Apoptotic Effects of C-82 and Naringenin on Menstrual Blood-derived Mesenchymal Stem Cells of Endometriosis Patients

Apoptotic Effects of C-82 and Naringenin on Menstrual Blood-derived Mesenchymal Stem Cells of Endometriosis Patients
Azar Sheikholeslami,¹ Hoda fazaeli,² Faezeh Davoodi Asl,³ Naser Kalhor,^4,* Rahil Jannatifar,⁵ Seyedeh Saeideh Sahraei,⁶
1. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
2. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
3. Department of Reproductive Biology, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
4. Department of Mesenchymal Stem Cells, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
5. Department of Reproductive Biology, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
6. Department of Reproductive Biology, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom, Iran
Introduction: One of the leading causes of endometriosis - a chronic estrogen-dependent disease associated with pelvic pain and infertility- is the return of menstrual blood flow into the pelvic cavity and the establishment of menstrual blood mesenchymal stem cells (MenSCs) in areas outside the uterine cavity. MenSCs from endometriosis patients (E-MenSCs) and healthy women have been shown to vary, particularly in terms of surface markers and gene expression, which may suggest the involvement of these cells in the development, expansion, and maintenance of ectopic lesions. The purpose of this study is to investigate the effect of small molecule C-82 and naringenin as inhibitors of involving pathways on endometriosis to modulate their gene expression and functional pattern.
Methods: The menstrual blood samples were obtained from endometriosis patients through the day 2-3 of menstruation cycle. Based on density gradient method, stromal cells were isolated from the samples by means of Ficoll-Paque and were cultivated in DMEM with 10% FBS and 1% penicillin/streptomycin and incubated at 37°C, 5% CO2, and 95% humidity.In the 3rd passage, E-MenSCs were treated with C-82 and naringenin. Then, cell behavior was studied using annexin V/PI assay. After reaching 70% confluence in passage 3, the cells were seeded in proper dishes for 24 hours. Then, the small molecule group and the naringenin group were cultured with fresh medium containing 2 μM small molecule C-82 (Sigma-Aldrich, Lyon, France) and 100 μM naringenin (Sigma-Aldrich), respectively. The same amount of C-82 and naringenin for SM and Nr groups, were used simultaneously for treating SM/Nr group. The control group received no treatment. The time of treatment for all groups was 72 hours.
Results: Annexin V/PI assay The effects of applied treatments (C-82 and Naringenin, alone or together) on the apoptosis of E-MenSCs were assessed through flow cytometry with annexin V and PI detection kit (Fig.2). As expected , the obtained data showed a significant increase in early apoptosis of Combination treatment of Nr with SM co-treated SM/Nr group in comparison with untreated E-MenSCs (p= 0.0232) . SM group (p= 0.0293), while although C-82 and Naringenin alone increased the early apoptosis percentage in treated cells but they did not elevate it in a statistical significant way (p= 0.9991 and p= 0.2141, respectively). However, it was demonstrated that none of the applying treatments could exert a significant change in the percentage of the late and total apoptosis (p>0.05) (Fig.2). BAX/BCL2 gene expression Next, we evaluated apoptotic related genes, that showed C-82 and combination of C-82/Naringenin significantly increase expression of BAX (proapoptotic gene) and BCL-2 (antiapoptotic gene) as compared with control group (p= 0.00), while no significant change was observed in Nr group (p>0.05). So, when the BAX/ BCL2 ratio was assessed, none of the treated group were different from the untreated E-MenSCs (p>0.05) (Fig.3).
Conclusion: In the present study, increased early apoptosis was observed in E-MenSCs after treatment. We observed that small molecule C-82 together with naringenin can promote apoptosis in E-MenSCs. These results are significant because they clarify the function of C-82 and naringenin in endometriosis. Further research is needed to analyze the precise effects of small molecule C-82 and naringenin on endometriosis E-MenSCs.
Keywords: endometriosis, mesenchymal stem cells, menstrual blood, small molecule C-82, naringenin