Anti-osteoporotic potential of Lawsonia inermis leaf extract: integration of network pharmacology, molecular docking,...

With an emphasis on modulation of the oestrogen receptor (ER)/osteoprotegerin (OPG)/receptor activator of nuclear factor-κβ ligand (RANKL) signalling pathway, the current study sought to explore the anti-osteoporotic potential of Lawsonia inermis leaves ethanolic extract (LIEE) using a unified method linking network pharmacology, molecular docking, and experimental validation in an ovariectomised (OVX) rat model. LC-MS/MS and HPTLC were applied for the phytochemical profiling of LIEE. To find possible targets and pathways connected to osteoporosis, network pharmacology analysis was carried out using Cytoscape and public databases (PubChem, BindingDB, DisGeNET, and GeneCards). To confirm interactions between important phytoconstituents and target proteins, molecular docking was used. Six groups (n = 8) of female Wistar rats were randomly assigned for in vivo evaluation: OVX Control, Sham Control, OVX + LIEE (100, 200, and 400 mg/kg, p.o.), and OVX + 17β-estradiol (30 µg/kg/week, s.c.). Bone turnover markers (parathyroid hormone, calcitonin, osteocalcin, tartrate-resistant acid phosphatase, osteoprotegerin, receptor activator of nuclear factor), lipid profile, oxidative stress markers (superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde), pro-inflammatory cytokines (Tumour necrosis factor-α, Interleukin-1β, Interleukin-6), and histopathology of the femur and reproductive tissues were assessed after ten weeks of treatment. RT-PCR was used to analyse the gene expression of epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), and matrix metalloproteinase-9 (MMP-9). Fourteen bioactive phytoconstituents, including gallic acid, rutin, quercetin, apigenin, and many others, were found by LC-MS/MS analysis. With important hub genes including MMP-9, mTOR, B-cell lymphoma-2, Hypoxia-Inducible Factor 1-alpha, and EGFR, network pharmacology identified 61 common targets between LIEE and osteoporosis. Enrichment analysis highlighted the involvement of ER/OPG/RANKL signalling pathways. Molecular docking demonstrated strong binding affinity of major phytoconstituents with target proteins. In vivo studies showed that LIEE significantly attenuated OVX-induced body weight gain and restored uterine weight and serum estrogen levels, indicating phytoestrogenic activity. LIEE significantly improved bone physical parameters and increased BMD. Treatment normalised serum calcium, phosphorus, and alkaline phosphatase levels, and favourably modulated bone turnover markers by decreasing tartrate-resistant acid phosphatase, parathyroid hormone and osteocalcin levels while increasing calcitonin and osteoprotegerin levels and reducing receptor activator of nuclear factor kappa-B Ligand expression. LIEE also significantly enhanced lipid profile by reducing total cholesterol, triglyceride, and low-density lipoprotein while enhancing high-density lipoprotein levels. Furthermore, LIEE markedly reduced oxidative stress by increasing superoxide dismutase, catalase, and glutathione peroxidase levels and decreasing malondialdehyde. Pro-inflammatory cytokines (Tumour necrosis factor-α, Interleukin-1β, Interleukin-6) were considerably suppressed. Reestablishment of trabecular bone architecture and normalisation of uterine and vaginal morphology were verified by histopathological examination. Gene expression analysis demonstrated downregulation of MMP-9, mTOR, and EGFR in LIEE-treated groups. LIEE exhibits significant anti-osteoporotic effects through a multi-targeted mechanism involving modulation of ER/OPG/RANKL signalling, suppression of inflammation and oxidative stress, and regulation of key molecular targets. These outcomes propose that LIEE could help as a capable phytotherapeutic candidate for the management of postmenopausal osteoporosis and warrant more clinical exploration.