Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (21): 4330-4343.doi: 10.3864/j.issn.0578-1752.2023.21.015

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

NAC Affects Proliferation and Differentiation of Adipose-Derived Mesenchymal Stem Cells by Regulating Reactive Oxygen Species

LIU PeiPei1(), DING ShiJie1, SONG WenJuan1, TANG ChangBo1, LI HuiXia1(), TANG Hong2()   

  1. 1 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
    2 Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural and Reclamation Sciences/State Key Laboratory for Sheep Genetic Improvement and Healthy Production, Shihezi 832000, Xinjiang
  • Received:2022-05-09 Accepted:2023-09-17 Online:2023-11-01 Published:2023-11-06
  • Contact: LI HuiXia, TANG Hong

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Abstract:

【Objective】Cells are sensitive to oxidative stress and elevated levels of intracellular reactive oxygen species during in vitro culture, which affects cell function. In this research, the regulation of reactive oxygen species in porcine adipose mesenchymal stem cells by N-acetyl-L cysteine (NAC) was evaluated, and the effects on their proliferation and differentiation were further clarified, which could provide a theoretical basis and reference for cultured fat seed cells to expand in vitro in large numbers and improve differentiation efficiency.【Method】In this research, to model oxidative stress, the different concentrations of H2O2 (0, 25, 50, and 100 μmol·L-1) were added during the proliferation of adipose-derived mesenchymal stem cells (ADSCs). The added concentration of H2O2 was identified by cell counting results, cell morphology, cell viability and intracellular reactive oxygen species levels detected by a High-throughput High-content Live Cells Confocal Imaging System. To select the appropriate addition concentration of NAC to promote the proliferation of ADSCs, the different concentrations of NAC (0, 1, 2, and 3 mmol·L-1) were added during the proliferation of ADSCs, and the appropriate addition concentration of NAC was determined by cell counting results and cell morphology. To further explore the effect of NAC on the proliferation of oxidatively stressed ADSCs, cell proliferation under different treatment conditions (Control, 1 mmol·L-1 NAC, 50 μmol·L-1 H2O2, and 1 mmol·L-1 NAC + 50 μmol·L-1 H2O2) was analyzed by EdU staining and cell counting. To investigate the level of reactive oxygen species in ADSCs under different treatment conditions, ADSCs proliferated under different treatment conditions for 3 d were stained by CellRox, and the intracellular reactive oxygen species content was detected by High-throughput High-inclusion Live Cell Confocal Imaging System to clarify the relationship between ADSCs proliferation and intracellular reactive oxygen species level. To explore the effect of reactive oxygen species levels within ADSCs on their differentiation, ADSCs were stained with Oil Red O after 10 d of differentiation under different treatment conditions, and the amount of differentiated lipid accumulation in ADSCs was assessed by Image J analysis of stained area, and the relative expression of ADSCs differentiation-related genes was examined by RT-qPCR.【Result】During the proliferation of ADSCs, ADSCs in the group with 50 μmol·L-1 H2O2 were shuttle-shaped and had significantly higher intracellular reactive oxygen species content compared with the control group (P<0.05), and the oxidative stress model was successfully established. Compared with the control group, when 50 μmol·L-1 H2O2 was added during proliferation of ADSCs, the number of ADSCs proliferation was significantly decreased (P<0.05), but the lipid accumulation of ADSCs was significantly higher (P<0.05) when 50 μmol·L-1 H2O2 was added during the differentiation of ADSCs. During the proliferation of ADSCs, ADSCs in the group with the addition of 1 mmol·L-1 NAC had a shuttle shape, the intracellular reactive oxygen species content was significantly lower (P<0.05), and proliferation number of ADSCs was significantly higher (P<0.05) compared with the control group, but the addition of 1 mmol·L-1 NAC during the differentiation of ADSCs had no significant effect on the lipid accumulation of ADSCs (P>0.05).【Conclusion】When ADSCs were affected by oxidative stress, the level of reactive oxygen species in ADSCs increases, which was detrimental to the massive expansion of ADSCs in vitro, inducing cell differentiation and accelerating cellular senescence. The addition of 1 mmol·L-1 NAC to the in vitro expansion system of ADSCs could reduce the oxidative stress damage brought about by long-term culture and exogenous stimulation, and had a protective effect on oxidatively stressed ADSCs, which could effectively promote cell proliferation and did not affect the differentiation ability of the cells.

