Ceratonia siliqua L. pod Effects on Viability Gene Expression of Endometrial Mesenchymal Stromal/Stem Cells Isolated from Women with Endometriosis-Associated Infertility

Abstract

Background: This study aims to investigate the effects of carob (Ceratonia siliqua L.) pod extract (CPE) on theviability of human endometrial mesenchymal stromal/stem cells (EnMSCs) and its impact on mRNA and protein expressionsof DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), histone deacetylase 1 (HDAC1), matrixmetalloproteinase-2 (MMP2), and cyclooxygenase-2 (COX-2) in endometriotic patients.Materials and Methods: In this experimental study, EnMSCs were derived from endometrium of patients with ovarianendometrioma (OMA-EnMSCs group) and deep infiltrative endometriosis (DIE) samples of 10 endometriosisassociatedinfertility (EAI) women (E-EnMSCs group) and compared to EnMSCs derived from the endometrium ofan endometriosis-free, normal woman as the control group (C-EnMSCs). The metabolic activity of the control andcase groups were evaluated by treating them with different concentrations of CPE. Cell viability was analysed byMTT. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to evaluatethe expression of specific genes at the mRNA and protein levels, respectively.Results: Treatment with 0.8 and 2 μg/mL of CPE downregulated COX-2 and HDAC1 in the E-EnMSC groupcompared to the C-EnMSCs group. Treatment with 0.8 μg/mL of CPE also decreased MMP2 and DNMT3B geneexpressions. The COX-2 and DNMT3A genes were significantly upregulated after treatment with 2 μg/mL of CPE.Expressions of the COX-2, HDAC1, DNMT1, DNMT3A, and DNMT3B peptides decreased in the all three groups after treatment with 0.8 and 2 μg/mL of CPE. Gas chromatography-mass spectroscopy (GC-MS) analysis of CPEidentified 14 bioactive compounds. Molecular docking showed the best position of each bioactive compound on thedifferent target proteins that are involved in the process of apoptosis in EnMSCs.Conclusion: In vitro and in silico analyses of CPE bioactive compounds show that they may downregulate the cellinflammatory pathway involved in the pathophysiology of endometriosis.

Keywords

• carob
• endometriosis
• gene expression
• mesenchymal
• stem cells