Bushen Jianpi Tiaoxue Decoction (BJTD) inhibits the LIF-mTOR signaling axis to regulate mitochondrial function and alleviate cyclophosphamide-induced diminished ovarian reserve.

Abstract

Diminished ovarian reserve (DOR) is a challenging obstacle impacting women' fertility globally with limited treatment option. Bushen Jianpi Tiaoxue Decoction (BJTD) has shown significant efficacy and safety in treating DOR patients, yet the molecular mechanisms behind its effect remain uncertain. Our study aimed to uncover the pharmacology and signaling pathway of BJTD in cyclophosphamide (Cy)-provoked DOR mice and 4-hydroperoxy cyclophosphamide (4-HC)-irritated KGN cells (human granulosa-like cell line) damage models. Granulosa cells from DOR patients and Cy-induced models were reanalyzed utilizing transcriptomics to predict differentially expressed genes and crucial signaling pathways. Validation experiments were conducted in vitro using KGN cells treated with 4-HC, a Cy metabolite, to establish a DOR model. Cell viability, apoptosis, oxidative stress, mitochondrial function, and ferroptosis markers were assessed using the cck8 assay, flow cytometry, fluorescence staining, RT-qPCR, and western blotting analyses. BJTD-serum was evaluated for its protective effects on 4-HC-triggered KGN damages. In vivo, a Cy-induced DOR mouse model was treated with BJTD to evaluate ovarian morphology, estrous cycle, follicle counts, hormone markers, mitochondrial apoptosis and ferroptosis levels, respectively via the vaginal smear, histological analysis, immunostaining, gene and protein expression experiments. The UPLC-MS analysis and network pharmacology were applied to identify BJTD's active ingredients, followed by molecular dockings to assess interactions with the target protein. To confirm the BJTD's mechanism of action, mTOR signaling modulation was analyzed using a specific inhibitor or activator in vitro. Transcriptomic reanalysis revealed significant gene expression differences, with LIF identified as a key target associated with apoptosis pathway. In vitro, 4-HC exposure induced apoptosis, mitochondrial dysfunction, and ferroptosis in KGN cells, accompanied by upregulation of LIF, mTOR, and FoxO3a signalings. BJTD-serum treatment significantly improved cell viability, reduced apoptosis, and alleviated oxidative stress by modulating mitochondrial function and ferroptosis markers, such as Nrf2, HO-1, and GPX4. In vivo, BJTD alleviated Cy-induced ovarian damage, improving ovarian index, estrous cycle, follicle development, and hormone levels, while reducing follicular atresia and granulosa cells apoptosis. Mechanically, BJTD suppressed Cy-induced activation of the LIF-mTOR axis and downstream mitochondrial apoptosis markers, including Cleaved Caspase 9/3, BAX, and γH2AX, while enhancing OPA1 and Bcl-2 expressions. The UPLC-MS outcome combining with network pharmacology identified mainly 20 active compounds in BJTD, with astragaloside IV exhibiting the strongest binding to the mTOR protein. The mTOR pathway modulation experiments confirmed that BJTD's protective effects are mediated through inhibition of hyperactivated mTOR phosphorylation and mitochondrial apoptosis cascades. BJTD demonstrates efficacy in alleviating Cy- and 4-HC-induced DOR models through targeting the LIF-mTOR signaling axis to suppress granulosa cells mitochondrial apoptosis and ferroptosis. These results might highlight promising therapeutic potential of BJTD for ovarian reserve preservation.