Podophyllum hexandrum Royle mitigates perimenopausal symptoms in an OVX rat model by activating the PI3K/AKT/mTOR pathway and enhancing estrogen receptor expression

Ethnopharmacological relevance: Podophyllum hexandrum Royle, recorded in the Pharmacopoeia of the People's Republic of China as Sinopodophyllum hexandrum (Royle) Ying (SH), is a nationally protected Tibetan medicinal plant in China, which has been traditionally used to regulate menstruation, enhance blood circulation, and treat blood stasis and dystocia. However, its potential role and mechanisms in managing perimenopausal syndrome (PMS) remain unclear.

Aim of the study: This study evaluates the therapeutic potential of Sinopodophyllum hexandrum (Royle) Ying rhizomes (SHR) and fruits (SHF) in PMS and investigates their underlying molecular mechanisms.

Materials and methods: The anti-PMS effects of SHR and SHF were examined in an ovariectomized (OVX) rat model by assessing uterine histopathology and hormone levels. Serum lipid profiles, including triglycerides (TG), total Cholesterol (TC), low-density lipoprotein Cholesterol (LDL-C), and high-density lipoprotein Cholesterol (HDL-C), as well as malondialdehyde (MDA) levels and superoxide dismutase (SOD) levels, were analyzed using biochemical assays. Astral-DIA proteomics identified differentially expressed proteins (DEPs) and key signaling pathways affected by SHF. Protein expression was evaluated via western blotting, immunohistochemistry, and RT-qPCR. Estrogenic activity was further assessed in vitro through MCF-7 cell viability and Estrogen receptor (ER) expression analysis.

Results: SHR and SHF treatment significantly improved uterine morphology in OVX rats, restoring endometrial and epithelial thickness. High-dose SHF (SHF-H) increased serum estradiol (E2) by 100.02 %, anti-Müllerian hormone (AMH) by 34.68 %, and progesterone (PROG) by 39.96 % while decreasing luteinizing hormone (LH) by 31.53 %. High-dose SHR (SHR-H) treatment resulted in a 112.89 % increase in E2 levels and a 23.88 % decrease in LH levels. Additionally, SHR-H and SHF-H regulated the level of oxidative stress and serum lipid levels, showing a significant decline in MDA (48.32 %, 65.52 %), TG (34.71 %, 33.30 %), TC (22.34 %, 27.77 %), and LDL-C (57.09 %, 42.96 %). Proteomic analysis identified the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway and apoptotic regulation as key mechanisms underlying SHF's effects. SHF reversed the OVX-induced suppression of p-PI3K/PI3K, p-AKT/Akt, and p-mTOR in the uterus. SHR and SHF also modulated apoptosis-related proteins, downregulating Bax and Cleaved Caspase-3/9 while upregulating Bcl-2. Moreover, SHF significantly increased uterine ERα and ERβ expression at both mRNA and protein levels. In vitro, SHR and SHF extracts promoted MCF-7 cell viability and upregulated ERα, ERβ, and proliferating cell nuclear antigen (PCNA), indicating estrogenic activity.

Conclusions: SH alleviates PMS by inhibiting uterine Apoptosis via PI3K/Akt/mTOR pathway activation and enhancing Estrogen receptor expression. It also regulates hormone levels, lipid metabolism, and oxidative stress, potentially slowing PMS progression. These findings highlight SH as a promising therapeutic agent for PMS and provide novel insights into the molecular mechanisms of Tibetan medicine.

Apoptosis; Estrogen receptor; PI3K/AKT/mTOR signaling pathway; PMS; Podophyllum hexandrum Royle; The rhizomes and fruits.