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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 779-784

Preventive effect of ethanolic extract of cactus (Opuntia ficus-indica) cladodes on methotrexate-induced oxidative damage of the small intestine in Wistar rats


1 Research Unit of “Valorization of Active Biomolecules”, Genie Biology Department, Higher Institute of Applied Biology Medenine, University of Gabes; Research Unit of Functional Neurophysiology and Pathology, Biology Department, Faculty of Sciences Tunis, University of Tunis El Manar, Tunis, Tunisia
2 Laboratory of Research on Histopathology, Histology Department, Faculty of Medicine Sfax, University Sfax, Tunis, Tunisia
3 Research Unit of “Valorization of Active Biomolecules”, Genie Biology Department, Higher Institute of Applied Biology Medenine, University of Gabes; Research Unit of Macromolecular Biochemistry and Genetics, Biology Department, Faculty of Sciences, Gafsa, University of Gafsa, Tunis, Tunisia
4 Research Unit of Functional Neurophysiology and Pathology, Biology Department, Faculty of Sciences Tunis, University of Tunis El Manar, Tunis, Tunisia

Date of Web Publication24-Sep-2018

Correspondence Address:
Lazhar Zourgui
Research Unit of “Valorization of Active Biomolecules”, Higher Institute of Applied Biology Medenine, Route El Jorf, P.O. Box 522, 4119 Medenine
Tunisia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.174555

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 > Abstract 


Context: Methotrexate (MTX) is a cytotoxic chemotherapeutic element for various inflammatory diseases. The cytotoxic effect of MTX is also seen in normal tissues having a high proliferation rate including gastrointestinal and bone marrow.
Aims: The aim of this study was to find out whether oxidative damage could be relevant for MTX-induced toxicity in vivo using Wistar rats and to investigate the preventive potential of cactus cladodes.
Materials and Methods: Adult and healthy male Wistar rats (200–250 g) were pretreated by ethanol fraction of cactus cladodes. Following a single dose of MTX (20 mg/kg), either vehicle (saline) or ethanolic (400 mg/kg) was administered intraperitoneally. All animals were killed 24 h after the intraperitoneal injection of MTX. Small intestine samples were collected for malondialdehyde (MDA) level, protein carbonyl generation, and peroxidase and catalase (CAT) activity measurement. The small intestine was also collected for histopathology analysis.
Statistical Analysis Used: Each experiment was conducted in triplicate separately. Values were presented as a mean ± standard deviation. Differences were considered significant at P < 0.05.
Results: Our results showed that MTX-induced significant alterations in oxidative stress markers noticed in the form of intestinal tissues damage, MDA level increased and protein carbonyls generation. CAT and peroxidase activities decreased with MTX administration. The combined treatment of MTX with cactus extracts showed a reduction of MTX-induced oxidative damage.
Conclusions: It could be concluded that cactus cladodes extract was effective in protecting the small intestine against MTX-induced damage.

Keywords: Cactus cladodes, catalase activity, malondialdehyde level induction, methotrexate, oxidative stress, peroxidase, proteins carbonyls


How to cite this article:
Akacha A, Rebai T, Zourgui L, Amri M. Preventive effect of ethanolic extract of cactus (Opuntia ficus-indica) cladodes on methotrexate-induced oxidative damage of the small intestine in Wistar rats. J Can Res Ther 2018;14, Suppl S3:779-84

How to cite this URL:
Akacha A, Rebai T, Zourgui L, Amri M. Preventive effect of ethanolic extract of cactus (Opuntia ficus-indica) cladodes on methotrexate-induced oxidative damage of the small intestine in Wistar rats. J Can Res Ther [serial online] 2018 [cited 2020 Oct 28];14:779-84. Available from: https://www.cancerjournal.net/text.asp?2018/14/10/779/174555




 > Introduction Top


Methotrexate (MTX) is a cytotoxic chemotherapeutic agent used for leukemia and other malignancies.[1],[2] However, its effectiveness is often limited by severe side effects and toxic conditions.[3] The chemical and morphological changes in the small intestine may possibly be triggered by crypt cells damage.[4],[5],[6] Recently, special interest has been focused on the cactus to Opuntia species. Indeed, prickly pear fruits are recommended for their beneficial and therapeutic properties.[7],[8] In fact, several studies have reported that both cactus its efficiency in the treatment of several diseases:[9],[10],[11] Cactus extract exhibit antitumoral,[12] antiviral,[13] anti-inflammatory,[14] antioxidant effects,[15],[16],[17] in addition to anti-hyperlipidemic properties[18] and an analgesic action.[19] These data have made cactus pear fruits and cladodes perfect candidates for cytoprotective investigations.


