The authors have declared that no competing interests exist.
Methotrexate (MTX) is an anti-metabolite in cancer chemotherapy and is associated with various toxicities assigned to inflammation and oxidative stress. The present study was undertaken to corroborate the therapeutic effects of bone marrow mesenchymal stem cells (BM-MSCs) and adipose-derived mesenchymal stem cells (AD-MSCs) in MTX-induced intestinal toxicity in experimental animals as compared with dexamethasone (Dex). Rats were divided into five groups: I-Normal control group, II- MTX (14 mg/kg, as a single dose/week for 2 weeks), III & IV- BM-MSCs & AD-MSCs (2 × 106 cells/rat, 1 week after last dose of MTX), respectively, plus V- Dex (0.5 mg/kg/ for 7 days, 1 week after last dose of MTX). MTX induced marked intestinal elevation of interleukin-6, total oxidant, and nitrite/ nitrate, caspase-3 contents and myeloperoxidase activity, along with the reduction of reduced glutathione content and catalase activity. In conclusion, the positive modulation of MTX toxicity could be attributed to the free radical scavenging, anti-inflammatory and antiapoptotic potential of BM-MSC and AD-MSCs which will possibly make them as remarkable hopeful for the treatment of intestinal injury.
Methotrexate (MTX) is an antagonist of folic acid
Nevertheless, MTX is restrained by its toxicity, including intestinal injury causes severe mucositis
Bone-marrow mesenchymal stem cells (BM-MSCs) are fibroblast-like, pluripotent adult stem cells. BM-MSCs can adhere to plastic and grow readily in the laboratory producing other types of cells, including new stem cells identical to mother cells. MSCs have been shown to have immunomodulatory capabilities due to the secretion of several growth factors
Adipose-derived mesenchymal stem cells (AD-MSCs) seem to be a promising regenerative therapeutic agent due to the minimally invasive approach of their harvest and multi-lineage differentiation potential. The harvested adipose tissues are further digested to extract stromal vascular fraction, which is cultured, and the anchorage-dependent cells are isolated in order to characterize their stemness, surface markers, and multi-differentiation potential
Dexamethasone (Dex) is a well-known steroid agent that regulates inflammation by downregulates expression of anti-inflammatory mediators such as TNF-α, IL-6 by decreasing the mRNA stability of these cytokine
The present study aimed to study the therapeutic effects of BM-MSCs and AD-MSCs in MTX-induced intestinal injury in rats as compared with Dex.
Male Wistar albino rats, weighing 150-200 g, obtained from the animal house of the National Organization for Drug Control and Research (NODCAR, Cairo, Egypt) were used in the present study. Animals were housed for at least one week in the laboratory room prior to testing under controlled environmental conditions; constant temperature (25 ± 2 ͦ C), humidity (60 ± 10%), and alternating 12 h light/dark cycles. Standard pellet diet and water was allowed
Forty rats were randomly allocated into 5 groups. Group 1 received normal saline orally and served as control group. The rest of animals received MTX (Orion Pharma, Finland) orally in a dose 14 mg/kg/week for 2 consecutive weeks
Bone marrow was harvested by flushing the tibiae and femurs of 6 weeks old male white albino rats, with Dulbecco’s modified Eagle’s medium (DMEM,GIBCO/BRL) supplemented with 10% fetal bovine medium (GIBCO/BRL). Nucleated cells were isolated with a density gradient Ficoll/Paque (Pharmacia) and resuspended in complete culture medium supplemented with 1% penicillin-streptomycin (GIBCO/BRL).
Cells were incubated at 37°C in 5% humidified CO2 for 12-14 days as primary culture or upon formation of large colonies. When large colonies developed (80-90% confluence), cultures were washed twice with phosphate buffer saline (PBS) and cells were trypsinized with 0.25% trypsin in 1mM EDTA (GIBCO/BRL) for 5 minutes at 37°C
Adipose tissue was excised from the inguinal fat pad (i.e., subcutaneous) under complete aseptic condition. Then adipose tissue underwent enzymatic digestion by 0.075% collagenase II (sigma) in Hank's Balanced Salt Solution for 60 minutes at 37°C with shaking. Digested tissue was filtered and centrifuged, and erythrocytes were removed by treatment with erythrocyte lysis buffer. The remaining cells were transferred to tissue culture flasks with Dulbecco Modified Eagle Medium (DMEM; Gibco/Invitrogen Corp., Grand Island, NY) plus supplement F12 (Gibco/Invitrogen). After an attachment period of 24 hours, non-adherent cells were removed by a phosphate buffered solution (PBS; Gibco/Invitrogen) wash. Attached cells were cultured in DMEM/F12 media, supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen), 0.1 µM dexamethasone (Sigma-Aldrich), streptomycin (Gibco/Invitrogen), and 1.25 mg/L amphotericin B (Gibco/Invitrogen), and expanded in vitro until passage three
Mesenchymal stem cells in culture were characterized by their adhesiveness and fusiform shape and by flow cytometric detection of cluster of differentiation (CD) 29, one of surface marker of rat mesenchymal stem cell
Determination of intestinal MPO activity was done using a kinetic colorimetric method described by Bradley
MPO was calculated in terms of (U/g) =
where; ΔA= change of the sample absorbance over 2 minutes.