Key words: NAC, ADSCs, reactive oxygen species, proliferation, differentiation, cultured fat

Table 1

The primer sequences used for RT-qPCR"

基因 Genes name 引物序列 Primer sequence
PPARγ 上游 Forward：TGGCCATTCGCATCTTTCAG 下游 Reverse：ATCTCGTGGACGCCATACTT
FABP4 上游 Forward：AGAAGTGGGAGTGGGCTTTG 下游Reverse：ATGATCAGGTTGGGTTTGGC
GAPDH 上游 Forward：GTCGGAGTGAACGGATTTGGC 下游Reverse：CTTGCCGTGGGTGGAATCAT
PLIN1 上游 Forward：CAGTTCACAGCTGCCAATGA 下游Reverse：TTCAGCTCAGAGGCGATCTT
CEBP/α 上游 Forward：GAGCCCGGCAACTCTAGTAT 下游Reverse：CCCTACTCGGTAGGAATCGG

Fig. 1

Construction of an oxidative stress model of cells A-D. Bright field images of cells when 0, 25, 50, and 100 μmol·L-1 H2O2 were added to the cell culture medium. E. Cell proliferation folds when different concentrations of H2O2 were added. F. Cell count when different concentrations of H2O2 were added. G-J. EdU staining graph of cells when different concentrations of H2O2 were added. K. Percentage of cells positive for EdU staining when different concentrations of H2O2 were added. L-M. CellRox staining in the control and 50 μmol·L-1 H2O2 addition group. N. CellRox: cell average intensity. O. All CellRox mean stain area. P. All CellRox stain integrated intensities"

Fig. 2

Screening of suitable NAC addition concentration A-D. Bright field images of cells when 0, 1, 2 and 3 mmol·L-1 NAC were added to the cell culture medium. E. Cell count when different concentrations of NAC were added. F. Cell proliferation folds when different concentrations of NAC were added"

Fig. 3

Results of EdU staining and cell counting under different treatment conditions of ADSCs A-D. EdU staining results of different treatment groups. E. Percentage of EdU staining positive cells of different treatment groups. F. Cell count of different treatment groups"

Fig. 4

Evaluation of the content of reactive oxygen species within ADSCs under different treatment conditions A-D. CellRox staining of different treatment groups. E. CellRox: cell average intensity in different treatment groups. F. All CellRox mean stain area in different treatment groups. G. All CellRox stain integrated intensities in different treatment groups"

Fig. 5

Differentiation effect of different treatment groups A-D. Oil red O staining plot of different treatment groups. E. Lipid accumulation analysis of different treatment groups. F. Oil red O staining area analysis of different treatments"

Fig. 6

Relative expression of genes related to fat differentiation"

[1]
周光宏, 丁世杰, 徐幸莲. 培养肉的研究进展与挑战. 中国食品学报, 2020, 20(5): 1-11.
ZHOU G H, DING S J, XU X L. Progress and challenges in cultured meat. Journal of Chinese Institute of Food Science and Technology, 2020, 20(5): 1-11. (in Chinese)
[2]
FISH K D, RUBIO N R, STOUT A J, YUEN J S K, KAPLAN D L. Prospects and challenges for cell-cultured fat as a novel food ingredient. Trends in Food Science & Technology, 2020, 98: 53-67.
[3]
KOZAKOWSKA M, PIETRASZEK-GREMPLEWICZ K, JOZKOWICZ A, DULAK J. The role of oxidative stress in skeletal muscle injury and regeneration: focus on antioxidant enzymes. Journal of Muscle Research and Cell Motility, 2015, 36(6): 377-393.

