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Rationale <p>Senile osteoporosis (SOP) is driven largely by bone marrow mesenchymal stem cell (BMSC) senescence and mitochondrial dysfunction. S-glutathionylation is an important redox modification regulating mitochondrial homeostasis, yet its role in skeletal aging remains unclear. The objective of this study is to systematically investigate the role of Grx2 deficiency-mediated CD36 S-glutathionylation in BMSC senescence and SOP development.</p> Methods <p>Grx2, p53, and p21 expression in human and mouse BMSCs were analyzed by immunohistochemistry (IHC), western blotting (WB), and polymerase chain reaction (PCR). Aging-related and ovariectomy-induced osteoporosis models were established in Grx2<sup>−</sup>/<sup>−</sup> mice for bone metabolism and senescence assessments. BMSC senescence, osteogenesis, and adipogenesis were evaluated by SA-<i>β</i>-Gal and WB, ALP/ARS staining, and ORO staining. Transcriptomic and S-glutathionylated proteomic analyses were performed to identify underlying mechanisms. Fatty acid uptake was quantified using two-color flow cytometry. DAG and MDA levels were measured to assess lipid overload and oxidative injury. Mitochondrial structure and function were evaluated by TEM, Mitotracker, qPCR, NAD<sup>+</sup>/NADH, ATP, ROS, JC-1, and Seahorse assays. The PI3K/AKT pathway was assessed by WB. Co-IP confirmed CD36 S-glutathionylation, and molecular docking predicted C272 as the key modification site. CD36-C272S mutation and Grx2 overexpression were applied to validate functional mechanisms in vitro and in vivo.</p> Results <p>Grx2 deficiency, in both male and ovariectomized female mice, accelerates bone loss, inhibits osteoblast formation without altering osteoclast function, and exacerbates BMSC senescence. Through integrated transcriptomic and S-glutathionylated proteomic analysis, we identified the fatty acid transporter CD36 as a critical downstream target of Grx2. Notably, Grx2 deficiency markedly increases the S-glutathionylation of CD36, which not only enhances its fatty acid uptake capacity, leading to the accumulation of toxic lipid metabolites and oxidative damage, but also impairs mitochondrial energy metabolism by inhibiting the PI3K/AKT signaling pathway. Overexpression of Grx2 or the C272S mutation in CD36, which disrupts its S-glutathionylation, can break this harmful cycle and inhibit BMSC senescence.</p> Conclusions <p>Grx2 deficiency-mediated CD36 S-glutathionylation drives BMSC senescence and SOP, providing new insight into the redox regulatory mechanisms underlying skeletal aging.</p>

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Grx2 deficiency accelerates BMSC senescence and senile osteoporosis via CD36 S-glutathionylation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月21日

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  • Muzhe Li,
  • Taxi Wumiti,
  • Jie Sun,
  • Tianchi Zhang,
  • Lining Wang,
  • Qinfeng Zhou,
  • Tianwei Xia,
  • Jirong Shen,
  • Yafeng Zhang,
  • Yong Ma,
  • Yang Guo

摘要

Rationale

Senile osteoporosis (SOP) is driven largely by bone marrow mesenchymal stem cell (BMSC) senescence and mitochondrial dysfunction. S-glutathionylation is an important redox modification regulating mitochondrial homeostasis, yet its role in skeletal aging remains unclear. The objective of this study is to systematically investigate the role of Grx2 deficiency-mediated CD36 S-glutathionylation in BMSC senescence and SOP development.

Methods

Grx2, p53, and p21 expression in human and mouse BMSCs were analyzed by immunohistochemistry (IHC), western blotting (WB), and polymerase chain reaction (PCR). Aging-related and ovariectomy-induced osteoporosis models were established in Grx2−/− mice for bone metabolism and senescence assessments. BMSC senescence, osteogenesis, and adipogenesis were evaluated by SA-β-Gal and WB, ALP/ARS staining, and ORO staining. Transcriptomic and S-glutathionylated proteomic analyses were performed to identify underlying mechanisms. Fatty acid uptake was quantified using two-color flow cytometry. DAG and MDA levels were measured to assess lipid overload and oxidative injury. Mitochondrial structure and function were evaluated by TEM, Mitotracker, qPCR, NAD+/NADH, ATP, ROS, JC-1, and Seahorse assays. The PI3K/AKT pathway was assessed by WB. Co-IP confirmed CD36 S-glutathionylation, and molecular docking predicted C272 as the key modification site. CD36-C272S mutation and Grx2 overexpression were applied to validate functional mechanisms in vitro and in vivo.

Results

Grx2 deficiency, in both male and ovariectomized female mice, accelerates bone loss, inhibits osteoblast formation without altering osteoclast function, and exacerbates BMSC senescence. Through integrated transcriptomic and S-glutathionylated proteomic analysis, we identified the fatty acid transporter CD36 as a critical downstream target of Grx2. Notably, Grx2 deficiency markedly increases the S-glutathionylation of CD36, which not only enhances its fatty acid uptake capacity, leading to the accumulation of toxic lipid metabolites and oxidative damage, but also impairs mitochondrial energy metabolism by inhibiting the PI3K/AKT signaling pathway. Overexpression of Grx2 or the C272S mutation in CD36, which disrupts its S-glutathionylation, can break this harmful cycle and inhibit BMSC senescence.

Conclusions

Grx2 deficiency-mediated CD36 S-glutathionylation drives BMSC senescence and SOP, providing new insight into the redox regulatory mechanisms underlying skeletal aging.