Mesenchymal stromal cell neuroprotection of hydrogen peroxide -challenged pheochromocytoma cells through reducing apoptosis and releasing cytokines

Abstract

Background aims. Immunoregulation of mesenchymal stromal cells (MSC) is more efficient at restoring biologic function of injured tissue than transdifferentiation during transplantation. However, the exact mechanisms and characteristics of MSC regarding immunomodulation are still unknown, especially in the damaged niche after hypoxic–ischemic insult. We investigated the anti-apoptotic actions of MSC against the neurotoxicity of hydrogen peroxide (H₂O₂) on pheochromocytoma (PC12) cells. Methods. To mimic hypoxic–ischemic brain injury in vivo, a relatively high H₂O₂ concentration and short period were used to treat PC12 cells. MSC were co-cultured directly with the injured PC12 cells, and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), lactose dehydrogenase (LDH) and nitric oxide (NO) assays, intracellular Ca²⁺ and resting membrane potential (RMP) were analyzed. Apoptotic-associated genes and cytokine releases were detected by reverse transcriptase–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results. After exposure to H₂O₂, the viability of PC12 cells was significantly decreased, while the levels of LDH and NO increased, resulting in intracellular Ca²⁺ accumulation and cell apoptosis. Co-culture of MSC with the H₂O₂-treated PC12 cells sustained viability of the PC12 cells, reduced LDH levels and NO release, and improved Ca²⁺ influx and cell apoptosis. The injured PC12 cells exhibited lower RMP following co-culture with MSC compared with the injured PC12 cells alone. The mRNA expression levels of Bcl-2 were up-regulated and caspase-3 levels down-regulated in the MSC co-culture groups. The release of interleukin (IL)-10 was gradually reduced by co-cultured MSC, while the release of IL-6 was sharply increased following MSC co-culture. Conclusions. These findings indicate that MSC have neuroprotective effects on suppressing cell apoptosis via regulation of the H₂O₂-impaired microenvironment, partly through IL-6 and IL-10 cytokine production.

Key Words::

• apoptosis
• cytokines
• hydrogen peroxide
• hypoxic–ischemic insult
• marrow-derived mesenchymal stromal cells
• pheochromocytoma cells

Acknowledgments

This work was supported by grants of the National Natural Science Foundation in China (numbers 30872670 and 30830106) and the Key Project of Chongqing Municipal Health Bureau, China (number 2009-1-41).

Conflict of interest: All authors declare no financial conflict.