Besides, interleukien-6 (IL-6) was determined quantitatively by ELISA using a test reagent kit (SinoGeneClon Biotech Co., Ltd; China) according to the manufacturer’s instructions. Intestinal IL-6 contents were estimated in the tested samples and determined as pg/g tissue from the standard curve constructed.
The total oxidant (TO) content of samples was determined as previously described in
Activity of caspase3 was determined using the caspase-3 colorimetric assay Kit (R&D systems, a bio-techne brand, Minneapolis, USA) according to the instructions of the manufacturer. Actually, cells that are suspected to or have been induced to undergo apoptosis are first lysed to collect their intracellular contents. The cell lysate can then be tested for protease activity by the addition of a caspase-specific peptide that is conjugated to the color reporter molecule p nitroaniline (pNA). The cleavage of the peptide by the caspase releases the chromophore pNA, which can be quantitated spectrophotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate is directly proportional to the color reaction
The obtained results were presented as mean ± standard error of the mean. Comparisons between means were carried out using One-Way ANOVA followed by Tukey-Kramer multiple comparisons test. Statistical analysis was performed using GraphPad Prism software (version 5); a probability level of less than 0.05 was accepted as statistically significant.
Isolated and cultured undifferentiated MSCs were typical of adherent spindle and fibroblast-like morphology and reached 70-80% confluence at 2 weeks culture (
The effects of BM-MSCs and AD-MSCs on inflammatory markers as compared with Dex in MTX-induced intestinal injury in rats
Administration of MTX produced a significant increase in intestinal content of MPO (0.755 ± 0.047, to 3 folds) as well as IL-6 (115 ± 3.59, to 4 folds) as compared with the control group (0.186 ± 0.0177) and (23.7 ± 1.99), respectively (
The effects of BM-MSCs and AD-MSCs on oxidative stress markers as compared with Dex in MTX-induced intestinal injury in rats
Administration of MTX produced a significant increase in intestinal content of proxidant, TO, (148 ± 7.17, to 3 folds) plus NOx (358 ± 18.7, to 1 fold) accompanied with significant reduction in the enzymatic antioxidant activity of CAT (0.199 ± 0.008, by 70%) and non-enzymatic antioxidant content of GSH in the intestine (0.150 ± 0.027, by 83%) compared with the control group (39.6 ± 3.25), (173 ± 10.4), (0.641 ± 0.046), and (0.895 ± 0.137), respectively.
In contrast, either treatment with BM-MSCs, AD-MSCs or Dex decreased intestinal contents of TO (70.5 ± 6.95, by 52%), (74.1 ± 5.39, by 50%), and (63.2 ± 2.61, by 57%), respectively, NOx (125 ± 13.1, by 65%), (166 ± 10.4, by 54%), and (110 ± 10.6, by 70%), respectively, accompanied with significant increase intestinal CAT activity (0.510 ± 0.0263, by 150%) , (0.339 ± 0.009, by 70%), (0.278 ± 0.01, by 40%), respectively, as well as GSH content (0.994 ± 0.127, to 6 folds), (0.651 ± 0.01, to 3 folds), and (0.855 ± 0.026, to 5 folds), respectively, compared to MTX group (
The effects of BM-MSCs and AD-MSCs on (apoptotic marker) caspase-3 as compared with Dex in MTX-induced intestinal injury in rats
Administration of MTX produced a significant increase in apoptotic content of Caspase-3 (2.33 ± 0.211, to 1 fold) as compared with the control group (
This study investigated the anti-apoptotic and antioxidant effects of two different types of mesenchymal stem cells (MSCs), BM-MSCs and AD-MSCs against MTX-induced intestinal injury (mucositis) as compared to Dex. The main mechanism behind the development of mucositis was considered to be the result of direct cytotoxic effects of chemotherapy or radiotherapy on the basal cells of the epithelium because of its high cell turnover rate
The present study confirmed that as MTX induced elevation in intestinal TO, and NOx contents, MPO, IL-6 and apoptotic marker caspase-3, in addition; it caused depletion of antioxidant defense GSH content and CAT activity as compared to control animals. This result in accordance with studies of Kolli et al. and Moghadam et al
MTX is well established that pro-inflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis alpha (TNF-a) and interleukin-6 (IL-6) are potent inducers of iNOS in a wide variety of cells types, with consequent production of NO
This work revealed a marked antioxidant potential of BM-MSCs and AD-MSCs as shown by replenishing intestinal GSH content and CAT activity associated with hampering MPO and NO contents as compared to the MTX group. The antioxidant effects of stem cells were formerly demonstrated in various organs injury models
Studies in intestinal injury in rodent models have demonstrated that MSCs can beneficially produce paracrine growth factors and anti-inflammatory cytokines to reduce intestinal injury in rats
In addition, the present study reported a prominent antioxidant as well as antiapoptotic effects for Dex. that probably exhibited elevated tissue GSH content and CAT activity in the settings of reduced MPO, TO, IL-6. Increasing of CAT activity may be by an enzymatic induction mechanism
We have demonstrated that treatment with MTX induced intestinal epithelial damage in wild type rats. BM-MSCs and AD-MSCs reduced oxidative stress markers as compared with Dex in MTX-induced intestinal injury in rats. These findings suggest that the curative effects of BM-MSCs and AD-MSCs against MTX may rely on their anti-apoptotic function.
The authors are grateful to Dr. Laila Ahmed Rashed, Professor of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, for her efforts in isolation of mesenchymal stem cells.