Evaluation of the Anti-Oxidative Effects of Monocyte Cells Treated with Bone Marrow Mesenchymal Stem Cells Supernatant on Experimental Colitis in BALB/c Mice

1Student Research Committee, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 3Baqiyatallah Research Center for Gastroenterology and Liver Diseases (BRCGL), Baqiyatallah University of Medical Sciences, Tehran, Iran 4Department of Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 5Medical Nanotechnology and Tissue engineering Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 6Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran GMJ.2021;10:e2131 www.gmj.ir


Introduction
A s a chronic inflammatory bowel disease, ulcerative colitis (UC) has an unknown cause that affects the colon and rectum [1]. UC pathogenicity is proposed to result from the contribution of several factors, including genetic context, environmental and luminal factors, and mucosal immune dysregulation [2]. UC has become an encumbrance globally due to a high prevalence in developed countries and a considerable rise in frequency in developing countries [3]. Achieving ideal disease management warrants developing innovative drugs and improving the therapeutic approach by implementing personalized medicine [4]. The bone marrow and most other tissues, including adipose tissue and umbilical cord blood, are normally rich in mesenchymal stem cells (MSCs), dermal tissue, and peri-endothelial areas [5]. During the last few decades, MSCs have been among the broadly discovered multipotent stem cells. Hypo-immunogenic and immunoregulatory properties of MSCs have been reported in their therapeutic impacts on various inflammatory diseases and kidney transplantation [6]. Prior studies established that MSCs were capable of controlling both innate and adaptive immune responses through the release of various mediators (such as immunosuppressant molecules, growth factors, exosomes, chemokines, complement components, and metabolites) upon exposure to inflammatory situations, which thereby promotes the reparation and restoration of injured tissues [7]. Similar to the MSCs, many investigations have demonstrated that the supernatant obtained from MSCs (MSCs-Sp) is therapeutically beneficial in various experimental diseases [7]. Monocytes have a significant contribution to initiating and resolving inflammatory situations [8]. Nonetheless, information is absent or scarce concerning the function of monocytes treated with MSCs-Sp in mitigating oxidative situations. Therefore, the current research aimed to examine whether the MSCs-Sp treatedmonocytes could reduce the oxidative condition of experimental colitis in BALB/c mice.

Ethical Statement
The procedure was supervised and approved by the Shahid Beheshti University of Medical Sciences ethics committee (approval code: I R . S B M U . R E T E C H . R E C . 1 3 9 8 . 2 5 9 ) . Animal treatment and general guidelines for animal handling were followed by the legislation of the Iranian Ministry of Health and Medical Education. Besides, the least number of mice was assigned to every group with a statistical significance.

Isolation of MSCs and Supernatant Preparation
In short, the femurs and tibias from the sacrificial BALB/c mice were extracted. The cells were rinsed twice and then developed to a concentration of 0.3-0.4 in the Dulbecco Modified Eagle medium (DMEM;Gibo, USA) in 25 cm2 tissue-cropping flake at a concentration of 0.3-0.4 per 106 cells/ cm2 enhanced by 15 `% fetal bovine serum (FBS;Gibco, USA) in a 5% CO2-enriched moistened incubator at 37°C. On the fourth day, unattached cells were washed, and full media were added two days a week to the attachments. After 80% confluence, the bone marrow-MSCs were separated with slight trypsinization using trypsin/EDTA 0.25%(Dacell, Iran), followed by counting and re-plating in a ratio of 1:3 (about 1 × 106 cells/25-cm2 flask). The cell lines have been pushed up to the third generation. Next, the MSCs were exposed to a medium without FBS for 24 hours. The supernatants were extracted and centrifuged for 10 minutes at 300 g to remove cell remnants. Afterward, a mixture was prepared from the harvested supernatants from every BALB/c mice, passed through a 0.2-μm filter orderly, and then kept frozen at -80ºC before tests [9].

Monocyte Isolation and Treatment
Blood samples were isolated from acutely anesthetized animals using a cardiac puncture in Roswell Park Memorial  [9].

