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"

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