg + methotrexate group (IV) showed mild tubular degeneration (Fig. 1.6). In female rats, control group revealed normal histoarchitecture details in the kidney section (Fig. 1.7). Methotrexate treated group (II) rats showed severe necrosis and tubular degeneration, presence of tubular cast along with infiltration of inflammatory cells and detachment of glomeruli from basement membrane (Fig. 1.8, Fig. 1.9). The kidney section of hesperidin 100 mg + methotrexate group (III) showed mild degeneration, infiltration of inflammatory cells and capillary congestion in cortex (Fig. 1.10). The kidney section of hesperidin 200 mg + methotrexate group (IV) revealed mild congestion in interstitial spaces of kidney (Fig. 1.11).

Liver

Histopathological lesion in the liver from male rats in control group revealed normal histoarchitecture details (Fig. 1.12). The liver of rats treated with methotrexate group (II) showed severe degeneration of hepatocytes, dilation of sinusoids near portal triad, fatty changes in hepatocyte (Fig. 1.13, Fig. 1.15), congestion of central vein (Fig. 1.14) and focal infiltration of inflammatory cells (Fig. 1.13). The liver section of hesperidin 100 mg + methotrexate group (III) rat revealed mild to moderate dilatation of sinusoids and fatty changes of hepatocyte (Fig. 1.16). The liver of rats treated with hesperidin 200 mg+ methotrexate group revealed only mild dilation of sinusoids (Fig. 1.17). In female rats, liver section showed normal histoarchitecture details in control group (I) (Fig. 1.18). The liver of rat treated with methotrexate group (II) revealed diffuse degeneration and fatty changes of hepatocyte (Fig. 1.19). The liver section of hesperidin 100 mg + methotrexate group (III) rats observed fatty changes in hepatocyte (Fig. 1.20). The liver of rats treated with hesperidin 200 mg + methotrexate group revealed mild congestion in central veins (Fig. 1.21).

Discussion

Similarly, to present findings, Jahovic et al. bserved extensive degeneration of glomeruli, swelling of proximal tubule in methotrexate treated rats. Vardi et al. (2013) [1] , Morsy et al. (2013) [14] and Erboga et al. (2015) noticed tubular dilation, tubular degeneration and glomerular congestion in methotrexate treated rats. Cakir et al. noticed tubular necrosis, tubular epithelial degeneration, narrowing of Bowman's space, congestion and interstitial inflammation in methotrexate treated rats. Ahmed and Abdulmajeed (2017) [3] observed focal degeneration of renal tubular epithelial cells, infiltration of inflammatory cells in interstitial spaces and necrosis in methotrexate treated rats. Moustafa et al. (2022) [15] noticed aluminum phosphide treated rats showed vacuolar degeneration, cloudy swelling, degeneration of renal tubular epithelial cells while coadministration of hesperidin and aluminium phosphide treated rats revealed restoring of the normal glomerular architecture with reappearance of a number of normal capillary loops and the proximal convoluted tubule in hesperidin treated rats. Kasem et al. (2022) [11] observed Bisphenol treated rats revealed infiltration of inflammatory cells in interstitial tissue and glomerular destruction while co administration of bisphenol and hesperidin treated rats revealed infiltration of few inflammatory cells with no congestion. The present findings were in accordance with by Tunali-Akbay et al. They noticed degeneration of hepatocytes, infiltration of inflammatory cells around portal vein and central vein in methotrexate rats. Abdul et al. (2012) [1] revealed scattered dysplastic hepatocytic changes, periportal inflammatory cell infiltration and sinusoidal dilation in methotrexate treated rats. Yucel et al. (2017), Ekinci-akdemir et al. (2018) [7] , Kalantari et al. (2018) [10] , Hoshyar et al. (2020) [9] , Azadnasab et al. (2021) and Abdel-Raheim et al. (2022) [15] also observed infiltration of inflammatory cells, fatty changes, congestion of central vein, necrosis and dilation of sinusoids in methotrexate treated rats. The hepato protective effect of hesperidin was also observed by Ansar et al. (2018) [4] . They observed expansion of portal tract, hyalinization of hepatocytes, infiltration of inflammatory cells, increased number of Kupffer cell and increased accumulation of cells around the central vein in zinc oxide treated rats. Co administration of hesperidin and zinc oxide in rats revealed mild degeneration of hepatocyte and dilation of sinusoids. Mesallam and Atef (2020) [13] noticed diazinon treated rats showed congestion of the sinusoids and central vein, hepatocytes had pyknotic nuclei and intracytoplasmic vacuolation, dilation of bile ducts, hyalinization and thickening of the arterial wall and infiltration of inflammatory cells. Co administration of hesperidin with diazinon-treated rats revealed improvement indicated by congestion and dilation of the central vein and sinusoids and the majority of the hepatocytes were polyhedral with acidophilic cytoplasm and rounded vesicular nuclei. Aboraya et al. (2022) [2] observed Cisplatin treated rats showed local extensive necrosis of hepatocytes, congestion of the sinusoids while hesperidin + cisplatin treated rats showed mild focal necrosis of the hepatocytes.

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