The role of SMER28 in increasing the life of BMSCs after exposure to oxidative stress induced by hydrogen peroxide

Bone marrow mesenchymal stem cells (BMSCs) have long been of therapeutic importance in tissue engineering and restorative medicine. Recently, introducing the autophagy inducer SMER28 as a molecule for increasing BMSCs viability has received special attention. We aimed to determine the role of SMER28 in increasing the life of BMSCs after exposure to oxidative stress induced by hydrogen peroxide. Methods Five female and adult Wistar eld samples prepared to extract BMSCs specimens. To prove the mesenchymal origin of BMSCs in the third passage, the expression of CD31, CD34, and CD106 were evaluated as the immunocytochemical markers. Trypan blue staining was performed on cells to evaluate the toxicity of H2O2 and the protective effect of SMER28.


Introduction
Due to their accessibility and development potential, bone marrow mesenchymal stem cells (BMSCs) have long been of therapeutic importance in tissue engineering and restorative medicine. Recently, it has been shown that not only BMSCs are able to participate directly in repair, but also have the capacity to migrate to distant areas of tissue damage and participate in tissue repair, either directly by differentiation or indirectly through paracrine mechanisms [1,2]. In addition, they can produce immune-regulating signals that weaken in ammatory and immune responses [3]. In fact, BMSCs have been clinically studied to treat a wide range of diseases, including bone defects, host graft disease, cardiovascular disease, autoimmune diseases, diabetes, neurological diseases, and liver and kidney diseases [4][5][6]. For this reason, these cells are of special importance, but these cells die quickly after transplantation to the lesion area due to the environment of oxidative stress or hypoxic conditions, as well as deprivation of serum [7,8]. These cells have a short lifetime after transplantation into a damaged spinal cord or ischemic area in the brain due to oxidative agents and in ammatory cells [9]. Therefore, ideas that increase the lifespan of these cells play an important role in increasing the survival of these cells after transplantation. In recent years, large-scale chemical screening has been performed to nd new drugs in the regulation of autophagy for the treatment of neurodegenerative diseases, some of which are 50729 compounds that increase and decrease the cytosolic effect of rapamycin [10]. In these large screenings, Saccharomyces cerevisiae was rst studied to nd a molecule that could inhibit the growth inhibition of rapamycin, and this study led to the identi cation of SMER molecules [11]. SMERs were initially identi ed as modulators of the effect of rapamycin on yeast growth, but eventually their effect on mammalian neuronal autophagy was also investigated [12]. SMER is actually a bromo-substituted quinazoline. In this study, the effect of this substance on increasing the life of BMSCS cells was investigated. In fact, in this study, the role of SMER28 in increasing the life of BMSCs exposed to hydrogen peroxide at different concentrations was investigated. In fact, we aimed to determine the role of SMER28 in increasing the life of BMSCs after exposure to oxidative stress induced by hydrogen peroxide.

Materials And Methods
Extraction and culture of bone marrow stromal cells In this study, 5 female and adult Wistar eld samples prepared from Pasteur Institute at the age of 6-8 weeks were used. The animals were kept in a 12-hour light and dark period in the standard conditions of the zoo of Qazvin University of Medical Sciences in accordance with the ethical rules. After separation from the lower bones of the lower limbs and washing with saline buffer phosphate, BMSCs were placed in Dulbecco modi ed Eagle's medium (Dulbecco modi ed Eagle's medium: F12 (DMEM/F12)) with fetal bovine serum (100 µg/mL), FBS 10%, penicillin (100U/mL) and streptomycin in an incubator with the condition of CO2 5%, humidity 95% and temperature of 37°C.

Immunohistochemistry assessment
To prove the mesenchymal origin of BMSCs in the third passage, the expression of CD31 (marker of endothelial cells), CD34 (marker of blood stem cells), and CD106 (marker of BMSCs) were evaluated as the immunocytochemical markers. After trypsinization, 5,000 cells were evenly spaced in each of the 6cell lamellar plate cells. Immunocytochemical steps were performed similar to the recommended protocols. The cells were immersed in 4% paraformaldehyde solution for 20 minutes. After washing with phosphate buffer, the cells were placed in Triton X 0.3% for 15 minutes. After re-washing with phosphate buffer, the cells were exposed to the initial antibody at 4 ° C for 24 hours. Primary antibodies included primary antibodies CD31, CD34, and CD106. The cells were washed with phosphate buffer and placed in a secondary antibody conjugated to FITC (1: 100; Chemicon), in which the cytoplasm is green, at room temperature for 2 hours. Propium iodine (PI) dye was used to count the cells, in which the cell nucleus turns red. Cells with a positive immune response were counted under a uorescent microscope.

Viability test
Prior to the tests, trypan blue was performed on cells to evaluate the toxicity of H2O2 and the protective effect of SMER28. The cells were divided into 1000 cells in a well of 96 cells. To evaluate the toxicity of H2O2, the durability and survival of BMSCs were measured at different doses of H2O2 (0, 50, 100, 200 and 400 µM) for 24 hours. To evaluate the protective effect of SMER28, cells were pretreated with SMER28 (5 µM) about two hours before exposure to H2O2. To evaluate cell survival, a volume of cell suspension and an equal volume of trypan blue dye are mixed and cell counts were performed using a neobar slide under a microscope. In this method, the dye penetrated into the dead cells and turned to blue color. Unstained cells represented living cells, which was obtained by counting the total number of cells and stained cells.