doi: 10.1007/s10974-015-9438-9 pmid: 26728750
[4]
DENU R A, HEMATTI P. Effects of oxidative stress on mesenchymal stem cell biology. Oxidative Medicine and Cellular Longevity, 2016, 2016: 2989076.
[5]
ETO H, KATO H, SUGA H, AOI N, DOI K, KUNO S, YOSHIMURA K. The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes. Plastic and Reconstructive Surgery, 2012, 129(5): 1081-1092.

doi: 10.1097/PRS.0b013e31824a2b19
[6]
TURKER I, ZHANG Y H, ZHANG Y M, REHMAN J. Oxidative stress as a regulator of adipogenesis. Faseb Journal, 2007, 21: 830.
[7]
RUSHWORTH G F, MEGSON I L. Existing and potential therapeutic uses for N-acetylcysteine: The need for conversion to intracellular glutathione for antioxidant benefits. Pharmacology & Therapeutics, 2014, 141(2): 150-159.
[8]
JARIYAMANA N, CHUVEERA P, DEWI A, LEELAPORNPISID W, ITTICHAICHAROEN J, CHATTIPAKORN S, SRISUWAN T. Effects of N-acetyl cysteine on mitochondrial ROS, mitochondrial dynamics, and inflammation on lipopolysaccharide-treated human apical papilla cells. Clinical Oral Investigations, 2021, 25(6): 3919-3928.

doi: 10.1007/s00784-020-03721-7 pmid: 33404763
[9]
DING S J, SWENNEN G N M, MESSMER T, GAGLIARDI M, MOLIN D G M, LI C B, ZHOU G H, POST M J. Maintaining bovine satellite cells stemness through p38 pathway. Scientific Reports, 2018, 8(1): 10808.

doi: 10.1038/s41598-018-28746-7 pmid: 30018348
[10]
胡荣蓉, 丁世杰, 郭赟, 朱浩哲, 陈益春, 刘政, 丁希, 唐长波, 周光宏. Trolox对猪肌肉干细胞增殖及分化的影响. 中国农业科学, 2021, 54(24): 5290-5301.

doi: 10.3864/j.issn.0578-1752.2021.24.011
HU R R, DING S J, GUO Y, ZHU H Z, CHEN Y C, LIU Z, DING X, TANG C B, ZHOU G H. Effects of trolox on proliferation and differentiation of pig muscle stem cells. Scientia Agricultura Sinica, 2021, 54(24): 5290-5301. (in Chinese)

doi: 10.3864/j.issn.0578-1752.2021.24.011
[11]
李惠侠, 罗肖, 刘荣鑫, 杨映娟, 杨公社. 激活Wnt/β-catenin信号通路抑制猪AMSCs向脂肪细胞分化. 畜牧兽医学报, 2010, 41(12): 1523-1528.
LI H X, LUO X, LIU R X, YANG Y J, YANG G S. Activation of Wnt/β-catenin signaling pathway inhibits the adipogenic differentiation of porcine adipose-derived mesenchymal stem cells. Chinese Journal of Animal and Veterinary Sciences, 2010, 41(12): 1523-1528. (in Chinese)
[12]
CHEN Y J, LIU H Y, CHANG Y T, CHENG Y H, MERSMANN H J, KUO W H, DING S T. Isolation and differentiation of adipose-derived stem cells from porcine subcutaneous adipose tissues. Journal of Visualized Experiments, 2016(109): e53886.
[13]
CALZADILLA P, SAPOCHNIK D, COSENTINO S, DIZ V, DICELIO L, CALVO J C, GUERRA L N. N-acetylcysteine reduces markers of differentiation in 3T3-L1 adipocytes. International Journal of Molecular Sciences, 2011, 12(10): 6936-6951.

