Basic fibroblast growth factor protects C17.2 cells from radiation-induced injury through ERK1/2

Abstract

Aims: To establish a radiation-induced neural injury model using C17.2 neural stem cells (NSCs) and to investigate whether basic fibroblast growth factor (bFGF) can protect the radiation-induced injury of C17.2 NSCs. Furthermore, we aim to identify the possible mechanisms involved in this model.

Methods: C17.2 NSCs received a single exposure (3, 6, and 9 Gy, respectively) at a dose rate of 300 cGy/min with a control group receiving 0 Gy. Different concentrations of bFGF were added for 24 h, 5 min postirradiation. The MTS assay and flow cytometry were used to detect cytotoxicity and apoptosis. Expression of FGFR1, ERK1/2, and p-ERK1/2 proteins was detected with or without U0126 was pretreated prior to C17.2 NSCs receiving irradiation.

Results: C17.2 NSCs showed a dose-dependent cell death as the dose of radiation was increased. Additionally, the rate of apoptosis in the C17.2 NSCs reached 31.2 ± 1.23% in the 6 Gy irradiation group, which was the most significant when compared to the other irradiation treated groups. bFGF showed protective effect on cell apoptosis in a dose-dependent manner. The mean percentage of apoptotic cells decreased to 7.83 ± 1.75% when 100 ng/mL bFGF was given. Furthermore, U0126 could block the protective effect of bFGF by inhibiting the phosphorylation of ERK1/2.

Conclusions: An in vitro cellular model of radiation-induced apoptosis of NSCs, in C17.2 NSCs, was developed successfully. Additionally, bFGF can protect neurons from radiation injury in vitro via the ERK1/2 signal transduction pathway.

Figure 1

Radiation‐induced neural toxicity on C17.2 NSCs. Panel A, C17.2 NSCs received different doses of irradiation treatment, and the cell viability was analyzed by the MTS assay. The effect of irradiation on C17.2 NSCs shows a dose‐dependent decrease in cell viability. Panel B, Data from flow cytometric analysis shown that when cells were exposed to the lower dose of irradiation (≤6 Gy), apoptosis was the leading cause of cell death. As the dose of irradiation increased to 9 Gy, necrosis began to play a prominent role in cell viability. NSC, neural stem cell.

Figure 2

Effect of bFGF on radiation‐induced apoptosis. Panel A, with increasing concentrations of bFGF, apoptosis of C17.2 cells decreased. The most protective concentration of bFGF for C17.2 NSCs against apoptosis was 100 ng/mL. Panel B, C17.2 NSCs pretreated with 10 μM of U0126 1 h before receiving 6 Gy irradiation could prevent the protective effect from the 100 ng/mL bFGF treatment on cell apoptosis. *P < 0.05, **P < 0.01 compared to control group. bFGF, basic fibroblast growth factor; NSC, neural stem cell.

Figure 3

Effect of bFGF on the expression of FGFR1 in C17.2 NSCs. Panel A, B, ICC staining of FGFR1 in C17.2 NSCs. Panel B, increased expression of FGFR1 after bFGF treatment. Panel C, Western blots for FGFR1 expression showing the average densitometric values after β‐actin normalization represents the protein levels for each data point (n = 3). #P < 0.001 compared to untreated group. Scale bar, 30 μm. bFGF, basic fibroblast growth factor; ICC, immunocytochemistry; NSC, neural stem cell.

Figure 4

Effects of U0126 on the expression of ERK1/2 in C17.2 NSCs. Western blots for ERK1/2 showed that U0126 reduced the phosphorylation of ERK1/2. The average densitometric values after β‐actin normalization represents the protein levels for each data point (n = 3). #P < 0.001 compared to U0126 untreated group. NSC, neural stem cell.