Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HE Pengjie , LI Yang , ZHANG Ruotian , REN Maoxian , LIU Hedong ,  YANG Min

Department of Traumatology and Orthopedics, Yijishan Hospital, Wannan Medical College, Wuhu Anhui, 241001, P.R.China;

Corresponding author：

YANG Min, Email: pkuyang@hotmail.com

Keywords：

Hypoxia; osteoblasts; p22phox; NOX5; reactive oxygen species; autophagy; apoptosis

DOI：

10.7507/1002-1892.202008039

Video：

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Objective To investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.Methods The skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.Results After identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.Conclusion Inhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.

Citation： HE Pengjie, LI Yang, ZHANG Ruotian, REN Maoxian, LIU Hedong, YANG Min. Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(7): 855-861. doi: 10.7507/1002-1892.202008039 Copy

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2.	Ducy P, Amling M, Takeda S, et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell, 2000, 100(2): 197-207.
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11.	张俊宇, 何鹏杰, 程傲, 等. 缺氧复氧环境对成骨细胞自噬水平的影响. 中华医学杂志, 2019, 99(11): 844-849.
12.	Prior KK, Wittig I, Leisegang MS, et al. The endoplasmic reticulum chaperone calnexin is a NADPH oxidase NOX4 interacting protein. J Biol Chem, 2016, 291(13): 7045-7059.
13.	Nowak-Sliwinska P, Alitalo K, Allen E, et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis, 2018, 21(3): 425-532.
14.	Yamamoto H, Saito M, Goto T, et al. Heme oxygenase-1 prevents glucocorticoid and hypoxia-induced apoptosis and necrosis of osteocyte-like cells. Med Mol Morphol, 2019, 52(3): 173-180.
15.	Hao Z, Ma Y, Wang J, et al. Hypoxia promotes AMP-activated protein kinase (AMPK) and induces apoptosis in mouse osteoblasts. Int J Clin Exp Pathol, 2015, 8(5): 4892-4902.
16.	Tan C, Day R, Bao S, et al. Group VIA phospholipase A2 mediates enhanced macrophage migration in diabetes mellitus by increasing expression of nicotinamide adenine dinucleotide phosphate oxidase 4. Arterioscler Thromb Vasc Biol, 2014, 34(4): 768-778.
17.	Zhou L, Chen X, Yan J, et al. Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS. Osteoporos Int, 2017, 28(12): 3325-3337.
18.	Joo JH, Huh JE, Lee JH, et al. A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase. Sci Rep, 2016, 6: 22389. doi: 10.1038/srep22389.
19.	Rosen CJ. Bone remodeling, energy metabolism, and the molecular clock. Cell Metab, 2008, 7(1): 7-10.
20.	Tseng PC, Hou SM, Chen RJ, et al. Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis. J Bone Miner Res, 2011, 26(10): 2552-2563.
21.	Zhou XJ, Klionsky DJ, Zhang H. Podocytes and autophagy: a potential therapeutic target in lupus nephritis. Autophagy, 2019, 15(5): 908-912.
22.	Kaushal GP, Chandrashekar K, Juncos LA. Molecular interactions between reactive oxygen species and autophagy in kidney disease. Int J Mol Sci, 2019, 20(15): 3791. doi: 10.1016/j.bbamcr.2021.119041.
23.	Li CF, Pan YK, Gao Y, et al. Autophagy protects HUVECs against ER stress-mediated apoptosis under simulated microgravity. Apoptosis, 2019, 24(9-10): 812-825.

