Mechanism of sodium valproate in inhibiting ferroptosis of bone marrow mesenchymal stem cells via the adenosine monophosphate-activated protein kinase/Sirtuin 1 axis_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GU Qingsong , LI Jianqiao , CHEN Yuhu , WANG Linhui , LI Yiheng , WANG Ziru , WANG Yicong ,  YANG Min

Department of Traumatic Orthopedics, the First Affiliated Hospital of Wannan Medical College/Yijishan Hospital, Wuhu Anhui, 241001, P. R. China;

Corresponding author：

YANG Min, Email: pkuyang@hotmail.com

Keywords：

Sodium valproate; bone marrow mesenchymal stem cells; ferroptosis; osteoporosis; adenosine monophosphate-activated protein kinase; Sirtuin 1

DOI：

10.7507/1002-1892.202411089

Video：

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Objective To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. Methods BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. Results The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. Conclusion VPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.

Citation： GU Qingsong, LI Jianqiao, CHEN Yuhu, WANG Linhui, LI Yiheng, WANG Ziru, WANG Yicong, YANG Min. Mechanism of sodium valproate in inhibiting ferroptosis of bone marrow mesenchymal stem cells via the adenosine monophosphate-activated protein kinase/Sirtuin 1 axis. Chinese Journal of Reparative and Reconstructive Surgery, 2025, 39(2): 215-223. doi: 10.7507/1002-1892.202411089 Copy

