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Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.

Wang W, Zhang Y, Lu W, Liu K - PLoS ONE (2015)

Bottom Line: ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye.The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase).Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Peking University People's Hospital, Beijing, China; Institute of Hematology, Peking University, Beijing, China.

ABSTRACT

Objective: The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.

Methods: The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.

Results: ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

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ROS mediate adipocyte differentiation of MSCs in vitro.(A) Fold increase in ROS on Day 2/Day 0 of differentiation. “NAC” and “NAC+diff” groups were treated with 5 mM NAC for 4 h prior to ROS measurement on Day 2. The NAC treatment was administered daily and lasted to Day 14. (B) Fold increase in ROS on Day 7/Day 0 of differentiation. (C) Fold increase in ROS on Day 14/Day 0 of differentiation. (D, E) NAC diminished lipid accumulation. Cells were fixed and stained with oil red O on Day 14. The oil red O was extracted with isopropanol and absorbance was measured at 518 nm. (F) Gene expression of PPARγ and adiponectin was decreased in the presence of NAC at Day 7 and Day 14 of differentiation compared to control. *P < 0.05.
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pone.0120629.g001: ROS mediate adipocyte differentiation of MSCs in vitro.(A) Fold increase in ROS on Day 2/Day 0 of differentiation. “NAC” and “NAC+diff” groups were treated with 5 mM NAC for 4 h prior to ROS measurement on Day 2. The NAC treatment was administered daily and lasted to Day 14. (B) Fold increase in ROS on Day 7/Day 0 of differentiation. (C) Fold increase in ROS on Day 14/Day 0 of differentiation. (D, E) NAC diminished lipid accumulation. Cells were fixed and stained with oil red O on Day 14. The oil red O was extracted with isopropanol and absorbance was measured at 518 nm. (F) Gene expression of PPARγ and adiponectin was decreased in the presence of NAC at Day 7 and Day 14 of differentiation compared to control. *P < 0.05.

Mentions: To investigate the role of ROS in adipocyte differentiation of MSCs, the change in ROS production during differentiation and the effect of the antioxidant NAC on differentiation in vitro was examined. The NAC treatment started on Day 2 of differentiation and lasted until Day 14. Antioxidant treatment began on Day 2 to avoid interference with mitotic clonal expansion during adipocyte differentiation, as previously described [18]. As shown in Fig. 1A, an increase in cellular ROS was observed in human MSCs on Day 2 of differentiation compared to Day 0. And this increase was abolished by treatment with the antioxidant NAC. The change in ROS levels was examined at Day 7 and Day 14. It was found that the generation of ROS in the early stage of differentiation was gradually attenuated at Day 7 and Day 14 (Fig. 1B-C) compared to Day 2, whereas NAC treatment had no effect. After cells were cultured with the Adipogenesis Differentiation Kit for 14 days, a significant increase in fat droplets and lipid accumulation was observed in differentiated cells compared to undifferentiated cells. Meanwhile, an inhibition effect on lipid accumulation was observed after NAC treatment (Fig. 1D-E). In conjunction with lipid accumulation, mRNA levels of the major adipogenic transcription factor PPARγ and its target gene adiponectin were also significantly decreased at Day 7 and Day 14 in the presence of NAC (Fig. 1F). These results indicate that ROS play an important role in regulating adipocyte differentiation of MSCs that can be regulated by the antioxidant NAC.


Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.

Wang W, Zhang Y, Lu W, Liu K - PLoS ONE (2015)

ROS mediate adipocyte differentiation of MSCs in vitro.(A) Fold increase in ROS on Day 2/Day 0 of differentiation. “NAC” and “NAC+diff” groups were treated with 5 mM NAC for 4 h prior to ROS measurement on Day 2. The NAC treatment was administered daily and lasted to Day 14. (B) Fold increase in ROS on Day 7/Day 0 of differentiation. (C) Fold increase in ROS on Day 14/Day 0 of differentiation. (D, E) NAC diminished lipid accumulation. Cells were fixed and stained with oil red O on Day 14. The oil red O was extracted with isopropanol and absorbance was measured at 518 nm. (F) Gene expression of PPARγ and adiponectin was decreased in the presence of NAC at Day 7 and Day 14 of differentiation compared to control. *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359087&req=5

pone.0120629.g001: ROS mediate adipocyte differentiation of MSCs in vitro.(A) Fold increase in ROS on Day 2/Day 0 of differentiation. “NAC” and “NAC+diff” groups were treated with 5 mM NAC for 4 h prior to ROS measurement on Day 2. The NAC treatment was administered daily and lasted to Day 14. (B) Fold increase in ROS on Day 7/Day 0 of differentiation. (C) Fold increase in ROS on Day 14/Day 0 of differentiation. (D, E) NAC diminished lipid accumulation. Cells were fixed and stained with oil red O on Day 14. The oil red O was extracted with isopropanol and absorbance was measured at 518 nm. (F) Gene expression of PPARγ and adiponectin was decreased in the presence of NAC at Day 7 and Day 14 of differentiation compared to control. *P < 0.05.
Mentions: To investigate the role of ROS in adipocyte differentiation of MSCs, the change in ROS production during differentiation and the effect of the antioxidant NAC on differentiation in vitro was examined. The NAC treatment started on Day 2 of differentiation and lasted until Day 14. Antioxidant treatment began on Day 2 to avoid interference with mitotic clonal expansion during adipocyte differentiation, as previously described [18]. As shown in Fig. 1A, an increase in cellular ROS was observed in human MSCs on Day 2 of differentiation compared to Day 0. And this increase was abolished by treatment with the antioxidant NAC. The change in ROS levels was examined at Day 7 and Day 14. It was found that the generation of ROS in the early stage of differentiation was gradually attenuated at Day 7 and Day 14 (Fig. 1B-C) compared to Day 2, whereas NAC treatment had no effect. After cells were cultured with the Adipogenesis Differentiation Kit for 14 days, a significant increase in fat droplets and lipid accumulation was observed in differentiated cells compared to undifferentiated cells. Meanwhile, an inhibition effect on lipid accumulation was observed after NAC treatment (Fig. 1D-E). In conjunction with lipid accumulation, mRNA levels of the major adipogenic transcription factor PPARγ and its target gene adiponectin were also significantly decreased at Day 7 and Day 14 in the presence of NAC (Fig. 1F). These results indicate that ROS play an important role in regulating adipocyte differentiation of MSCs that can be regulated by the antioxidant NAC.

Bottom Line: ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye.The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase).Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Peking University People's Hospital, Beijing, China; Institute of Hematology, Peking University, Beijing, China.

ABSTRACT

Objective: The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.

Methods: The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.

Results: ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

Show MeSH
Related in: MedlinePlus