1. Fata JE, Kong YY, Li J, et al. The osteoclast differentiation factor osteoprotegerin-ligand is essential for mammary gland development. Cell, 2000, 103(1): 41-50.
2. Ducy P, Amling M, Takeda S, et al. Leptin inhibits bone formation through a hypothalamic relay: a central control of bone mass. Cell, 2000, 100(2): 197-207.
3. Bechor E, Zahavi A, Berdichevsky Y, et al. p67 phox -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism. J Leukoc Biol, 2021, 109(3): 657-673.
4. Mandal CC, Ganapathy S, Gorin Y, et al. Reactive oxygen species derived from Nox4 mediate BMP2 gene transcription and osteoblast differentiation. Biochem J, 2011, 433(2): 393-402.
5. Kim YR, Kim JS, Gu SJ, et al. Identification of highly potent and selective inhibitor, TIPTP, of the p22phox-Rubicon axis as a therapeutic agent for rheumatoid arthritis. Sci Rep, 2020, 10(1): 4570. doi: 10.1038/s41598-020-61630-x.
6. Chen JR, Lazarenko OP, Blackburn ML, et al. p47phox-Nox2-dependent ROS signaling inhibits early bone development in mice but protects against skeletal aging. J Biol Chem, 2015, 290(23): 14692-14704.
7. Shimada M, Greer PA, McMahon AP, et al. In vivo targeted deletion of calpain small subunit, Capn4, in cells of the osteoblast lineage impairs cell proliferation, differentiation, and bone formation. J Biol Chem, 2008, 283(30): 21002-21010.
8. Gregory CA, Gunn WG, Peister A, et al. An alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem, 2004, 329(1): 77-84.
9. van Gastel N, Stegen S, Eelen G, et al. Lipid availability determines fate of skeletal progenitor cells via SOX9. Nature, 2020, 579(7797): 111-117.
10. Merceron C, Ranganathan K, Wang E, et al. Hypoxia-inducible factor 2α is a negative regulator of osteoblastogenesis and bone mass accrual. Bone Res, 2019, 7: 91-104.
11. 张俊宇, 何鹏杰, 程傲, 等. 缺氧复氧环境对成骨细胞自噬水平的影响. 中华医学杂志, 2019, 99(11): 844-849.
12. Prior KK, Wittig I, Leisegang MS, et al. The endoplasmic reticulum chaperone calnexin is a NADPH oxidase NOX4 interacting protein. J Biol Chem, 2016, 291(13): 7045-7059.
13. Nowak-Sliwinska P, Alitalo K, Allen E, et al. Consensus guidelines for the use and interpretation of angiogenesis assays. Angiogenesis, 2018, 21(3): 425-532.
14. Yamamoto H, Saito M, Goto T, et al. Heme oxygenase-1 prevents glucocorticoid and hypoxia-induced apoptosis and necrosis of osteocyte-like cells. Med Mol Morphol, 2019, 52(3): 173-180.
15. Hao Z, Ma Y, Wang J, et al. Hypoxia promotes AMP-activated protein kinase (AMPK) and induces apoptosis in mouse osteoblasts. Int J Clin Exp Pathol, 2015, 8(5): 4892-4902.
16. Tan C, Day R, Bao S, et al. Group VIA phospholipase A2 mediates enhanced macrophage migration in diabetes mellitus by increasing expression of nicotinamide adenine dinucleotide phosphate oxidase 4. Arterioscler Thromb Vasc Biol, 2014, 34(4): 768-778.
17. Zhou L, Chen X, Yan J, et al. Melatonin at pharmacological concentrations suppresses osteoclastogenesis via the attenuation of intracellular ROS. Osteoporos Int, 2017, 28(12): 3325-3337.
18. Joo JH, Huh JE, Lee JH, et al. A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase. Sci Rep, 2016, 6: 22389. doi: 10.1038/srep22389.
19. Rosen CJ. Bone remodeling, energy metabolism, and the molecular clock. Cell Metab, 2008, 7(1): 7-10.
20. Tseng PC, Hou SM, Chen RJ, et al. Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis. J Bone Miner Res, 2011, 26(10): 2552-2563.
21. Zhou XJ, Klionsky DJ, Zhang H. Podocytes and autophagy: a potential therapeutic target in lupus nephritis. Autophagy, 2019, 15(5): 908-912.
22. Kaushal GP, Chandrashekar K, Juncos LA. Molecular interactions between reactive oxygen species and autophagy in kidney disease. Int J Mol Sci, 2019, 20(15): 3791. doi: 10.1016/j.bbamcr.2021.119041.
23. Li CF, Pan YK, Gao Y, et al. Autophagy protects HUVECs against ER stress-mediated apoptosis under simulated microgravity. Apoptosis, 2019, 24(9-10): 812-825.

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Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia

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