1.	Yang R, Ma Q, Zhang X, et al. A study on the prevalence of osteoporosis in people with different altitudes in Sichuan, China. Clin Interv Aging, 2024, 19: 1819-1828.
2.	Martin AR, Sornay-Rendu E, Chandler JM, et al. The impact of osteoporosis on quality-of-life: the OFELY cohort. Bone, 2002, 31(1): 32-36.
3.	刘合栋, 任茂贤, 李杨, 等. 褪黑素对H₂O₂诱导的成骨细胞氧化应激损伤的保护作用. 中国骨质疏松杂志, 2022, 28(8): 1093-1098.
4.	Marques-Carvalho A, Kim HN, Almeida M. The role of reactive oxygen species in bone cell physiology and pathophysiology. Bone Rep, 2023, 19: 101664. doi: 10.1016/j.bonr.2023.101664.
5.	Riegger J, Schoppa A, Ruths L, et al. Oxidative stress as a key modulator of cell fate decision in osteoarthritis and osteoporosis: a narrative review. Cell Mol Biol Lett, 2023, 28(1): 76. doi: 10.1186/s11658-023-00489-y.
6.	Liu Z, Li T, Deng S, et al. Radiation induces apoptosis and osteogenic impairment through miR-22-mediated intracellular oxidative stress in bone marrow mesenchymal stem cells. Stem Cells Int, 2018, 2018: 5845402. doi: 10.1155/2018/5845402.
7.	Xia Y, Zhang H, Wang H, et al. Identification and validation of ferroptosis key genes in bone mesenchymal stromal cells of primary osteoporosis based on bioinformatics analysis. Front Endocrinol (Lausanne), 2022, 13: 980867. doi: 10.3389/fendo.2022.980867.
8.	Long SW, Li SH, Li J, et al. Identification of osteoporosis ferroptosis-related markers and potential therapeutic compounds based on bioinformatics methods and molecular docking technology. BMC Med Genomics, 2024, 17(1): 99. doi: 10.1186/s12920-024-01872-0.
9.	Li J, Cao F, Yin HL, et al. Ferroptosis: past, present and future. Cell Death Dis, 2020, 11(2): 88. doi: 10.1038/s41419-020-2298-2.
10.	Lane DJ, Merlot AM, Huang ML, et al. Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease. Biochim Biophys Acta, 2015, 1853(5): 1130-1144.
11.	Latunde-Dada GO. Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochim Biophys Acta Gen Subj, 2017, 1861(8): 1893-1900.
12.	Ursini F, Maiorino M. Lipid peroxidation and ferroptosis: The role of GSH and GPx4. Free Radic Biol Med, 2020, 152: 175-185.
13.	Zhang Y, Swanda RV, Nie L, et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun, 2021, 12(1): 1589. doi: 10.1038/s41467-021-21841-w.
14.	Ru Q, Li Y, Chen L, et al. Iron homeostasis and ferroptosis in human diseases: mechanisms and therapeutic prospects. Signal Transduct Target Ther, 2024, 9(1): 271. doi: 10.1038/s41392-024-01969-z.
15.	Kim BJ, Ahn SH, Bae SJ, et al. Iron overload accelerates bone loss in healthy postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J Bone Miner Res, 2012, 27(11): 2279-2290.
16.	Wang Z, Yan Q, Wang Z, et al. Ferroptosis and its implications in bone-related diseases. PeerJ, 2024, 12: e18626. doi: 10.7717/peerj.18626.
17.	Zhang H, Wang A, Li G, et al. Osteoporotic bone loss from excess iron accumulation is driven by NOX4-triggered ferroptosis in osteoblasts. Free Radic Biol Med, 2023, 198: 123-136.
18.	Drzewiecka M, Gajos-Michniewicz A, Hoser G, et al. Histone deacetylases (HDAC) inhibitor-valproic acid sensitizes human melanoma cells to dacarbazine and PARP inhibitor. Genes (Basel), 2023, 14(6): 1295. doi: 10.3390/genes14061295.
19.	Uzel G, Oylumlu E, Durmus L, et al. Duality of valproic acid effects on inflammation, oxidative stress and autophagy in human eosinophilic cells. Int J Mol Sci, 2023, 24(17): 13446. doi: 10.3390/ijms241713446.
20.	Seet LF, Toh LZ, Finger SN, et al. Valproic acid exerts specific cellular and molecular anti-inflammatory effects in post-operative conjunctiva. J Mol Med (Berl), 2019, 97(1): 63-75.
21.	蒋文凯, 周志, 刘合栋, 等. 丙戊酸钠对羰基氰化物间氯苯腙诱导的成骨细胞氧化应激损伤的保护作用研究. 中国修复重建外科杂志, 2023, 37(3): 316-323.
