Transcriptomic, artificial neural network and Chou-Talalay analysis based investigation of potential mechanism and
Xi-Yang Tang1, Qing-Chang Wu1, Lv-Qi Xie1
1State Key Laboratory of Bioactive Molecules and Druggability Assessment, Guangdong Basic Research Center of Excellence for Natural Bioactive Molecules and Discovery of Innovative Drugs, Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, and International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Ministry of Education (MOE) of China, Jinan University, Guangzhou, 510632, PR China.
Ethnopharmacological Relevance:
Qi-Lin pill (QLP), which is derived from the renowned Wu-Zi-Yan-Zong formulation of the Tang Dynasty, has been used for tonifying the kidneys, replenishing vital essence, and treating infertility and menstrual disorders. However, its mechanisms and active components for treating diminished ovarian reserve (DOR) remain unclear.
Aim Of The Study:
Herein, we integrated transcriptomics, artificial neural network (ANN), and Chou-Talalay analysis to elucidate the potential mechanisms and active components responsible for the effects of QLP.
Materials And Methods:
First, QLP was orally administered to a cisplatin (CDDP)-induced mouse model of DOR; ovarian index, estrous cycle, ovarian tissue morphology, and biochemical parameters were observed to assess efficacy. Second, transcriptomics analysis was performed to reveal the possible mechanisms of QLP, and qRT-PCR and western blotting were used to validate the expressions of key signaling molecules in vivo. Third, the aforementioned mechanisms of QLP were further investigated in CDDP-induced functional impairment of estradiol (E2) synthesis in ovarian granulosa-like KGN cells via using siRNA-mediated depletion and specific inhibition. Subsequently, UPLC-QQQ-MS was used to quantify 33 compounds in 13 batches of QLP. Then, the ANN with feature importance analysis was used to correlate the chemical constituents in the content assay with pharmacological indicators to screen for potential active ingredients. Finally, the synergistic effect of the selected components was investigated using the Chou-Talalay index method in the CDDP-induced functional damage model of human ovarian granulosa-like tumor cells (KGN).
Results:
QLP restored estrous cyclicity, ovarian index, serum hormones (follicle-stimulating hormone, anti-Müllerian hormone, E2), and follicular development (antral follicles and corpora lutea). Mechanistically, QLP ameliorated CDDP-induced functional damage of the ovaries in vivo and in vitro by upregulating follicle-stimulating hormone receptor (FSHR) expression and activating the ADCY1/cAMP/PKA/CREB signaling pathway, which resulted in upregulated aromatase expression and increased E2 synthesis. Subsequently, ten potentially active compounds, including paeoniflorin, albiflorin, danshensu, gallic acid, betaine, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, salvianolic acid A, hesperidin, nobiletin, and epimedin C, were selected by ANN combined with feature importance analysis. Finally, these ten components were divided into two mixture forms, one of which comprised monoterpenoids (paeoniflorin and albiflorin) in a mixture. Two optimized mixtures synergistically amplified FSHR expression and cAMP/PKA/CREB signaling via Chou-Talalay analysis, thus boosting E2 and aromatase in KGN cells.
Conclusion:
These findings reveal that QLP could exert its effects through a multicomponent synergistic interaction involving cAMP-mediated steroidogenesis, thus providing new insights into the mechanism by which this traditional Chinese medicine prescription treats ovarian dysfunction.