 > Materials and Methods Top


Chemicals

MTX, thiobarbituric acid (TBA); trichloroacetic acid (TCA); and butyl hydroxyl toluene (BHT) were obtained from Sigma Chemical Co. (St. Louis, MO, USA), 2,4-dinitro-phenylhydrazine (2,4-DNPH) and guanidine were purchased from Prolabo (France).

Preparation of the extracts of cactus cladodes

Young cactus cladodes of Opuntia ficus-indica (2–3 weeks of age) collected from the local area were washed with water, chopped into small pieces, and dried. Air-dried cladodes were ground to fine powder and successively extracted with solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, ethanol, and then methanol). Thus, 20 g of cladodes powder were placed in hexane (200 ml) for 18 h under frequent agitation at ambient pressure and temperature. The mixture was filtered using Whatman paper (GF/A, 110 mm) and the experience was repeated twice. The solvent was evaporated using a rotary evaporator under vacuum at 35°C. Then, the firstly extracted powder was extracted again with dichloromethane under the same conditions as with hexane. The same procedure was applied for the following solvents: ethyl acetate, ethanol, and methanol.

Animals treatment

Adult and healthy male albinos rats (200–250 g) obtained from an Animal Breeding Centre (SEXAL St. Doulchard, France following the agreement of the Ethics Committee named National Committee of Medical Ethics CNEM, BP 74 - Pasteur Institute, Tunis 1002, Tunisia) were used in this study. The animals were kept 1 week for acclimatization under constant conditions of temperature and a 12 h light/dark cycle. Animals had free access to standard granulated chow and drinking water. Following a single dose of MTX (20 mg/kg) given intraperitoneally, either vehicle (saline) or ethanol fraction of cactus cladode (400 mg/kg) was administered intraperitoneally. Treatment was continued daily for 10 consecutive days. In control rats, following a single dose of saline injection, either saline or ethanol fraction of cactus cladode was administered for 10 days. Each group consisted of six rats. After treatment, animals were sacrificed by decapitation. Blood and small intestine were dissected out.

Preparation of small intestine extract

The small intestine was homogenized with a Potter (glass-Teflon) in the presence of 10 mM Tris-HCl, pH 7.4 at 4°C and centrifuged at 4000 rpm for 30 min at 4°C. The supernatant was collected for analysis, and the protein concentration was determined in the intestinal extract using Protein BioRad assay.[20]

Evaluation of lipid peroxidation status

Lipid peroxidation was determined indirectly by measuring the production of malondialdehyde (MDA) level in the intestinal extracts following the method of Buege and Aust.[21] Briefly, 200 μl of intestinal extracts were mixed with 150 μl of TBS (Tris 50 mM and NaCl 150 mM, pH 7.4) and 250 μl TCA-BHT (20% TCA and BHT 1%). The mixture was vigorously vortexed and centrifuged at 1500 rpm for 10 min. A volume of 400μl of the supernatant was added to HCl 0.6 N and 320 μl Tris-TBA (Tris 26 mM and TBA 120 mM); the content was mixed and incubated for 10 min at 80°C. Absorbance was measured at 535 nm. The optic density corresponding to the complex formed with the TBA-MDA was proportional to MDA concentration of lipid peroxide. The concentration of μmol of MDA/mg of proteins is calculated from the absorbance at 530 nm using the MDA molar extinction coefficient of MDA 1.56 × 105 M-1 cm-1.