22.	Marin TL, Gongol B, Zhang F, et al. AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1. Sci Signal, 2017, 10(464): eaaf7478. doi: 10.1126/scisignal.aaf7478.
23.	Cantó C, Gerhart-Hines Z, Feige JN, et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature, 2009, 458(7241): 1056-1060.
24.	Ibrahim WW, Kamel AS, Wahid A, et al. Dapagliflozin as an autophagic enhancer via LKB1/AMPK/SIRT1 pathway in ovariectomized/D-galactose Alzheimer’s rat model. Inflammopharmacology, 2022, 30(6): 2505-2520.
25.	Tang D, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications. Cell Res, 2021, 31(2): 107-125.
26.	Chen P, Li Z, Hu Y. Prevalence of osteoporosis in China: a meta-analysis and systematic review. BMC Public Health, 2016, 16(1): 1039. doi: 10.1186/s12889-016-3712-7.
27.	周志, 蒋文凯, 刘之义, 等. 曲古抑菌素A促进去卵巢大鼠钛植入物骨整合. 中国骨质疏松杂志, 2023, 29(9): 1266-1271.
28.	Wu Z, Li W, Jiang K, et al. Regulation of bone homeostasis: signaling pathways and therapeutic targets. MedComm (2020), 2024, 5(8): e657. doi: 10.1002/mco2.657.
29.	Teitelbaum SL. Bone resorption by osteoclasts. Science, 2000, 289(5484): 1504-1508.
30.	León-Reyes G, Argoty-Pantoja AD, Becerra-Cervera A, et al. Oxidative-stress-related genes in osteoporosis: A systematic review. Antioxidants (Basel), 2023, 12(4): 915. doi: 10.3390/antiox12040915.
31.	Li X, Li B, Shi Y, et al. Targeting reactive oxygen species in stem cells for bone therapy. Drug Discov Today, 2021, 26(5): 1226-1244.
32.	Wu C, Li A, Leng Y, et al. Histone deacetylase inhibition by sodium valproate regulates polarization of macrophage subsets. DNA Cell Biol, 2012, 31(4): 592-599.
33.	Chen PS, Peng GS, Li G, et al. Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes. Mol Psychiatry, 2006, 11(12): 1116-1125.
34.	Luís PB, Ruiter JP, Aires CC, et al. Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation. Biochim Biophys Acta, 2007, 1767(9): 1126-1133.
35.	Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J, 2011, 434(3): 365-381.
36.	Tao ZS, Zhou WS, Xu HG, et al. Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats. Eur J Pharmacol, 2021, 906: 174268. doi: 10.1016/j.ejphar.2021.174268.
37.	Liu Y, Lu S, Wu LL, et al. The diversified role of mitochondria in ferroptosis in cancer. Cell Death Dis, 2023, 14(8): 519. doi: 10.1038/s41419-023-06045-y.
38.	Li Q, Peng F, Yan X, et al. Inhibition of SLC7A11-GPX4 signal pathway is involved in aconitine-induced ferroptosis in vivo and in vitro. J Ethnopharmacol, 2023, 303: 116029. doi: 10.1016/j.jep.2022.116029.
39.	Tian Y, Lu J, Hao X, et al. FTH1 inhibits ferroptosis through ferritinophagy in the 6-OHDA model of Parkinson’s disease. Neurotherapeutics, 2020, 17(4): 1796-1812.
40.	Sharma A, Anand SK, Singh N, et al. AMP-activated protein kinase: An energy sensor and survival mechanism in the reinstatement of metabolic homeostasis. Exp Cell Res, 2023, 428(1): 113614. doi: 10.1016/j.yexcr.2023.113614.
41.	Qu Q, Chen Y, Wang Y, et al. Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing. Nature, 2024. doi: 10.1038/s41586-024-08348-2.
42.	Chen Y, Xiao H, Liu Z, et al. Sirt1: An increasingly interesting molecule with a potential role in bone metabolism and osteoporosis. Biomolecules, 2024, 14(8): 970. doi: 10.3390/biom14080970.
43.	Fu C, Cao N, Zeng S, et al. Role of mitochondria in the regulation of ferroptosis and disease. Front Med (Lausanne), 2023, 10: 1301822. doi: 10.3389/fmed.2023.1301822.
44.	Li Y, Wang Y, Liu Q, et al. Kaempferol promotes osteogenic differentiation in bone marrow mesenchymal stem cells by inhibiting CAV-1. J Orthop Surg Res, 2024, 19(1): 678. doi: 10.1186/s13018-024-05174-0.

