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. 2019 Feb 28:10:163.
doi: 10.3389/fphar.2019.00163. eCollection 2019.

VSports最新版本 - Icariin Protects Bone Marrow Mesenchymal Stem Cells Against Iron Overload Induced Dysfunction Through Mitochondrial Fusion and Fission, PI3K/AKT/mTOR and MAPK Pathways

Xudong Yao  1 , Xingzhi Jing  1 , Jiachao Guo  1 , Kai Sun  1 , Yi Deng  1 , Yong Zhang  1 , Fengjing Guo  1 , Yaping Ye  1
Affiliations

Icariin Protects Bone Marrow Mesenchymal Stem Cells Against Iron Overload Induced Dysfunction Through Mitochondrial Fusion and Fission, PI3K/AKT/mTOR and MAPK Pathways

"VSports最新版本" Xudong Yao et al. Front Pharmacol. .

Abstract

Iron overload has been reported to contribute to bone marrow mesenchymal stem cells (BMSCs) damage, but the precise mechanism still remains elusive. Icariin, a major bioactive monomer belonging to flavonoid glucosides isolated from Herba Epimedii, has been shown to protect cells from oxidative stress induced apoptosis. The aim of this study was to investigate whether icariin protected against iron overload induced dysfunction of BMSCs and its underlying mechanism. In this study, we found that iron overload induced by 100 μM ferric ammonium citrate (FAC) caused apoptosis of BMSCs, promoted cleaved caspase-3 and BAX protein expressions while inhibited Bcl-2 protein expression, which effects were significantly attenuated by icariin treatment. In addition, iron overload induced significant depolarization of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation and inhibition of mitochondrial fusion/fission, which effects were also attenuated by icariin treatment. Meanwhile, we found that iron overload induced by 100 μM FAC significantly inhibited mitochondrial fission protein FIS1 and fusion protein MFN2 expressions, inhibited DRP1 and Cytochrome C protein translocation from the cytoplasm to mitochondria. Icariin at concentration of 1 μM was able to promote mitochondrial fission protein FIS1 and fusion protein MFN2 expressions, and increase DRP1 and cytochrome C protein translocation from the cytoplasm to mitochondria. Further, osteogenic differentiation and proliferation of BMSCs was significantly inhibited by iron overload, but icariin treatment rescued both osteogenic differentiation and proliferation of BMSCs. Further studies showed that icariin attenuated iron overload induced inactivation of the PI3K/AKT/mTOR pathway and activation of the ERK1/2 and JNK pathways. In summary, our study indicated that icariin was able to protect against iron overload induced dysfunction of BMSCs. These effects were potentially related to the modulation of mitochondrial fusion and fission, activation of the PI3K/AKT/mTOR pathway and inhibition of ERK1/2 and JNK pathways VSports手机版. .

Keywords: MAPK pathway; PI3K/AKT/mTOR pathway; bone marrow mesenchymal stem cells; icariin; iron overload; mitochondrial fusion and fission V体育安卓版. .

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Figures (V体育ios版)