Results
Recognize the nature of BMSCs BMSCs had a rounded appearance after separation (Figure 1), but after 48 hours, they adhered to the bottom of the ask, and after rinsing with PBS, the oating cells that did not adhere to the bottom of the ask were washed (Figure 2). There are large populations of different stem cells in the bone marrow, such as endothelial stem cells, blood stem cells, and bone marrow stromal stem cells, which are usually washed away after 48 hours after separation and rinsing with PBS, except for BMSCs. However, to ensure the identity of the BMSCs, the cultured cells were evaluated after the third passage for super ciality and mesenchymal origin by immunocytochemical analysis of surface markers. BMSCs were negative for CD31 antibody, which is a marker of endothelial cells (Figure 3), and CD34, which is a marker of blood stem cells (Figure 4), but the cells responded positively to the CD106 antibody (marker of BMSCs) ( Figure  5). Fluorescence light is associated with the FITC-conjugated secondary antibody, which appears green.
PI dye is used to count the cells, and the nuclei of the cells emit red light.

Check the viability of BMSCs
In the viability test review, BMSCs in the third passage without H2O2 were considered as the control group. The cells were then treated with H2O2 at different concentrations for 24 hours. Viability rate after 24 hours was 88%, 75%, 48%, 38% of the control group after receiving 50, 100, 200, 400 µM of H2O2, respectively. The highest cellular death was revealed in the cell groups that received H2O2 with the doses of 200 µM and 400 µM, indicating a signi cant difference as the control group without exposing H2O2 (p < 0.001).

Investigation of SMER28 protective effect
To evaluate the protective effect of SMER28, BMSCs cells were pretreated with SMER28 at a concentration of 5 µM, two hours before exposure to H2O2. The results showed that pretreatment with SMER28 at a dose of 5 µM increased cell viability. In this regard, cell survival in medium containing SMER28 (5 µM) and H2O2 at concentrations of 50, 100, 200, 400 µM were 94%, 86%, 78%, and 43%, respectively. Compared between cells exposed to H2O2 with and without SMER28, SMER28 signi cantly increased cell viability and decreased cell death due to H2O2.

Discussion
Due to the recent discovery of these molecules, little research has been done on their effect only on PC12 cell line and Drosophila. But the same studies conducted by FLOTO et al. In 2007 show that SMERs are good candidates for increasing autophagy (13). In Alzheimer's disease, SMER 28 is one of the small molecules that cause a signi cant reduction in the accumulation of Aβ peptide and APP-CTF, and the presence of Atg5 is essential in this pathway (13). A study by RAVIKUMAR et al (14) on PC12 cell line found that four SMERs of 16, 18, 28, and 10 could be good candidates for increasing α-syn A53T cell clearance through autophagy and is able to reduce the amount of α-syn by up to 50%. In this regard, when rapamycin and 28 SMER are used together, the clearance effect of α-syn A53T is much higher than when used alone. According to the present information, the increase in SMER 28 autophagy is through a non-mTOR-dependent pathway and possibly depends on a downstream composition of the pathway. On the other hand, in the same study, the SMER s mentioned in Huntington's disease increase the autophagy of Huntington's protein. Today, the structural analogues of these SMERs have been identi ed and tested as new drug candidates for activating autophagy, and according to the results obtained, they are also effective. Due to the above, SMER28 molecule with the highest ability to induce autophagy has a high potential for further studies in the treatment of neurodegenerative diseases. Another advantage of SMER28 is that in su cient doses to increase autophagy they do not produce any apparent toxicity to the PC12 cell line (15). In a study conducted by KOUKOURAKI et al in 2018, it was revealed that rst SMER28 increased the rate of autophagy ow leading elevation of the survival rate of liver cells and thus it has a protective effect on stem cells, which is only on normal cells but not on cancer cells (106). In another study conducted by Darabi et al in 2018 (17), SMER28 has been shown to prevent the destruction of snc cells, weaken microgliosis, and improve motor function.

Conclusion
According to previous researches and studies on SMER28, in this study, its effect on increasing the viability of stem cells was investigated. The results showed that pretreatment with SMER28 at a dose of 5 µM increased cell survival. Cell survival in medium containing SMER28 (5µM) and H2O2 at concentrations of 50, 100, 200, 400 µM were 94%, 86%, 78%, and 43%, respectively. In other words, compared to cells that were not previously pretreated with SMER28 in the face of oxidative stress induction by H2O2 with the same concentrations, the percentage of viable cells in the state of pretreatment with SMER28 could be successfully increased.

RESEARCH FUNDING
This research received no speci c grant from any funding agency in the public, commercial, or not-forpro t sectors.
AUTHOR CONTRIBUTIONS SD designed the study planning, SM1 and SM3 collected the data, SM1 analyzed the data and drafted the paper. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

COMPETING INTERESTS
Authors state no con ict of interest.

INFORMED CONSENT
Informed consent was obtained from all individuals included in this study.

ETHICAL APPROVAL
The study was ethically approved by the Qazvin University of medical Sciences.

AVAILABILITY OF DATA AND MATERIALS
The study data can be availible for the editor if necessary.  Identi cation of bone marrow stromal cells. BMSC cells isolated from bone marrow in culture medium lacked endothelial stem cells. Immunocytochemical staining of primary CD31 antibody (endothelial cell marker) in BMSC cells after third passage. FITC-bound secondary antibody (green) and nuclei stained with PI (red).

Figure 4
Identi cation of bone marrow stromal cells. BMSC cells isolated from bone marrow in culture medium lacked blood stem cells. Immunocytochemical staining is the primary CD34 antibody (marker of hematopoietic cells) in BMSC cells after the third passage. FITC-attached secondary antibodies (green) and nuclei stained with PI (red).

Figure 5
Identi cation of bone marrow stromal cells. BMSC cells isolated from bone marrow in marker culture medium expressed bone marrow stromal stem cells, which appear green. Immunocytochemical staining is the primary antibody to CD 106 (bone marrow stromal stem cell marker) in BMSC cells after the third passage. FITC-attached secondary antibodies (green) and nuclei stained with PI (red)