1. Yang R, Ma Q, Zhang X, et al. A study on the prevalence of osteoporosis in people with different altitudes in Sichuan, China. Clin Interv Aging, 2024, 19: 1819-1828.
2. Martin AR, Sornay-Rendu E, Chandler JM, et al. The impact of osteoporosis on quality-of-life: the OFELY cohort. Bone, 2002, 31(1): 32-36.
3. 刘合栋, 任茂贤, 李杨, 等. 褪黑素对H₂O₂诱导的成骨细胞氧化应激损伤的保护作用. 中国骨质疏松杂志, 2022, 28(8): 1093-1098.
4. Marques-Carvalho A, Kim HN, Almeida M. The role of reactive oxygen species in bone cell physiology and pathophysiology. Bone Rep, 2023, 19: 101664. doi: 10.1016/j.bonr.2023.101664.
5. Riegger J, Schoppa A, Ruths L, et al. Oxidative stress as a key modulator of cell fate decision in osteoarthritis and osteoporosis: a narrative review. Cell Mol Biol Lett, 2023, 28(1): 76. doi: 10.1186/s11658-023-00489-y.
6. Liu Z, Li T, Deng S, et al. Radiation induces apoptosis and osteogenic impairment through miR-22-mediated intracellular oxidative stress in bone marrow mesenchymal stem cells. Stem Cells Int, 2018, 2018: 5845402. doi: 10.1155/2018/5845402.
7. Xia Y, Zhang H, Wang H, et al. Identification and validation of ferroptosis key genes in bone mesenchymal stromal cells of primary osteoporosis based on bioinformatics analysis. Front Endocrinol (Lausanne), 2022, 13: 980867. doi: 10.3389/fendo.2022.980867.
8. Long SW, Li SH, Li J, et al. Identification of osteoporosis ferroptosis-related markers and potential therapeutic compounds based on bioinformatics methods and molecular docking technology. BMC Med Genomics, 2024, 17(1): 99. doi: 10.1186/s12920-024-01872-0.
9. Li J, Cao F, Yin HL, et al. Ferroptosis: past, present and future. Cell Death Dis, 2020, 11(2): 88. doi: 10.1038/s41419-020-2298-2.
10. Lane DJ, Merlot AM, Huang ML, et al. Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease. Biochim Biophys Acta, 2015, 1853(5): 1130-1144.
11. Latunde-Dada GO. Ferroptosis: Role of lipid peroxidation, iron and ferritinophagy. Biochim Biophys Acta Gen Subj, 2017, 1861(8): 1893-1900.
12. Ursini F, Maiorino M. Lipid peroxidation and ferroptosis: The role of GSH and GPx4. Free Radic Biol Med, 2020, 152: 175-185.
13. Zhang Y, Swanda RV, Nie L, et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun, 2021, 12(1): 1589. doi: 10.1038/s41467-021-21841-w.
14. Ru Q, Li Y, Chen L, et al. Iron homeostasis and ferroptosis in human diseases: mechanisms and therapeutic prospects. Signal Transduct Target Ther, 2024, 9(1): 271. doi: 10.1038/s41392-024-01969-z.
15. Kim BJ, Ahn SH, Bae SJ, et al. Iron overload accelerates bone loss in healthy postmenopausal women and middle-aged men: a 3-year retrospective longitudinal study. J Bone Miner Res, 2012, 27(11): 2279-2290.
16. Wang Z, Yan Q, Wang Z, et al. Ferroptosis and its implications in bone-related diseases. PeerJ, 2024, 12: e18626. doi: 10.7717/peerj.18626.
17. Zhang H, Wang A, Li G, et al. Osteoporotic bone loss from excess iron accumulation is driven by NOX4-triggered ferroptosis in osteoblasts. Free Radic Biol Med, 2023, 198: 123-136.
18. Drzewiecka M, Gajos-Michniewicz A, Hoser G, et al. Histone deacetylases (HDAC) inhibitor-valproic acid sensitizes human melanoma cells to dacarbazine and PARP inhibitor. Genes (Basel), 2023, 14(6): 1295. doi: 10.3390/genes14061295.
19. Uzel G, Oylumlu E, Durmus L, et al. Duality of valproic acid effects on inflammation, oxidative stress and autophagy in human eosinophilic cells. Int J Mol Sci, 2023, 24(17): 13446. doi: 10.3390/ijms241713446.
20. Seet LF, Toh LZ, Finger SN, et al. Valproic acid exerts specific cellular and molecular anti-inflammatory effects in post-operative conjunctiva. J Mol Med (Berl), 2019, 97(1): 63-75.
21. 蒋文凯, 周志, 刘合栋, 等. 丙戊酸钠对羰基氰化物间氯苯腙诱导的成骨细胞氧化应激损伤的保护作用研究. 中国修复重建外科杂志, 2023, 37(3): 316-323.
22. Marin TL, Gongol B, Zhang F, et al. AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1. Sci Signal, 2017, 10(464): eaaf7478. doi: 10.1126/scisignal.aaf7478.
