Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Tao ¹ ,  PENG Wuxun ² , ZHANG Fei ² , ZHENG Yinggang ¹ , WANG Zhenwen ¹ , YUAN Dajiang ¹

1. Clinical Medical College, Medical University of Guizhou, Guiyang Guizhou, 550004, P.R.China;
2. Department of Emergency Orthopedics, Affiliated Hospital of Guizhou Medical University, Guiyang Guizhou, 550004, P.R.China;

Corresponding author：

PENG Wuxun, Email: 528591441@qq.com

Keywords：

Nicotinamide mononucleotide adenylyl transferase 3; nicotinamide adenine dinucleotide; mitochondrion; bone marrow mesenchymal stem cells; oxidative stress; rabbit

DOI：

10.7507/1002-1892.201910037

Video：

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Objective To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD⁺) levels.Methods The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H₂O₂ to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H₂O₂ treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H₂O₂ simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD⁺ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.Results The rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H₂O₂, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD⁺ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).Conclusion NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD⁺ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.

Citation： WANG Tao, PENG Wuxun, ZHANG Fei, ZHENG Yinggang, WANG Zhenwen, YUAN Dajiang. Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(5): 621-629. doi: 10.7507/1002-1892.201910037 Copy

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12.	Son MJ, Kwon Y, Son T, et al. Restoration of mitochondrial NAD⁺ levels delays stem cell senescence and facilitates reprogramming of aged somatic cells. Stem Cells, 2016, 34(12): 2840-2851.
13.	Gulshan M, Yaku K, Okabe K, et al. Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age-associated insulin resistance. Aging Cell, 2018, 17(4): e12798.
14.	Dai SH, Chen T, Wang YH, et al. Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells. Int J Mol Med, 2014, 34(4): 1159-1168.
15.	Kariminekoo S, Movassaghpour A, Rahimzadeh A, et al. Implications of mesenchymal stem cells in regenerative medicine. Artif Cells Nanomed Biotechnol, 2016, 44(3): 749-757.
16.	Zhang W, Zhang F, Shi H, et al. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One, 2014, 9(2): e88794.
17.	Tan SL, Ahmad TS, Selvaratnam L, et al. Isolation, characterization and the multi‐lineage differentiation potential of rabbit bone marrow‐derived mesenchymal stem cells. J Anat, 2013, 222(4): 437-450.
18.	Kiriyama Y, Nochi H. Intra-and intercellular quality control mechanisms of mitochondria. Cells, 2018, 7(1): E1.
19.	Lisowski P, Kannan P, Mlody B, et al. Mitochondria and the dynamic control of stem cell homeostasis. EMBO Rep, 2018, 19(5): e45432.
20.	Mouthuy PA, Snelling SJB, Dakin SG, et al. Biocompatibility of implantable materials: An oxidative stress viewpoint. Biomaterials, 2016, 109: 55-68.
21.	Wauquier F, Leotoing L, Coxam V, et al. Oxidative stress in bone remodelling and disease. Trends Mol Med, 2009, 15(10): 468-477.
22.	Stein LR, Imai S. The dynamic regulation of NAD metabolism in mitochondria. Trends Endocrinol Metab, 2012, 23(9): 420-428.
23.	Galindo R, Banks Greenberg M, Araki T, et al. NMNAT3 is protective against the effects of neonatal cerebral hypoxia‐ischemia. Ann Clin Transl Neurol, 2017, 4(10): 722-738.
24.	Cheng Z, Ristow M. Mitochondria and metabolic homeostasis. Antioxid Redox Signal, 2013, 19(3): 240-242.
25.	Abate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence. Semin Cell Dev Biol, 2020, 98: 139-153.
26.	Sorrentino V, Menzies KJ, Auwerx J. Repairing mitochondrial dysfunction in disease. Annu Rev Pharmacol Toxicol, 2018, 58: 353-389.
27.	Hershberger KA, Martin AS, Hirschey MD. Role of NAD⁺ and mitochondrial sirtuins in cardiac and renal diseases. Nat Rev Nephrol, 2017, 13(4): 213-225.

