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. 2018 Jul 18;18(1):84.
doi: 10.1186/s12871-018-0553-1.

Mesenchymal stromal cells attenuate sevoflurane-induced apoptosis in human neuroglioma H4 cells

Yanyong Cheng  1 , Yunfeng Jiang  1 , Lei Zhang  1 , Jiayi Wang  1 , Dongdong Chai  1 , Rong Hu  1 , Chunzhu Li  1 , Yu Sun  2 , Hong Jiang  3
Affiliations

Mesenchymal stromal cells attenuate sevoflurane-induced apoptosis in human neuroglioma H4 cells

Yanyong Cheng et al. BMC Anesthesiol. .

Abstract

Background: Inhalation of sevoflurane can induce neuronal apoptosis, cognitive impairment and abnormal behaviors. Bone marrow mesenchymal stem cells (MSCs) can secret neurotrophic factors and cytokines to protect from oxidative stress-related neuronal apoptosis. However, whether MSCs can protect from sevoflurane-induced neuronal apoptosis and the potential mechanisms are unclear.

Methods: A non-contact co-culture of MSCs with human neuroglioma H4 cells (H4 cells) was built. H4 cells were co-cultured with MSCs or without MSCs (control) for 24 h. The co-cultured H4 cells were exposed to 4% sevoflurane for 6 h. The levels of caspase-3, reactive oxygen species (ROS), adenosine triphosphate (ATP), and the release of cytochrome C were determined by Western blot and fluorescence assay.

Results: Sevoflurane exposure significantly elevated the levels of cleaved caspase 3 and Bax in H4 cells. However, these phenomena were significantly offset by the co-culture with MSCs in H4 cells. Co-culture with MSCs before, but not after, sevoflurane exposure, significantly attenuated sevoflurane-induced ROS production in H4 cells. MSCs prevented sevoflurane-mediated release of cytochrome C from the mitochondria and production of ATP in H4 cells.

Conclusions: Our study indicated that soluble factors secreted by MSCs attenuated the sevoflurane-induced oxidative stress and apoptosis of neuronal cells by preserving their mitochondrial function.

Keywords: Apoptosis, mitochondrial dysfunction; Mesenchymal stem cells; Reactive oxygen species; Sevoflurane.

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Conflict of interest statement

Ethics approval and consent to participate

All experimental protocols were approved by the Animal Care and Use of Shanghai Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Flowchart of the experimental design
Fig. 2
Fig. 2
MSCs mitigate the sevoflurane-induced caspase 3 cleavage and Bax expression in H4 cells. H4 cells were cultured alone or co-cultured with MSCs for 24 h and then exposed to 4% sevoflurane for 6 h in transwell plates. Subsequently, some cells from each group were cultured for another 24 h. The relative levels of cleaved caspase 3 and Bax expression in H4 cells were determined after sevoflurane exposure at 0 and 24 h by Western blot. Data are representative images or expressed as the mean ± SD of each group (n = 6) from three separate experiments. The levels of each protein expression in the control cells were designated as 100%. a. Western blot analysis; (b-c) Quantitative analysis
Fig. 3
Fig. 3
MSCs attenuate the sevoflurane-induced ROS production in H4 cells. The levels of ROS in the different groups of cells were characterized after staining with DCFH-DA by fluorescent microscopy and spectrum. Data are representative images or expressed as the mean ± SD of each group (n = 6) from three separate experiments. a. Microscopy characterization of ROS levels in H4 cells. b. Quantitative analysis. c. NAC scavenges the sevoflurane-induced ROS in H4 cells. H4 cells were treated with, or without, oxidant or exposed to sevoflurane for 6 h in the presence of NAC. The levels of intracellular ROS were measured by fluorescent spectrum. d. Quantitative analysis. The levels of ROS in the control cells were designated as 100%
Fig. 4
Fig. 4
NAC attenuates the sevoflurane-elevated caspase 3 cleavage in H4 cells. H4 cells were treated with vehicle (CON), SEV, Oxidant alone or together with MSCs (SEV + MSCs) or SEV + NAC. The relative levels of cleaved caspase 3 in the different groups of cells were determined by Western blot. Data are representative images or expressed as the mean ± SD of each group (n = 6) from three separate experiments. a. Western blot analysis. b. Quantitative analysis. The levels of each protein expression in the control cells were designated as 100%
Fig. 5
Fig. 5
MSCs inhibit the sevoflurane-induced mitochondrial cytochrome C release in H4 cells. The mitochondria and cytosol of the different groups of H4 cells were extracted and the relative levels of cytochrome C were determined by Western blot. Furthermore, the levels of ATP in the different groups of cells were measured. Data are representative images or expressed as the mean ± SD of each group (n = 6) from three separate experiments. a. Western blot analysis. b and c. Quantitative analysis. d. The levels of ATP. The levels of each protein expression or ATP in the control cells were designated as 100%
Fig. 6
Fig. 6
Co-culture with MSCs after sevoflurane exposure does not alter the sevoflurane-induced ROS production in H4 cells. H4 cells were cultured for 24 h and exposed to sevoflurane, followed by co-cultured with MSCs or cultured alone for 24 h. The control H4 cells were cultured alone throughout the experimental period. The cells were stained with DCFH-DA and the levels of ROS were determined by fluorescent microscopy and spectrum. Data are representative images or expressed as the mean ± SD of each group (n = 6) from three separate experiments. a. Fluorescent microscopy analysis of ROS in H4 cells. b. Fluorescent spectrum analysis of the ROS levels in H4 cells. The levels of ROS in the control cells were designated as 100%
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