23. Cantó C, Gerhart-Hines Z, Feige JN, et al. AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature, 2009, 458(7241): 1056-1060.
24. Ibrahim WW, Kamel AS, Wahid A, et al. Dapagliflozin as an autophagic enhancer via LKB1/AMPK/SIRT1 pathway in ovariectomized/D-galactose Alzheimer’s rat model. Inflammopharmacology, 2022, 30(6): 2505-2520.
25. Tang D, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications. Cell Res, 2021, 31(2): 107-125.
26. Chen P, Li Z, Hu Y. Prevalence of osteoporosis in China: a meta-analysis and systematic review. BMC Public Health, 2016, 16(1): 1039. doi: 10.1186/s12889-016-3712-7.
27. 周志, 蒋文凯, 刘之义, 等. 曲古抑菌素A促进去卵巢大鼠钛植入物骨整合. 中国骨质疏松杂志, 2023, 29(9): 1266-1271.
28. Wu Z, Li W, Jiang K, et al. Regulation of bone homeostasis: signaling pathways and therapeutic targets. MedComm (2020), 2024, 5(8): e657. doi: 10.1002/mco2.657.
29. Teitelbaum SL. Bone resorption by osteoclasts. Science, 2000, 289(5484): 1504-1508.
30. León-Reyes G, Argoty-Pantoja AD, Becerra-Cervera A, et al. Oxidative-stress-related genes in osteoporosis: A systematic review. Antioxidants (Basel), 2023, 12(4): 915. doi: 10.3390/antiox12040915.
31. Li X, Li B, Shi Y, et al. Targeting reactive oxygen species in stem cells for bone therapy. Drug Discov Today, 2021, 26(5): 1226-1244.
32. Wu C, Li A, Leng Y, et al. Histone deacetylase inhibition by sodium valproate regulates polarization of macrophage subsets. DNA Cell Biol, 2012, 31(4): 592-599.
33. Chen PS, Peng GS, Li G, et al. Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes. Mol Psychiatry, 2006, 11(12): 1116-1125.
34. Luís PB, Ruiter JP, Aires CC, et al. Valproic acid metabolites inhibit dihydrolipoyl dehydrogenase activity leading to impaired 2-oxoglutarate-driven oxidative phosphorylation. Biochim Biophys Acta, 2007, 1767(9): 1126-1133.
35. Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J, 2011, 434(3): 365-381.
36. Tao ZS, Zhou WS, Xu HG, et al. Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats. Eur J Pharmacol, 2021, 906: 174268. doi: 10.1016/j.ejphar.2021.174268.
37. Liu Y, Lu S, Wu LL, et al. The diversified role of mitochondria in ferroptosis in cancer. Cell Death Dis, 2023, 14(8): 519. doi: 10.1038/s41419-023-06045-y.
38. Li Q, Peng F, Yan X, et al. Inhibition of SLC7A11-GPX4 signal pathway is involved in aconitine-induced ferroptosis in vivo and in vitro. J Ethnopharmacol, 2023, 303: 116029. doi: 10.1016/j.jep.2022.116029.
39. Tian Y, Lu J, Hao X, et al. FTH1 inhibits ferroptosis through ferritinophagy in the 6-OHDA model of Parkinson’s disease. Neurotherapeutics, 2020, 17(4): 1796-1812.
40. Sharma A, Anand SK, Singh N, et al. AMP-activated protein kinase: An energy sensor and survival mechanism in the reinstatement of metabolic homeostasis. Exp Cell Res, 2023, 428(1): 113614. doi: 10.1016/j.yexcr.2023.113614.
41. Qu Q, Chen Y, Wang Y, et al. Lithocholic acid binds TULP3 to activate sirtuins and AMPK to slow down ageing. Nature, 2024. doi: 10.1038/s41586-024-08348-2.
42. Chen Y, Xiao H, Liu Z, et al. Sirt1: An increasingly interesting molecule with a potential role in bone metabolism and osteoporosis. Biomolecules, 2024, 14(8): 970. doi: 10.3390/biom14080970.
43. Fu C, Cao N, Zeng S, et al. Role of mitochondria in the regulation of ferroptosis and disease. Front Med (Lausanne), 2023, 10: 1301822. doi: 10.3389/fmed.2023.1301822.
44. Li Y, Wang Y, Liu Q, et al. Kaempferol promotes osteogenic differentiation in bone marrow mesenchymal stem cells by inhibiting CAV-1. J Orthop Surg Res, 2024, 19(1): 678. doi: 10.1186/s13018-024-05174-0.

Chinese Journal of Reparative and Reconstructive Surgery

Mechanism of sodium valproate in inhibiting ferroptosis of bone marrow mesenchymal stem cells via the adenosine monophosphate-activated protein kinase/Sirtuin 1 axis

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