Protein carbonyl assay

Protein carbonyls content was determined as described by Mercier et al.[22] in intestinal homogenates by measuring the carbonyl groups reactivity with 2,4-DNPH. Thus, 200 μl of small intestine supernatant were placed in two glass tubes. A volume 800 μl of 10 mM DNPH in 2.5 M HCl was added. Tubes were left in the dark for 1 h incubation at room temperature. Samples were vortexed every 15 min. Then, 1 ml of 20% TCA was added to samples, and the tubes were left in an ice bucket for 10 min and centrifuged for 5 min in a tabletop centrifuge to collect the protein precipitates, and the supernatants were discarded. Another wash was then performed using 1 ml of 10% TCA, and protein pellets were broken mechanically with the aid of a glass rod. Finally, the pellets were washed with 1 ml of ethanol-ethyl acetate (v/v) to remove the free DNPH. The final precipitates were dissolved in 500 μl of guanidine hydrochloride 6 M and left for 10 min at 37°C with general vortex mixing. Any insoluble materials were removed by additional centrifugation. Protein carbonyls concentration was determined from the absorbance at 370 nm, applying the molar extinction coefficient of 22.0/mM/cm. A range of moles of carbonyls per ml is usually obtained for most proteins and is related to the protein content in the pellets.

Catalase activity determination

Catalase (CAT) activity was measured in the intestinal extract at 240 nm, 25°C according to Clairbone.[23] Briefly, 20 μl of the extract was added to the quartz cube containing 780 μl phosphate buffer and 200 μl of H2O2 0.5 M. CAT activity was calculated using the molar extinction coefficient (0.04/mM/cm). The results were expressed as μmol of H2O2/min/mg of proteins.

Peroxidase activity determination

Peroxidase activity was measured at 25°C using guaiacol as hydrogen donor.[24] The reaction mixture contained 9 mM guaiacol, 19 mM H2O2 in 50 mM phosphate buffer pH 7, and 50 μl of enzyme extract in 1 ml final volume. The reaction was initiated by the addition of H2O2 and monitored by measuring the increase in absorbance at 470 nm. Peroxidase activity was expressed in nmol of guaiacol oxidized per min with a molecular extinction coefficient of 26.2/mM for calculation.

Histopathological analysis

The animals were killed by decapitation after urethane anesthesia. Three tissue samples of the small intestine were cut-off at a distance of 5 cm from the proximal end of the small intestine of each animal, fixed with 10% neutral formalin, embedded in paraffin and cut with a microtome set at a thickness of 5–6 μm. The tissue sections were stained with hematoxylin and eosin for histopathological analysis and examined with a light microscope.

Statistical analysis

Each experiment was conducted in triplicate separately. Values were presented as a mean ± standard deviation. Differences were considered significant at P < 0.05.


 > Results Top


Lipid peroxidation induction

Results of MTX effect alone and together with ethanol fraction of cactus cladode on lipid peroxidation induction in the intestinal extract as determined by MDA level are shown in [Figure 1]. A volume of 20 mg/kg b.w. of MTX-induced a significant increase in MDA formation as compared to control groups. Interestingly, when animals were treated with cladodes extract (400 mg/kg b.w.), 10 days prior to MTX treatment, a sharp decrease in MDA level was noticed in tissues. MDA level was found to be similar to that of control groups (ethanol fraction of cactus cladode and saline group).
Figure 1: Lipid peroxidation as determined by malondialdehyde level in the small intestine of Wistar rat. *P < 0.05 compared with saline treated control group

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Protein carbonyl assay

Protein carbonyls formation, indicating the severe protein oxidation, was assayed in the small intestine tissue homogenate, and results are illustrated in [Figure 2]. MTX-induced protein carbonyls formation in the small intestine compared to control groups. Ethanol fraction of cactus cladodes remarkably decreased protein carbonyls formation induced by a fixed dose of MTX (20 mg/kg b.w.).
Figure 2: Concentrations of protein carbonyls in the small intestine of treated rats. *P ≤ 0.05 compared with saline-treated control group, #P ≤ 0.05 compared with methotrexate-cactus-treated group