Grouping
Forty male BALB/c mice have abstained for 24 hours and then instilled 4% acetic acid (1 mL, Merk, Germany) into the rectal to induce experimental colitis. After the random assignment of mice to five equivalent isolated groups, the group I was excluded as a negative control without colitis induction.
Other BALB/c mice groups received acetic acid for UC induction, which were treated differently after ten days. Groups II, III, IV, and V were treated with normal saline (100 µL, intravenous [IV] injection) as normal colitis, treated-monocytes (105 cells/100 µL/ IV injection), non-treated-monocyte (105 cells/100 µL), and 30 mg/kg of oral mesalazine, respectively. The entire mice underwent euthanasia by CO2 inhalation, and cervical dislocation was performed on all mice and was sampled (blood sampling from hearts and isolation of colon tissue) on day 20.

Evaluation of Myeloperoxidase (MPO) Levels
After adding 2 mL of phosphate buffer comprising 0.5% hexadecyl trimethyl ammonium bromide (HTAB;Sigma-Aldrich, USA) to 100 mg of colon tissue, homogenization on ice was performed six times for 45 seconds, accompanied by sonicating for 10 seconds and freezing with liquid nitrogen three times, centrifugation at 3000 rpm and 4°C for 30 minutes obtained the supernatant. After decanting 2.9 mL of phosphate buffer comprising o-Dianisidine and 0.005% hydrogen peroxide to 0.1 mL of supernatant, 0.1 mL of 1.2 M hydrogen chloride was poured into a funnel until it appeared orange in color. The absorbance of the specimens was determined using the traditional package diagram at 460 nm using an ELISA reader (Biotek, USA).

Measurement of Colon Tissue Nitric Oxide (NO) Level
NO concentrations produced by homogenizing colon tissue were determined in supernatant cells with the Griess reaction. The residues of microscopic cells were discarded via centrifugation of the homogenized colon tissue specimens. The specimens were distilled by PBS contains 0.5% HTAB with a ratio of 2:1, followed by decanting 100 mL of individual specimens into the 96-wells plates triplicated according to the manufacturer protocol (Karmania pars gene, Iran) along with blank and standard samples (Nos. KPG-NO). The absorbance of the sample at 460 nm was measured using a UV-visible spectrophotometer (Eppendorf, Germany) [10].

Malondialdehyde
(MDA) Level Measurement Briefly, tissue (500 mg) was homogenized in 5 mL of 1.15% cold potassium chloride, and then 3 mL of 1% phosphoric acid and 1 mL of 0.6% thiobarbituric acid (Sigma-Aldrich, USA) were added to 500l of homogenate with sufficient shaking. Then it was heated indirectly to 100o C for 45 minutes, centrifuged at 10,000 rpm for 10 minutes, and the supernatant's absorbance at 532 nm was determined using a UV-visible spectrophotometer (Eppendorf, Germany) [10].

Histological Study
Samples were stained with Hematoxylin and Eosin (H&E; Sigma-Aldrich, USA ) stain for inspection under a light microscope of GMJ.2021;10:e2131 www.gmj.ir the histopathologic pattern. The H&E stain is one of the most often encountered tissue stains in histology. It is staining cell nuclei blue, extracellular matrix, and cytoplasm pink, with other structures exhibiting varying tones, hues, and variations of these colors. Thus, a pathologist can clearly distinguish between a cell's nuclear and cytoplasmic components. The staining findings were graded on a scale of 0 to 3. The ratings were as follows: 0=normal; 1=mucosal erythema alone; 2=moderate mucosal edema, minor bleeding, or slight erosion; and 3=extreme ulceration, erosions, edema, and necrosis of the tissue. Finally, the magnitude of the lesions was determined using the numbers derived from each of the parameters collected and analyzed.

Statistical Analysis
The mean and standard deviation (SD) of the whole set of data were recorded. The different findings from the groups were subjected to statistically meaningful analyses using analysis of variance (ANOVA) and the Student-Newman-Keuls test with a statistical significance level of P<0.05 using SPSS21(IBM, USA) software. After scoring tissue (Figure-4), the pessimistic control group received a score of 0 (the lowest possible score), and the control colitis group received a score of 3 (the highest score possible). The groups receiving treated monocytes and mesalazine demonstrated that the epithelium was not entirely destroyed; hence, they obtained a score of 2 for epithelium loss. Additionally, the community obtaining treated monocytes and mesalazine had a significantly greater reduction in acetic acid-induced ulcers (P<0.05). Also, the non-treated monocyte group had less colon damage than the control colitis group.  GMJ.2021;10:e2131 www.gmj.ir