doi: 10.3390/ijms12106936 pmid: 22072928
[14]
KURI-HARCUCH W, VELEZ-DELVALLE C, VAZQUEZ- SANDOVAL A, HERNÁNDEZ-MOSQUEIRA C, FERNANDEZ-SANCHEZ V. A cellular perspective of adipogenesis transcriptional regulation. Journal of Cellular Physiology, 2019, 234(2): 1111-1129.

doi: 10.1002/jcp.v234.2
[15]
SATISH L, KRILL-BURGER J M, GALLO P H, DES ETAGES S, LIU F, PHILIPS B J, RAVURI S, MARRA K G, LAFRAMBOISE W A, KATHJU S, RUBIN J P. Expression analysis of human adipose- derived stem cells during in vitro differentiation to an adipocyte lineage. BMC Medical Genomics, 2015, 8: 41.

doi: 10.1186/s12920-015-0119-8
[16]
LI S J, RAZA S H A, ZHAO C P, CHENG G, ZAN L S. Overexpression of PLIN1 promotes lipid metabolism in bovine adipocytes. Animals, 2020, 10(11): 1944.

doi: 10.3390/ani10111944
[17]
DING S J, WANG F, LIU Y, LI S, ZHOU G H, HU P. Characterization and isolation of highly purified porcine satellite cells. Cell Death Discovery, 2017, 3: 17003.

doi: 10.1038/cddiscovery.2017.3 pmid: 28417015
[18]
L'HONORÉ A, COMMÈRE P H, NEGRONI E, PALLAFACCHINA G, FRIGUET B, DROUIN J, BUCKINGHAM M, MONTARRAS D. The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration. eLife, 2018, 7: e32991.
[19]
WANG X, HAI C X. Novel insights into redox system and the mechanism of redox regulation. Molecular Biology Reports, 2016, 43(7): 607-628.

doi: 10.1007/s11033-016-4022-y pmid: 27255468
[20]
JIANG L, KON N, LI T Y, WANG S J, SU T, HIBSHOOSH H, BAER R, GU W. Ferroptosis as a p53-mediated activity during tumour suppression. Nature, 2015, 520(7545): 57-62.

doi: 10.1038/nature14344
[21]
RODRIGUES M, TURNER O, STOLZ D, GRIFFITH L G, WELLS A. Production of reactive oxygen species by multipotent stromal cells/mesenchymal stem cells upon exposure to fas ligand. Cell Transplantation, 2012, 21(10): 2171-2187.

doi: 10.3727/096368912X639035 pmid: 22526333
[22]
YAHATA T, TAKANASHI T, MUGURUMA Y, IBRAHIM A A, MATSUZAWA H, UNO T, SHENG Y, ONIZUKA M, ITO M, KATO S, ANDO K. Accumulation of oxidative DNA damage restricts the self-renewal capacity of human hematopoietic stem cells. Blood, 2011, 118(11): 2941-2950.

doi: 10.1182/blood-2011-01-330050 pmid: 21734240
[23]
BHATTI F U R, KIM S J, YI A K, HASTY K A, CHO H. Cytoprotective role of vitamin E in porcine adipose-tissue-derived mesenchymal stem cells against hydrogen-peroxide-induced oxidative stress. Cell and Tissue Research, 2018, 374(1): 111-120.

doi: 10.1007/s00441-018-2857-3 pmid: 29951700
[24]
BAI T, LI J Q, SINCLAIR A, IMREN S, MERRIAM F, SUN F, O'KELLY M B, NOURIGAT C, JAIN P, DELROW J J, BASOM R S, HUNG H C, ZHANG P, LI B W, HEIMFELD S, JIANG S Y, DELANEY C. Expansion of primitive human hematopoietic stem cells by culture in a zwitterionic hydrogel. Nature Medicine, 2019, 25(10): 1566-1575.