FIGURE 1
FIGURE 1
Icariin attenuated iron overload induced apoptosis of BMSCs. (A) The cytotoxicity of FAC on BMSCs viability was evaluated using the concentration of 0, 10, 50, and 100 μM after 24 and 48 h. ∗P < 0.05 versus control. (B) Icariin effectively attenuated the detrimental effects of FAC on BMSCs viability at concentrations of 0.1, 1, and 10 μM. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (C) Cleaved caspase-3, Bcl-2 and BAX protein levels were determined by Western blot analysis at 48 h. (D) Band density ratios of cleaved caspase-3 to β-actin and BAX to Bcl-2 in the Western blots were quantified by densitometry. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (E) Flow cytometric analysis of BMSCs stained with Annexin V-FITC/PI. (F) Percentage of apoptosis rates were expressed as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
FIGURE 2
FIGURE 2
Icariin protected BMSCs against iron overload induced collapse in mitochondrial membrane potential (MMP). BMSCs were treated with 100 μM FAC or 100 μM FAC combined with icariin (0.01, 0.1, 1, and 10 μM) for 48 h. (A) Representative fluorescence images of MMP after incubating with JC-1. Red fluorescence represents JC-1 aggregates in healthy mitochondria whereas green fluorescence represents JC-1 monomer indicating MMP dissipation. Merged images represent co-localization of the JC-1 aggregates and JC-1 monomers. (B) Representative graphs of the flow cytometric analysis after incubating with JC-1. FL1 represents JC-1 green and FL2 represents JC-1 red. (C) Changes in the MMP were represented as the red fluorescence ratio and data were presented as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
FIGURE 3
FIGURE 3
Icariin protected BMSCs against iron overload induced reactive oxygen species (ROS) generation. (A) Representative images of BMSCs with intracellular ROS stained by the fluorescence probe DCFH-DA after treatment with 100 μM FAC or 100 μM FAC combined with 1 μM icariin for 48 h. (B) Flow cytometric analysis of ROS production after staining with DCFH-DA. (C) Bar graphs showing the mean fluorescence intensity (MFI) of ROS levels in BMSCs. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
FIGURE 4
FIGURE 4
Icariin protected BMSCs against iron overload induced damage to the mitochondria through modulating mitochondrial fusion and fission. (A) Representative fluorescence images of the mitochondria (top row) and local amplification images of the selected area in the top row. Morphology of the mitochondria is stained with Mito-Tracker Green. The magnification time for the top row fluorescence images is 400. (B) BMSCs were treated with 10, 50, and 100 μM FAC for 48 h. The protein expression levels of FIS1 and MFN2 were evaluated with Western blot. (C) BMSCs were treated with 100 μM FAC and 100 μM FAC combined with different concentrations of icariin (0.01–10 μM) for 48 h. The protein expression levels of FIS1 and MFN2 were evaluated with Western blot. (D) The densitometric analysis of FIS1 and MFN2 protein expressions were normalized to β-actin. Data are shown as means ± SD. ∗P < 0.05 versus control. (E) The densitometric analysis of FIS1 and MFN2 protein expressions were normalized to β-actin. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (F) BMSCs were treated with 10, 100 μM FAC and 100 μM FAC combined with 1 μM icariin for 48 h. Cytosolic and mitochondrial proteins were separately extracted, relative expression of DRP1 and cytochrome C (cyt c) were evaluated with Western blot. COX IV was selected as the internal control for the mitochondrial protein. (G) The densitometric analysis of DRP1 and cyt c protein expressions was normalized to β-actin (cytosolic proteins) or COX IV (mitochondrial proteins). Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
FIGURE 5
FIGURE 5
Icariin exerted protective effects on iron overload induced osteogenic differentiation of BMSCs. (A) BMSCs were cultured in osteogenic medium or osteogenic medium supplemented with 100 μM FAC or osteogenic medium supplemented with 100 μM FAC and 1 μM icariin. Alizarin Red staining was performed 21 days after cell culture to demonstrate extracellular mineralization. (B) BMSCs were cultured with osteogenic medium or osteogenic medium supplemented with 100 μM FAC or osteogenic medium supplemented with 100 μM FAC combined with different concentrations of icariin. RUNX2, OPN and active β-catenin protein expressions were investigated with Western blot after cell culture for 72 h. (C) The densitometric analysis of RUNX2, OPN and active β-catenin expressions were normalized to β-actin. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (D) BMSCs were cultured with osteogenic medium or osteogenic medium supplemented with 100 μM FAC or osteogenic medium supplemented with 100 μM FAC combined with 1 μM icariin. Relative expression levels of RUNX2 and OPN were evaluated by RT-PCR after 72 h. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
FIGURE 6
FIGURE 6
(A) BMSCs were treated with 100 μM FAC and 100 μM FAC combined with different concentrations of icariin (0.01–10 μM) for 48 h. Cyclin D1 protein expression was evaluated with Western blot. (B) The densitometric analysis of cyclin D1 protein expression was normalized to β-actin. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (C) Representative fluorescence images of Edu staining of BMSCs. Proliferating BMSCs positively stained with EdU showed red color. Cell nuclei stained with DAPI showed blue color.
FIGURE 7
FIGURE 7
Effect of iron overload and icariin on the PI3K/AKT/mTOR pathway and MAPK pathway. BMSCs were treated with 100 μM FAC and 100 μM FAC combined with 1 μM icariin for 30 min. (A) Protein expression levels of t-PI3K, t-AKT, t-mTOR, p-PI3K, p-AKT, and p-mTOR were evaluated with Western blot. (B) Protein expression levels of t-ERK1/2, t-P38, t-JNK, p-ERK1/2, p-P38, and p-JNK were evaluated with Western blot. (C) The densitometric analysis of p-PI3K expression was normalized to t-PI3K, the densitometric analysis of p-AKT expression was normalized to t-AKT, and the densitometric analysis of p-mTOR expression was normalized to t-mTOR. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group. (D) The densitometric analysis of p-ERK expression was normalized to t-ERK, the densitometric analysis of p-P38 expression was normalized to t-P38, and the densitometric analysis of p-JNK expression was normalized to t-JNK. Data are shown as means ± SD. ∗P < 0.05 versus control, #P < 0.05 versus 100 μM FAC group.
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