1. Galipeau J, Sensébé L. Mesenchymal stromal cells: clinical challenges and therapeutic opportunities. Cell Stem Cell, 2018, 22(6): 824-833.
2. Sugimura R. Bioengineering hematopoietic stem cell niche toward regenerative medicine. Adv Drug Deliv Rev, 2016, 99(Pt B): 212-220.
3. 谭可可, 王秀秀, 张君, 等. 壳聚糖多孔支架复合 BMSCs 移植修复大鼠创伤性脑损伤的实验研究. 中国修复重建外科杂志, 2018, 32(6): 745-752.
4. Xia P, Wang S, Qi Z, et al. BMP-2-releasing gelatin microspheres/PLGA scaffolds for bone repairment of X-ray-radiated rabbit radius defects. Artif Cells Nanomed Biotechnol, 2019, 47(1): 1662-1673.
5. 邢飞, 李浪, 刘明, 等. 原位组织工程技术在骨与软骨修复领域的应用进展. 中国修复重建外科杂志, 2018, 32(10): 1358-1363.
6. Zhang HX, Zhang XP, Xiao GY, et al. In vitro and in vivo evaluation of calcium phosphate composite scaffolds containing BMP-VEGF loaded PLGA microspheres for the treatment of avascular necrosis of the femoral head. Mater Sci Eng C Mater Biol Appl, 2016, 60: 298-307.
7. Yao X, Liu Y, Gao J, et al. Nitric oxide releasing hydrogel enhances the therapeutic efficacy of mesenchymal stem cells for myocardial infarction. Biomaterials, 2015, 60: 130-140.
8. Lee S, Choi E, Cha MJ, et al. Cell adhesion and long-term survival of transplanted mesenchymal stem cells: a prerequisite for cell therapy. Oxid Med Cell Longev, 2015, 2015: 632902.
9. Scheibye-Knudsen M, Fang EF, Croteau DL, et al. Protecting the mitochondrial powerhouse. Trends Cell Biol, 2015, 25(3): 158-170.
10. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev, 2014, 94(3): 909-950.
11. Zhang H, Ryu D, Wu Y, et al. NAD⁺ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science, 2016, 352(6292): 1436-1443.
12. Son MJ, Kwon Y, Son T, et al. Restoration of mitochondrial NAD⁺ levels delays stem cell senescence and facilitates reprogramming of aged somatic cells. Stem Cells, 2016, 34(12): 2840-2851.
13. Gulshan M, Yaku K, Okabe K, et al. Overexpression of Nmnat3 efficiently increases NAD and NGD levels and ameliorates age-associated insulin resistance. Aging Cell, 2018, 17(4): e12798.
14. Dai SH, Chen T, Wang YH, et al. Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells. Int J Mol Med, 2014, 34(4): 1159-1168.
15. Kariminekoo S, Movassaghpour A, Rahimzadeh A, et al. Implications of mesenchymal stem cells in regenerative medicine. Artif Cells Nanomed Biotechnol, 2016, 44(3): 749-757.
16. Zhang W, Zhang F, Shi H, et al. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One, 2014, 9(2): e88794.
17. Tan SL, Ahmad TS, Selvaratnam L, et al. Isolation, characterization and the multi‐lineage differentiation potential of rabbit bone marrow‐derived mesenchymal stem cells. J Anat, 2013, 222(4): 437-450.
18. Kiriyama Y, Nochi H. Intra-and intercellular quality control mechanisms of mitochondria. Cells, 2018, 7(1): E1.
19. Lisowski P, Kannan P, Mlody B, et al. Mitochondria and the dynamic control of stem cell homeostasis. EMBO Rep, 2018, 19(5): e45432.
20. Mouthuy PA, Snelling SJB, Dakin SG, et al. Biocompatibility of implantable materials: An oxidative stress viewpoint. Biomaterials, 2016, 109: 55-68.
21. Wauquier F, Leotoing L, Coxam V, et al. Oxidative stress in bone remodelling and disease. Trends Mol Med, 2009, 15(10): 468-477.
22. Stein LR, Imai S. The dynamic regulation of NAD metabolism in mitochondria. Trends Endocrinol Metab, 2012, 23(9): 420-428.
23. Galindo R, Banks Greenberg M, Araki T, et al. NMNAT3 is protective against the effects of neonatal cerebral hypoxia‐ischemia. Ann Clin Transl Neurol, 2017, 4(10): 722-738.
24. Cheng Z, Ristow M. Mitochondria and metabolic homeostasis. Antioxid Redox Signal, 2013, 19(3): 240-242.
25. Abate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence. Semin Cell Dev Biol, 2020, 98: 139-153.
26. Sorrentino V, Menzies KJ, Auwerx J. Repairing mitochondrial dysfunction in disease. Annu Rev Pharmacol Toxicol, 2018, 58: 353-389.
27. Hershberger KA, Martin AS, Hirschey MD. Role of NAD⁺ and mitochondrial sirtuins in cardiac and renal diseases. Nat Rev Nephrol, 2017, 13(4): 213-225.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels

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