Ann Surg Treat Res.  2019 Oct;97(4):159-167. 10.4174/astr.2019.97.4.159.

Antioxidant action of hypoxic conditioned media from adipose-derived stem cells in the hepatic injury of expressing higher reactive oxygen species

• Hong HE
• Kim OH
• Kwak BJ
• Choi HJ
• im KH
• Ahn J
• Kim SJ

• KMID: 2459313
• DOI: http://doi.org/10.4174/astr.2019.97.4.159

Abstract

PURPOSE
Almost all liver diseases are known to be accompanied by increased levels of reactive oxygen species (ROS), regardless of the cause of the liver disorder. However, little is known about the role of hypoxic conditioned media (HCM) in the view of pro-oxidative/antioxidative balance.
METHODS
Normoxic conditioned media (NCM) and HCM were obtained after culturing adipose-derived stem cells in 20% Oâ‚‚ or 1% Oâ‚‚ for 24 hours, respectively. Their effects on the expression of various markers reflecting pro-oxidative/antioxidative balance were investigated in both in vitro (thioacetamide-treated AML12 cells) and in vivo (partially hepatectomized mice) models of liver injury, respectively.
RESULTS
HCM treatment induced the higher expression of antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase than did NCM in the in vitro model of liver injury. We also found that HCM increased the expression of nuclear factor erythroid 2-related factor (NRF2). The in vivo models of liver injury consistently validated the phenomenon of upregulated expression of antioxidant enzymes by HCM.
CONCLUSION
We thus could conclude that HCM provides protection against ROS-related toxicity by increasing the expression of antioxidant enzymes, in part by releasing NRF2 in the injured liver.

Keyword

Antioxidants; Conditioned culture media; Mesenchymal stem cell; Reactive oxygen species; Secretome

MeSH Terms

Antioxidants
Catalase
Culture Media, Conditioned*
Glutathione Peroxidase
In Vitro Techniques
Liver
Liver Diseases
Mesenchymal Stromal Cells
Reactive Oxygen Species*
Stem Cells*
Superoxide Dismutase
Antioxidants
Catalase
Culture Media, Conditioned
Glutathione Peroxidase
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 Validation of the in vitro effects of HCM. (A) Cell viability assay showing that HCM most significantly increased the cell viability of TAA-treated AML12 cells. (B) ROS assay showing that HCM most significantly downregulated intracellular ROS, especially in the TAA-treated AML12 cells. (C) Real-time polymerase chain reaction results showing that HCM most significantly increased the mRNA levels of 3 antioxidant genes, such as SOD, GPx, and catalase, in the TAA-treated AML12 cells. Values are presented as mean ± standard deviation of 3 independent experiments. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ct, control; HCM, hypoxic conditioned media that means the secretomes obtained from 5% hypoxic culturing of adipose-derived stem cells (ASCs) for 24 hours; NCM, normoxic conditioned media that means the secretomes obtained from normoxic culturing of ASCs for 24 hours; TAA, thioacetamide; ROS, reactive oxygen species; SOD, superoxide dismutase; GPx, glutathione peroxidase. *P < 0.05.

• 

  Fig. 2 Western blot analyses showing in vitro effects of HCM at the protein level. HCM was found to significantly increased NRF2, antioxidant proteins (SOD, GPx, and catalase), and an antioxidant protein (Bcl-xL), whiling decreasing the expression of a pro-apoptotic protein (BIM). Values are presented as mean ± standard deviation of 3 independent experiments. Ct, control; HCM, hypoxic conditioned media that means the secretomes obtained from 5% hypoxic culturing of adipose-derived stem cells (ASCs) for 24 hours; NCM, normoxic conditioned media that means the secretomes obtained from normoxic culturing of ASCs for 24 hours; TAA, thioacetamide; NRF2, nuclear factor erythroid 2-related factor 2; SOD, superoxide dismutase; GPx, glutathione peroxidase; BIM, Bcl-2-like protein 11; Bcl-xL, B-cell lymphoma-extra large. *P < 0.05.

• 

  Fig. 3 Western blot analyses showing in vivo effects of HCM in the liver specimens of 70% partially hepatectomized mice. HCM administration most significantly increased the expression of 3 antioxidant proteins, such as SOD, GPx, and catalase in the liver. In addition, HCM administration most significantly decreased the expression of BIM, while most significantly increasing the expression of Bcl-xL in the liver. Values are presented as mean ± standard deviation of 3 independent experiments. PH, partial hepatectomy; HCM, hypoxic conditioned media that means the secretomes obtained from 5% hypoxic culturing of adipose-derived stem cells (ASCs) for 24 hours; NCM, normoxic conditioned media that means the secretomes obtained from normoxic culturing of ASCs for 24 hours; SOD, superoxide dismutase; GPx, glutathione peroxidase; BIM, Bcl-2-like protein 11; Bcl-xL, B-cell lymphoma-extra large. *P < 0.05.

• 

  Fig. 4 Immunohistochemical stains of the liver specimens of 70% partially hepatectomized mice. (A) The immunohistochemical stains showing that HCM administration most significantly increased the expression of 3 antioxidant proteins, such as SOD, GPx, and catalase in the liver. Values are presented as mean ± standard deviation of 3 independent experiments. (B) LC/MS analysis of the components of HCM. LC/MS analysis identified 20 proteins that were not identified in NCM but only in in HCM. Of the 20 secretary proteins, decorin was found to be most prominently elevated compared with other components. PH, partial hepatectomy; HCM, hypoxic conditioned media that means the secretomes obtained from 5% hypoxic culturing of adipose-derived stem cells (ASCs) for 24 hours; NCM, normoxic conditioned media that means the secretomes obtained from normoxic culturing of ASCs for 24 hours; SOD, superoxide dismutase; GPx, glutathione peroxidase. *P < 0.05.

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  Fig. 5 Proposed mechanisms by which HCM reduces liver injury by upregulating the expression of proteins with higher antioxidant activities. HCM, hypoxic conditioned media that means the secretomes obtained from 5% hypoxic culturing of adipose-derived stem cells for 24 hours; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species.

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