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Background <p>To investigate the effects and mechanism of action of the main component, Stigmasterol (STG), of Liuwei Dihuang Pill (LP) on postmenopausal osteoporosis (PMOP) in ovariectomized (OVX) rats.</p> Methods <p>Cell viability was assessed using the cell counting kit 8 (CCK-8) assay. Osteoblast differentiation was evaluated through alkaline phosphatase (ALP) activity assay, ALP staining, and Alizarin Red S staining. Expression levels were assessed by real-time quantitative PCR (RT-qPCR), Western blot, and immunohistochemistry. Immunofluorescence was used to observe protein colocalization. Protein–protein interactions were detected using co-immunoprecipitation (Co-IP) and Molecular docking and drug affinity responsive target stability (DARTS) assays. Additionally, an OVX rat model was established through ovariectomy surgery. Utilizing micro-computed tomography (CT), we scanned and analyzed the fine structure of the rat femoral trabeculae across various groups, thereby gathering pertinent data. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of E2, key bone immune regulatory markers, bone turnover markers, and pro-inflammatory factor in serum.</p> Results <p>The main components of LP, including Quercetin, Kaempferol, β-sitosterol, and STG, significantly promote osteogenic differentiation of osteoblasts, with STG having the most significant effect. Moreover, STG also notably enhances the upregulation of the Akt/mTOR/ p70S6 kinase (p70S6K) signaling pathway. Bioinformatics analysis predicted the potential ubiquitin ligase synoviolin 1 (SYVN1) for Akt, which was experimentally validated. Molecular docking between STG and SYVN1 verified their binding activity, and STG downregulated the expression of SYVN1. Overexpression of SYVN1 reversed the promoting effect of STG on osteoblast differentiation and activation of the Akt/mTOR/p70S6K pathway. Furthermore, STG upregulated uterine index and serum levels of E2, OPG, BALP, PINP, OCN, and CTX-1, and downregulated levels of RANKL, IL-6, and TNF-α in OVX rats. Meanwhile, STG promoted the expression of OCN and inhibited the expression of NFATc1 and cathepsin-K in the femurs of OVX rats. Additionally, STG activated the Akt/mTOR/p70S6K pathway in the femurs of OVX rats. However, overexpressing SYVN1 reversed the effects of STG on OVX rats.</p> Conclusion <p>STG upregulated the Akt/mTOR/p70S6K signaling via SYVN1 to alleviate osteoporosis in OVX rats. Therefore, this study will provide new insights into discovering novel approaches for treating PMOP.</p>

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Stigmasterol upregulates Akt/mTOR/p70S6K signaling through SYVN1 to alleviate osteoporosis in ovariectomized rats

  • 期刊论文

  • 发表日期：

    2026年3月26日

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  • Fuping Zhu,
  • Wuping Li,
  • Yinsheng Cao,
  • Bing Dai,
  • Hang Wu,
  • Yutong Zhu,
  • Hui Liu

摘要

Background

To investigate the effects and mechanism of action of the main component, Stigmasterol (STG), of Liuwei Dihuang Pill (LP) on postmenopausal osteoporosis (PMOP) in ovariectomized (OVX) rats.

Methods

Cell viability was assessed using the cell counting kit 8 (CCK-8) assay. Osteoblast differentiation was evaluated through alkaline phosphatase (ALP) activity assay, ALP staining, and Alizarin Red S staining. Expression levels were assessed by real-time quantitative PCR (RT-qPCR), Western blot, and immunohistochemistry. Immunofluorescence was used to observe protein colocalization. Protein–protein interactions were detected using co-immunoprecipitation (Co-IP) and Molecular docking and drug affinity responsive target stability (DARTS) assays. Additionally, an OVX rat model was established through ovariectomy surgery. Utilizing micro-computed tomography (CT), we scanned and analyzed the fine structure of the rat femoral trabeculae across various groups, thereby gathering pertinent data. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of E2, key bone immune regulatory markers, bone turnover markers, and pro-inflammatory factor in serum.

Results

The main components of LP, including Quercetin, Kaempferol, β-sitosterol, and STG, significantly promote osteogenic differentiation of osteoblasts, with STG having the most significant effect. Moreover, STG also notably enhances the upregulation of the Akt/mTOR/ p70S6 kinase (p70S6K) signaling pathway. Bioinformatics analysis predicted the potential ubiquitin ligase synoviolin 1 (SYVN1) for Akt, which was experimentally validated. Molecular docking between STG and SYVN1 verified their binding activity, and STG downregulated the expression of SYVN1. Overexpression of SYVN1 reversed the promoting effect of STG on osteoblast differentiation and activation of the Akt/mTOR/p70S6K pathway. Furthermore, STG upregulated uterine index and serum levels of E2, OPG, BALP, PINP, OCN, and CTX-1, and downregulated levels of RANKL, IL-6, and TNF-α in OVX rats. Meanwhile, STG promoted the expression of OCN and inhibited the expression of NFATc1 and cathepsin-K in the femurs of OVX rats. Additionally, STG activated the Akt/mTOR/p70S6K pathway in the femurs of OVX rats. However, overexpressing SYVN1 reversed the effects of STG on OVX rats.

Conclusion

STG upregulated the Akt/mTOR/p70S6K signaling via SYVN1 to alleviate osteoporosis in OVX rats. Therefore, this study will provide new insights into discovering novel approaches for treating PMOP.