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Catalase activity

[Figure 3] illustrates the effect of MTX and cladodes extract on CAT activity. MTX-induced a marked increase in CAT activity in the intestinal extract. When animals were given cactus ethanol fraction of cactus cladodes, 8 days prior to MTX treatment a striking decrease of this activity in tissue was noticed.
Figure 3: Catalase enzyme activity in rat intestine. *P ≤ 0.05 compared with saline treated control group, #P ≤ 0.05 compared with methotrexate-treated group

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Peroxidase activity determination

MTX treatment significantly decreased glutathione peroxidase level in the intestinal tissue. However, the supplementation of ethanol fraction of cactus cladodes to MTX-treated rats significantly decreased this antioxidant level in the tissue [Figure 4].
Figure 4: Concentrations of peroxidase activity in the small intestine. *P ≤ 0.05 compared with saline-treated control group, #P ≤ 0.05 compared with methotrexate-cactus-treated group

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Weight loss

The weight of the animals in groups was compared; there was no significant change in the cactus-treated and control groups. When the body weight in the other group was compared, it was lower in the MTX-treated group than the cactus group (P < 0.05) [Figure 5].
Figure 5: Gain in body weight of rats. *P ≤ 0.05 compared with saline-treated control group, #P ≤ 0.05 compared with methotrexate-cactus-treated group

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Histological studies

MTX treatment induced serious damage to the intestine. The results revealed that MTX-induced small intestinal injury was characterized by villous shortening, variable fusion rates, and epithelial atrophy [Figure 6]a,[Figure 6]b,[Figure 6]c. Clear improvement in the intestine was noticed when the rats were treated with MTX and the ethanol fraction of cactus cladode. The histopathological findings of our study revealed that ethanol fraction ameliorated mucosal destruction and preserved the intestinal epithelium morphology [Figure 6]d.
Figure 6: Histological changes of the small intestine. (a) Treated-cactus group. (b) Control group. (c) Methotrexate - group: Villous shortening (black arrow), variable degrees of fusion (white arrow), epithelial atrophy. These structural alterations of the intestinal mucosa (cell loss and altered crypt integrity) suggest that methotrexate induces pathomorphology, dysfunctions, and structural changes in the small intestine (either change in subcellular, cellular, and histological structure). (d) Methotrexate + cactus group

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 > Discussion Top


MTX, a widely used drug in antimetabolite cancer therapy or in various forms of arthritis, may have seriously unpredictable side effects causing significant clinical problems. Cyclical high doses of MTX used for leukemia[25] or severe psoriasis have been associated with organ toxicity including hepatic fibrosis, cirrhosis, and renal failure.[26] Therefore, the cytotoxic effect of MTX does not select cancer cells, but it also affects normal tissues having a high proliferation rate, including the hematopoietic cells of the bone marrow and the actively dividing cells of the gut mucosa.[23] Enterocolitis used by intestinal damage is one of the most frequent and severe side effects of MTX for which no effective preventive method been defined so far.[27] This can lead to forcing reduction of chemotherapy intensity, thereby potentially reducing the efficacy of anti-cancer treatment.[28]

Recent advances in medicine have demonstrated that oxygen radicals and hydrogen peroxidase have been associated with MTX side effects.[29] Free radicals trigger cell damage through binding to cellular macromolecules, particularly membrane lipids and polyunsaturated fatty acids in the endoplasmic reticulum.[30] Hence, lipid peroxidation may be an important cause of destruction and damage to cell membranes and can contribute to the development of MTX-mediated tissue damage. It is increasingly apparent that lipid hydroperoxide may play an important role in mediating cellular and molecular events in degenerative pathophysiological processes that lead to intestinal disorders.[31] To further assess MTX-induced oxidative damage to Wistar rats, protein carbonyls generation was monitored. Protein carbonylation is a sign of irreversible oxidative damage, often leading to a loss of protein function, which may have lasting detrimental effects on cells and tissues.[32],[33] Our results clearly showed that MTX-induced a marked increase in protein carbonyls generation in small intestine extracts which was totally canceled with ethanol fraction of cactus cladodes in both organs [Figure 3]. CAT and peroxidase activities were also studied in this paper. In our study, we have shown that MTX treatment significantly reduced antioxidant enzyme capacity. Decreased CAT activities were probably due to the decrease in their synthesis.[34] Our results are in accordance with the study of Vardi et al.[35] who reported that MTX caused a significant decrease in antioxidant enzyme parameters (CAT) in the rat's small intestine. On the other hand, Uzar et al.[36] reported that MTX caused a significant increase in the antioxidant enzyme parameter (CAT) in the rat's small intestine and cerebellum. When cells are exposed to oxidative stress, they increase the activity and expression of antioxidant enzymes as a compensatory mechanism to be protected from the moderate level damage of toxic reactants inducing a rise in antioxidant enzymes.[37] The weight loss documented in the present experiments and noted by others[38] emphasizes the importance of evaluating nutritional factors in studying the response of the intestine to this drug.

