Potential mechanism of Ziyin Tongluo Formula in the treatment of postmenopausal osteoporosis: based on network pharmacology and ovariectomized rat model

Background Amending from ancient classic, Ziyin Tongluo Formula (ZYTLF) has been prescribed to treat postmenopausal osteoporosis (PMOP) for decades with good curative effect. However, the possible mechanisms of it are still unknown. Methods Ovariectomized rat model was established to validate the therapeutic effect of ZYTLF on PMOP by Micro-CT bone analysis and pathological observation. Subsequently, active ingredients of ZYTLF and corresponding putative targets were identified by online databases. Overlapping genes were first obtained from mining genes associated with PMOP and then overlapped them with the putative targets. Key genes were selected from the multiple constructed and analyzed networks. GO and KEGG pathway enrichment analysis were performed by importing the key genes to the DAVID database. Moreover, validation of the binding association between key targets and their corresponding active compounds were accomplished by AutoDock Tools and other software. Lastly, Enzyme linked immunosorbent assay (Elisa) detection and Western blot analysis were utilized to further explore the possible mechanism of ZYTLF on PMOP. Results With 129 target genes interacting with PMOP, 92 active compounds of ZYTLF corresponded to 243 targets, and 50 key genes were chosen. Network analysis revealed the top 10 active ingredients, such as quercetin and kaempferol and the top 50 key genes, such as ERα, p38 MAPK, p-AKT and TGF-β1. Enrichment analysis uncovered multiple signaling pathways, including estrogen signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway and MAPK signaling pathway. Furthermore, our finding of the foremost active compounds was tightly bound to the core proteins, which were verified by molecular docking analysis. Through experimental studies, we confirmed that the prescription of ZYTLF could ameliorate the OVX-induced bone loss, suppress the osteoclast activity and boost osteoblast ability through experimental studies. Conclusion The potential mechanisms and therapeutic effects of ZYTLF against PMOP may be ascribed to inhibition of osteoclast activity, boost of osteoblast activity and enhancement of the expression of ERα.

Phytoestrogen in cowpea (Vigna unguiculata L. Walp) (Fabaceae) extract reduces vaginal oxidative stress and increases proliferation of fibroblast in ovariectomized rats

Purpose: To evaluate the effects of a phytoestrogen extract of cowpea (Vigna unguiculata) on malondialdehyde (MDA) and superoxide dismutase (SOD) levels, mitogen-activated protein kinase (MAPK) activity, as well as proliferation of fibroblasts in ovariectomized rat vagina. This is with a view to identify the optimal dose of a phytoestrogen supplement for use during the menopausal period of women. Methods: Wistar rats (Rattus norvegicus) were divided into five groups: negative control, ovariectomy, and ovariectomy groups treated with 1.25, 2.5, or 5 mg/kg of cowpea extract, respectively. The three doses were used to identify the optimal dose required to reduce vaginal oxidative stress and increase fibroblast proliferation in ovariectomized rats. After treatment, the ovariectomized rat vagina was assessed for SOD and MDA levels, MAPK activity, and fibroblast proliferation. Results: Cowpea extract at a dose of 2.5 and 5 mg/kg caused significant reduction in SOD levels in the vaginal tissue of rats compared with control (p < 0.05). In contrast, cowpea extract at 1.25 mg/kg resulted in MDA levels similar to that of control rats (p < 0.05). Cowpea extract treatment had positive effects on SOD and MDA levels and on fibroblast proliferation. Increase in SOD levels in rat vagina was induced by increased MDA levels, which is characteristic of oxidative stress, while ovariectomy resulted in decreased MAPK activity. Conclusion: Overall, the cowpea extract has no significant effect on MAPK activity or fibroblast proliferation. However, it has a significant effect on SOD and MDA levels and on fibroblast proliferation at a dose of 2.5 mg/kg.