Marrubium alysson L. Ameliorated Methotrexate-Induced Testicular Damage in Mice through Regulation of Apoptosis and miRNA-29a Expression: LC-MS/MS Metabolic Profiling

Despite the efficient anti-cancer capabilities of methotrexate (MTX), it may induce myelosup-pression, liver dysfunction and testicular toxicity. The purpose of this investigation was to determinewhether Marrubium alysson L. (M. alysson L.) methanolic extract and its polyphenol fraction could pro-tect mouse testicles from MTX-induced damage. We also investigated the protective effects of threeselected pure flavonoid components of M. alysson L. extract. Mice were divided into seven groups(n = 8): (1) normal control, (2) MTX, (3) Methanolic extract + MTX, (4) Polyphenolic fraction + MTX,(5) Kaempferol + MTX, (6) Quercetin + MTX, and (7) Rutin + MTX. Pre-treatment of mice with themethanolic extract, the polyphenolic fraction of M. alysson L. and the selected pure compoundsameliorated the testicular histopathological damage and induced a significant increase in the serumtestosterone level and testicular antioxidant enzymes along with a remarkable decline in the malon-dialdehyde (MDA) level versus MTX alone. Significant down-regulation of nuclear factor kappa B(NF-κB), tumor necrosis factor-alpha (TNF-α), p53 and miRNA-29a testicular expression was alsoobserved in all the protected groups. Notably, the polyphenolic fraction of M. alysson L. displayed amore pronounced decline in the testicular levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) andMDA, with higher testosterone levels relative to the methanolic extract. Further improvementsin the Johnsen score, histopathological results and all biochemical assays were achieved by pre-treatment with the three selected pure compounds kaempferol, quercetin and rutin. In conclusion, M.alysson L. could protect against MTX-induced testicular injury by its antioxidant, anti-inflammatory,antiapoptotic activities and through the regulation of the miRNA-29a testicular expression. Thepresent study also included chemical profiling of M. alysson L. extract, which was accomplished byLC-ESI-TOF-MS/MS analysis. Forty compounds were provisionally assigned, comprising twentycompounds discovered in the positive mode and seventeen detected in the negative mode.