Gastrointestinal toxicity is one of the most serious side effects of MTX treatment. It has been known that MTX causes severe damage to the intestinal mucosa.[39],[40] It increases the incidence of apoptosis in the area of rapidly dividing cells, including gastrointestinal tract mucosa.[41] The proliferation of small intestinal epithelial cells occurs in the crypts. The crypt cells, which rapidly regenerate, migrate to the villus tip, and replacement of intestinal epithelium is complete in about 2 days in rats and about 3 days in humans.[42],[43] The histopathological changes in the small intestine occurring after MTX administration may be triggered by damage to the crypt cells.[40],[44],[45] The obtained results revealed that MTX-induced small intestinal injury is characterized by villous shortening, variable fusion degrees, and epithelial atrophy. Our results are similar to other studies in which MTX was reported to cause severe damage to the small intestine.[4],[46] The results of this experimental study showed that cactus administration protected against the oxidative and morphological intestinal damage caused by MTX-treatment.

Over the last decades, oxidative stress has been shown to be a major component of several biological and pathological processes like aging, inflammation, carcinogenesis, wound healing activity,[12] and several other diseases including Parkinson's and Huntington's.[47],[48] Interestingly, the study of natural products may counteract the detrimental effects of oxidative stress and prevent multiple human diseases. In this line, different types of fruits and vegetables have been re-evaluated and recognized as valuable sources of nutraceuticals. Cactus should attract great interest because of its nutritional and antioxidant properties. According to several studies, cactus pear (Opuntia ssp.) yield high values of important nutrients and exhibit antioxidant functions.[11],[19],[48] Our study showed that ethanol fraction of cactus cladodes has ameliorated effect against MTX-induced oxidative damage. This protection was demonstrated in all tested oxidative damage endpoints. The mechanism of this protection by cactus cladodes was probably related to the decrease in oxidative stress caused by MTX. Moreover, it is certainly associated with the presence of several antioxidants such as ascorbic acid, Vitamin E, carotenoids, reduced glutathione, flavonoids, and phenolic acids actually detected in the fruits and vegetables of various cactus.[19],[48],[49] In addition, more recently, significant antioxidant properties of the most frequent cactus betalains have been identified, and numerous in vitro studies have demonstrated their ability to neutralize reactive oxygen species.[50],[51],[52] Lee et al.[53] investigated the antioxidant activity of cactus cladodes and concluded that this antioxidant property was due to several compounds, particularly flavonoids (quercetin, myricetin) and vitamins. Our results are in accordance with other published studies, which underlined the relevant preventive potential of cactus extracts.[54],[55],[56],[57]


 > Conclusion Top


It could be concluded that cactus cladodes extract was effective in protecting the small intestine against MTX-induced damage, probably acting by promoting the antioxidant defence systems.

Financial support and sponsorship

This research was funded by the Tunisian Ministry of Scientific Research and Technology through the Research Unit of Macromolecular Biochemistry and Genetics (BMG), Faculty of Sciences of Gafsa. (cmcu 10G0807) and the Research Unit of “Valorization of Actives Biomolecules”(VBA), Higher Institute of Applied Biology Medenine University of Gabe, Tunisia.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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