Discussion
As a chronic, incapacitating, and persistent illness, the characteristic of UC is a lifetime therapy and detrimentally influences patients' quality of life. For some decades, UC has been treated using anti-inflammatory medicines (e.g., 5-aminosalicylic acid and glucocorticoids) [11]. Although, a variety of treatments (e.g., stem cell therapy) have been offered to promote our understanding of the pathologic mechanisms of UC exactly. There are reports on the regenerative and immunomodulatory activities of MSCs in UC models [12]. Our research focused on the anti-oxidant impacts of monocyte cells subjected to treatment by bone marrow MSCs-Sp experimental colitis in BALB/c mice. Through delivering treated monocytes to BALB/c mice, we found an effective reduction in colonic oxidative stress.
Colitis is caused experimentally in animal models with several compounds, including 2, 4, 6-trinitrobenzene sulfonic acid, disuccinimidyl suberate, oxazolone, and acetic acid [13]. Acetic acid colitis induction is used as an experimental colitis model since it causes acute inflammation and colonic ulceration like those seen in patients. It is described by bloody diarrhea, weight loss, inflammatory cell invasion, and epithelial cell loss on the surface [14]. Moreover, oxidative stress has a major contribution to the pathogenicity of UC. MPO, NO, and MDA are essential components of cell protection from oxidative injury [15]. Elevated MPO, NO, and MDA activities were found in the colonic homogenate of BALB/c mice with UC compared to the control animals. Also, the use of monocytes treated with bone marrow MSCs-Sp resulted in significantly declined MPO, NO, and MDA activities as opposed to normal colitis.
GMJ.2021;10:e2131 www.gmj.ir 7 However, there are unknown modes of action behind the immune inhibition by MSCs. Broadly available evidence indicates that MSCs inhibit the multiplication of T-cells once stimulated allogeneically or mitogenically [16]. It has been proposed that soluble factors such as indoleamine 2,3-dioxygenase, prostaglandin E2, transforming growth factor-1, interleukin (IL)-6, and heme oxygenase 1 are responsible for this effect [16]. MSCs have also been shown to prevent the proliferation of B cells and IL-2-stimulated natural killer cells and to facilitate the development of regulatory T cells (T reg) [17]. MSCs may also influence immune responses by inhibiting the differentiation of monocyte-derived immature dendritic cells (iDCs) [12]. IL-6 and IL-10 have been shown to suppress monocyte differentiation to iDC. Numerous studies have shown that IL-6 may contribute to MSCs' inhibitory impact on monocyte differentiation to dendritic cells [18]. Nonetheless, these data come from in vitro co-culture assays of MSCs and monocytes that do not recognize the cellular source of cytokine production. To be more precise, the function of these cytokines in suppressing MSC behavior is unknown at the moment [18]. Based on the pathogenicity of disease, neutrophils are the essential immune cells migrating into the colonic tissue after luminal instilment of acetic acid and induce inflammatory damages by the formation of oxygen and nitrogen species [19]. It was previously found that local inflammation would diminish by inserting MSCs into the inflamed tissue through suppression of MPO and NO generation, which improves tissue healing by the reduction of lipid peroxidation. The generation of reactive aldehydes (e.g., MDA) represents lipid peroxidation by oxygen and nitrogen species [20]. Melief et al. indicated that multipotent stromal cells influence monocytes to produce anti-inflammatory IL-10 through IL-6 production [21].
Esmaeili et al. reported that monocytes grown with supernatant of MSCs might potentially be an approach for cell-based treatments of mice model of asthma [9]. Accordingly, it was formerly documented that MSCs could increase the anti-oxidant capability of small bowel tissue after intestinal ischemia-reperfusion damage [22]. A leading observation in this research was elevated MDA levels in the colonic homogenates of BALB/c mice with colitis and treated monocytes' capability to reduce its levels. Here, it was confirmed that the migration of treated-monocytes into the reperfused small intestine resulted in declined oxidative stress by these cells due to the impacts of superoxide dismutase, catalase, and glutathione peroxidase, as well as reduction of the MDA levels. Also, histopathological results in parallel with the above results showed that the rate of degradation in the groups receiving mesalazine and treated monocytes were less than the normal group of colitis.

Conclusion
This study indicated that the monocytes co-cultured with bone marrow MSCs-Sp could inhibit the oxidative condition of experimental colitis. Overall, this procedure can potentially be an approach for cellbased treatments of colitis.