doi: 10.1038/s41591-019-0601-5 pmid: 31591594
[25]
LI Y, ZHANG W Z, CHANG L, HAN Y, SUN L, GONG X J, TANG H, LIU Z P, DENG H C, YE Y X, WANG Y, LI J, QIAO J, QU J, ZHANG W Q, LIU G H. Vitamin C alleviates aging defects in a stem cell model for Werner syndrome. Protein & Cell, 2016, 7(7): 478-488.
[26]
LA FATA G, WEBER P, MOHAJERI M H. Effects of vitamin E on cognitive performance during ageing and in Alzheimer’s disease. Nutrients, 2014, 6(12): 5453-5472.

doi: 10.3390/nu6125453
[27]
GUO Y, DING S J, DING X, LIU Z, WANG J L, CHEN Y, LIU P P, LI H X, ZHOU G H, TANG C B. Effects of selected flavonoids oncellproliferation and differentiation of porcine muscle stem cells for cultured meat production. Food Research International, 2022, 160: 111459.

doi: 10.1016/j.foodres.2022.111459
[28]
YIN R C, MAO S Q, ZHAO B L, CHONG Z C, YANG Y, ZHAO C, ZHANG D P, HUANG H, GAO J, LI Z, JIAO Y, LI C P, LIU S Q, WU D N, GU W K, YANG Y G, XU G L, WANG H L. Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. Journal of the American Chemical Society, 2013, 135(28): 10396-10403.

doi: 10.1021/ja4028346 pmid: 23768208
[29]
MARTACIC J, FILIPOVIC M K, BOROZAN S, CVETKOVIC Z, POPOVIC T, ARSIC A, TAKIC M, VUCIC V, GLIBETIC M. N-acetyl-L-cysteine protects dental tissue stem cells against oxidative stress in vitro. Clinical Oral Investigations, 2018, 22(8): 2897-2903.

doi: 10.1007/s00784-018-2377-2
[30]
XIONG L Y, SUN J M, HIRCHE C, YANG J, YANG Y Q, XIA Y, LEHNHARDT M, WANG R R, FU X. In vitro N-acetyl-L-cysteine promotes proliferation and suppresses interleukin-8 expression in adipose-derived stem cells. Aesthetic Plastic Surgery, 2012, 36(5): 1260-1265.

doi: 10.1007/s00266-012-9960-8
[31]
林芷昕, 谢秋萍, 王长康, 高玉云. N-乙酰半胱氨酸的生理功能及其在畜禽生产中的应用. 动物营养学报, 2022, 34(8): 4857-4866.

doi: 10.3969/j.issn.1006-267x.2022.08.012
LIN Z X, XIE Q P, WANG C K, GAO Y Y. Physiological function of N-acetylcysteine and its application in livestock and poultry production. Chinese Journal of Animal Nutrition, 2022, 34(8): 4857-4866. (in Chinese)

doi: 10.3969/j.issn.1006-267x.2022.08.012
[32]
SUGII S, WONG C Y Q, LWIN A K O, CHEW L J M. Alternative fat: Redefining adipocytes for biomanufacturing cultivated meat. Trends in Biotechnology, 2023, 41(5): 686-700.

doi: 10.1016/j.tibtech.2022.08.005
[33]
HIGUCHI M, DUSTING G J, PESHAVARIYA H, JIANG F, HSIAO S T F, CHAN E C, LIU G S. Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and Forkhead box O1 mediated upregulation of antioxidant enzymes. Stem Cells and Development, 2013, 22(6): 878-888.

doi: 10.1089/scd.2012.0306 pmid: 23025577
[34]
BALAJI S N, TRIVEDI V. Suicidal inactivation of methemoglobin by generation of thiyl radical: Insight into NAC mediated protection in RBC. Current Molecular Medicine, 2013, 13(6): 1000-1009.

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