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. 2019 Dec 12:2019:6493081.
doi: 10.1155/2019/6493081. eCollection 2019.

Coenzyme Q10 Regulation of Apoptosis and Oxidative Stress in H2O2 Induced BMSC Death by Modulating the Nrf-2/NQO-1 Signaling Pathway and Its Application in a Model of Spinal Cord Injury

Xing Li  1   2   3 , Jiheng Zhan  1   2   3 , Yu Hou  1   2   3 , Yonghui Hou  1   2   3 , Shudong Chen  1   2   3 , Dan Luo  1   2   3 , Jiyao Luan  1   2   3 , Le Wang  4 , Dingkun Lin  1   2   3
Affiliations

Coenzyme Q10 Regulation of Apoptosis and Oxidative Stress in H2O2 Induced BMSC Death by Modulating the Nrf-2/NQO-1 Signaling Pathway and Its Application in a Model of Spinal Cord Injury

Xing Li et al. Oxid Med Cell Longev. .

Abstract

Spinal cord injury (SCI) has always been considered to be a devastating problem that results in catastrophic dysfunction, high disability rate, low mortality rate, and huge cost for the patient. Stem cell-based therapy, especially using bone marrow mesenchymal stem cells (BMSCs), is a promising strategy for the treatment of SCI. However, SCI results in low rates of cell survival and a poor microenvironment, which limits the therapeutic efficiency of BMSC transplantation. Coenzyme Q10 (CoQ10) is known as a powerful antioxidant, which inhibits lipid peroxidation and scavenges free radicals, and its combined effect with BMSC transplantation has been shown to have a powerful impact on protecting the vitality of cells, as well as antioxidant and antiapoptotic compounds in SCI. Therefore, we aimed to evaluate whether CoQ10 could decrease oxidative stress against the apoptosis of BMSCs in vitro and explored its molecular mechanisms. Furthermore, we investigated the protective effect of CoQ10 combined with BMSCs transplanted into a SCI model to verify its ability. Our results demonstrate that CoQ10 treatment significantly decreases the expression of the proapoptotic proteins Bax and Caspase-3, as shown through TUNEL-positive staining and the products of oxidative stress (ROS), while increasing the expression of the antiapoptotic protein Bcl-2 and the products of antioxidation, such as glutathione (GSH), against apoptosis and oxidative stress, in a H2O2-induced model. We also identified consistent results from the CoQ10 treatment of BMSCs transplanted into SCI rats in vivo. Moreover, the Nrf-2 signaling pathway was also investigated in order to detail its molecular mechanism, and the results show that it plays an important role, both in vitro and in vivo. Thus, CoQ10 exerts an antiapoptotic and antioxidant effect, as well as improves the microenvironment in vitro and in vivo. It may also protect BMSCs from oxidative stress and enhance their therapeutic efficiency when transplanted for SCI treatment.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Figure 1
Figure 1
Morphologies, phenotypic characterizations, and differentiation of BMSCs. (a) Representative fields of BMSC morphologies at the primary passage (A) and passage 3 (B). (b) The phenotypic characterizations of BMSCs were identified with the corresponding isotype control (C), CD11 (0.2%) (D), and CD73 (92.8%) (F). (c) The differentiation of BMSC analysis: alcian blue staining (F), alizarin red staining (G), and alkaline phosphatase staining (H).
Figure 2
Figure 2
Effects of CoQ10 on the viability of H2O2-induced BMSCs. Cells were pretreated with different concentrations (20, 40, and 80 μM) of CoQ10 for 2 h followed with H2O2 (300 μM) for 24 h. (a) Structure of CoQ10. (b) Cells were cultured with different concentrations (100-500 μM) of H2O2 for 24 h. (c) Cells were cultured with different times (12-24 h) of H2O2 (300 μM). (d) The cell viability was determined by WST-1 assay. (e) The cell death was determined by LDH assay. Data are presented as mean ± SEM. ∗P < 0.05 or ∗∗P < 0.01 compared with the control group. #P < 0.05 compared with the H2O2-induced alone group.
Figure 3
Figure 3
CoQ10 alleviated H2O2-induced BMSC apoptosis. Cells were pretreated with CoQ10 (40 μM) for 2 h followed with H2O2 (300 μM) for 24 h. (a) Annexin V-FITC/PI assay was used to evaluate the apoptosis (A) control group, (B) CoQ10 group, (C) H2O2 group, (D) H2O2+CoQ10 group, and (E) H2O2+CoQ10+brusatol group. (b) TUNEL assays (with green fluorescence) measured the cell apoptosis. (c) The protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (d) The gene expression of Bax, Bcl-2, and Caspase-3 was determined by real-time quantitative PCR. Data are presented as mean ± SEM. ∗P < 0.05 compared with the control group, #P < 0.05 compared with the H2O2-induced alone group, and &P < 0.05 compared with the CoQ10+H2O2-induced group.
Figure 4
Figure 4
CoQ10 scavenges ROS produced by H2O2. (a) H2DCF-DA was used to determine the level of ROS induced by H2O2. (b) Quantitative analysis of DCF fluorescent intensity. (c) GSH assay kit was used to measure the level of GSH. Data are presented as mean ± SEM. ∗P < 0.05 or ∗∗P < 0.01 compared with the control group, #P < 0.05 compared with the H2O2-induced alone group, and &P < 0.05 compared with the CoQ10+H2O2-induced group.
Figure 5
Figure 5
CoQ10 protected BMSCs from H2O2-induced apoptosis and oxidative stress via the Nrf-2/NQO-1 signaling pathway. Cells were pretreated with CoQ10 (40 μM) for 2 h, followed by coincubation with H2O2 (300 μM) for 24 h. Data are presented as mean ± SEM. ∗P < 0.05 compared with the control group, #P < 0.05 compared with the H2O2-induced alone group, and &P < 0.05 compared with the CoQ10+H2O2-induced group.
Figure 6
Figure 6
CoQ10 decreased oxidative stress in a rat SCI model. (a) The level of SOD was measured by a SOD assay kit (b) The level of MDA was measured by a MDA assay kit. (c) The level of GSH was measured by a GSH assay kit. Data are presented as mean ± SEM. ∗P < 0.05 compared with the control group, #P < 0.05 compared with the SCI group, and &P < 0.05 compared with the BMSC group (n = 6).
Figure 7
Figure 7
CoQ10 reduced apoptosis in a rat SCI model. (a) The spine tissue sample protein expression of Bax, Bcl-2, and Caspase-3 was measured by Western blot. (b) Immunohistochemical analysis determined the Caspase-3 protein expression. (c) TUNEL staining of the cell apoptosis rate. Scale bar = 50 μm. Data are presented as mean ± SEM. ∗P < 0.05 compared with the control group, #P < 0.05 compared with the SCI group, and &P < 0.05 compared with the BMSC group (n = 6).
Figure 8
Figure 8
Treatment with CoQ10 combining BMSC transplantation affects the Nrf-2/NQO-1 signaling pathway after SCI. The spine tissue sample protein expression of Nrf-2 and NQO-1 was measured by Western blot. Data are presented as mean ± SEM. ∗P < 0.05 compared with the control group, #P < 0.05 compared with the SCI group, and &P < 0.05 compared with the BMSC group (n = 6).
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