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. 2018 Jul 6;19(1):12.
doi: 10.1186/s12860-018-0163-2.

mTOR and ROS Regulation by Anethole on Adipogenic Differentiation in Human Mesenchymal Stem Cells

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Free PMC article

mTOR and ROS Regulation by Anethole on Adipogenic Differentiation in Human Mesenchymal Stem Cells

Yun-Hee Rhee et al. BMC Cell Biol. .
Free PMC article

Abstract

Background: Adipocyte differentiation of human mesenchymal stem cells (hMSCs) is dependent on mitochondrial metabolism and reactive oxygen species (ROS) to initiate adipocyte differentiation. Although anethole has been known as an anti-oxidant and lipid peroxidation inhibitor, there is little investigated about its role in adipogenic differentiation.

Methods: The effects on cytotoxicity and proliferation of anethole in hMSCs were measured by the MTT assay. The anti-adipogenic effect of anethole on hMSCs was analyzed by Oil Red O staining and western blot analysis. The anti-oxidant activity of anethole on hMSC was assessed by flowcytometry and fluorescence staining using 2',7' -dichlorofluorescin diacetate (DCFDA). The western blotting was used to detect of phospho-Akt, phospho-mTOR, phospho-p70S6K, PPARγ, and phsopho-AMP-activated kinase (AMPK).

Results: Anethole suppressed the adipogenic differentiation of hMSCs through down-regulation of Akt-mTOR-p70S6K-PPARγ and up-regulation of AMPK. Anethole affected oxidative conditions through ROS generation. Anethole also rescued AMPK activity and reduced activation of mTOR-p70S6K-PPARγ under oxidative conditions in presence of exogenous hydrogen peroxide.

Conclusion: ROS and mTOR regulation is a crucial factor in adipogenic differentiation, anethole has an important role in regulating activities of mTOR/PPARγ and ROS control in adipogenic differentiation of hMSCs.

Keywords: AMPK; Adipogenesis; Anethole; ROS; hMSC; mTOR.

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The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Effect of anethole on cytotoxicity and proliferation in hMSCs. a The structure of anethole (1-methoxy-4-(1-propenyl) benzene). b Cells were seeded onto a 96-well plate at a density of 5000 cell/well, and were treated at various concetrations (0, 50, 100, 150, 200, and 250 μM) for 24 h. The cell viability was calculated as a percentage of viable cells in anethole-treated group vesus the untreated control. c For the proliferation assay, every step was performed as described previously and assessment was made at days 2, 4, and 6 post anethole treatment. Each experiment was repeated three times and the values are presented as mean ± S.D. d hMSCs were incubated in adipogenic differentiation media for 4 weeks in presence or absense of 50 μM anethole. Cells were fixed with 4% formaldehyde and stained with 0.5% Oil Red O staining solution at first day of each week for 4 weeks and phostographed by microscopy. e The stained-lipid accumulation was measured by dissoloving the cell contents in isopropanol and reading their absorbace at 405 nm by a microplate reader. Each experiment was repeated four times and the values are presented as mean ± S.D. *p < 0.1, **p < 0.05, and ***p < 0.001
Fig. 2
Fig. 2
Western blot analysis of anethole on adipogenic differentiation in hMSCs. a hMSCs were pretreated with 50 μM anethole and incubated in adipogenic media for 4 weeks. Once a week, whole cell pellets were lysed in lysis buffer and immnoblotted for p-mTOR, mTOR, p-p70S6K, p70S6K, PPAR-γ, p-Akt, Akt, p-AMPKand AMPK. b The phosphorylated expression of each protein was caculated by dividing the total form of corresponding protein, and was normalized by β-actin. The relative folds were measured and calculated using Image J. Each experiment was repeated three times and the values are presented as mean ± S.D. **p < 0.05, and ***p < 0.001
Fig. 3
Fig. 3
Effect of anethole on excessive ROS generation in hMSCs. a hMSCs were exposed at 2 mM H2O2 for 30 min and incubated for 2 days in presence or absence of 50 μM anethole. ROS was measured by staining the cells with DCFDA cellular ROS detection assay kit according to the manufacturer’s instructions. After staining, cells were strained briefly and analyzed using Accuri-C6. b ROS generation was also observed under a fluorescence microscope at 200× magnification after same treatment previously described. c Whole cell pellets underwent western blot analysis for p-mTOR and p-AMPK. d Each protein expression was exhibited in the same manner as described in Fig. 3b. Each experiment was repeated three times and the values are presented as mean ± S.D. **p < 0.05, and ***p < 0.001
Fig. 4
Fig. 4
Western blot analysis of theffect of anethole on transcriptional factors with excessive ROS during adipogenic differentiation. a 2 mM H2O2 was added for 30 min on the first day of adipogenic differentiation. After exposure to H2O2, hMSCs were incubated with adipogenic media for 2 weeks. Whole cell lysates were prepared equally at day 14 as described in Fig. 3. b Each protein expression was exhibited in the same manner as described in Fig. 3b. Each experiment was repeated three times and the values are presented as mean ± S.D. **p < 0.05, and ***p < 0.001

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