Effect of lead on ERK activity and the protective function of bFGF in rat primary culture astroglia

Abstract

Objective: To observe the effects of lead on levels of phosphorylated extracellular signal regulated kinase (p-ERK) in the cytoplasm of primary cultures of rat astroglial cells and the possible protective effect of basic fibroblast growth factor (bFGF) on lead-induced effects.

Methods: The primary astroglia cells from 1~6 d old Wistar rats were cultured. The cells pretreated with the MEK1 (mitogen-activated protein kinase kinase 1) inhibitor PD98059 and bFGF, respectively, were exposed to Pb acetate of different concentrations for different times. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) methods were used to detect the protein and mRNA expressions of ERK.

Results: mRNA expression for ERK peaked 15 min after initiation of lead exposure (P<0.05) and protein expression of p-ERK peaked at 30 min (P<0.05). ERK mRNA levels and p-ERK protein levels returned to baseline after 60 and 120 min of lead exposure, respectively (P>0.05). The increase in p-ERK levels in lead-treated cells could be inhibited by PD098059. Activation of ERK in the cells by lead was prevented by pretreatment with bFGF. Total ERK protein levels did not change under the same experimental conditions (P>0.05).

Conclusion: Low-level lead exposure resulted in transient activation of ERK through the MEK pathway, which then returned to basal levels in the continued presence of lead. Exogenous bFGF protected ERK signaling components in astroglia from lead poisoning.

Fig. 1

Immunohistochemical test for cell purity. (a) Negative control; (b) GFAP immunohistochemical stain Cells were incubated with GFAP antibody (PBS was used as substitute of GFAP in negative control) and observed under the microscope. Almost all cells were stained with brown in the cytoplasm and blue in the karyon (cells were not stained with brown in the cytoplasm in negative control)

Fig. 1

Immunohistochemical test for cell purity. (a) Negative control; (b) GFAP immunohistochemical stain Cells were incubated with GFAP antibody (PBS was used as substitute of GFAP in negative control) and observed under the microscope. Almost all cells were stained with brown in the cytoplasm and blue in the karyon (cells were not stained with brown in the cytoplasm in negative control)

Fig. 2

ERK2 and β-actin simultaneous gel electrophoresis of lead exposure group 1: Blank control; 2~6: 1.0 μmol/L Pb acetate treated for 5, 15, 30, 60 or 120 min. Compared with that of the control, the mRNA expression of ERK2 began to increase at beginning 5 min after the initiation of lead exposure, peaking at 15 min and returning to the basal level thereafter. The experiment was conducted 3 times and a representative gel is shown

Fig. 3

ERK2 and β-actin simultaneous gel electrophoresis of bFGF pretreated group 1~6: 1.0 μmol/L Pb acetate treated for 0, 5, 15, 30, 60 or 120 min after bFGF incubation for 1 h. Compared with the control, the level of ERK2 mRNA expression had no significant change. The experiment was conducted 3 times and a representative gel is shown

Fig. 4

p-ERK and β-actin simultaneous SDS-PAGE gel electrophoresis of groups exposed to different concentrations of lead for 30 min 0: Blank control; 1~3: Concentrations of Pb acetate were 0.2, 1.0 and 10 μmol/L in order. Compared with that of the control, the results showed an inverted U shaped curve. The experiment was conducted 3 times and a representative gel is shown

Fig. 5

p-ERK and β-actin simultaneous SDS-PAGE gel electrophoresis of groups exposed to 1.0 μmol/L lead for various time 0: Blank control; 1~5: 1.0 μmol/L Pb acetate treated for 5, 15, 30, 60 or 120 min respectively. Compared with that of the control, the level of p-ERK protein expression began to increase from the 5 min point to the peak at 30 min, and then came back to the normal level. The experiment was conducted 3 times and a representative gel is shown

Fig. 6

p-ERK and β-actin simultaneous SDS-PAGE gel electrophoresis for PD98059 pretreated group 0~5: 1.0 μmol/L Pb acetate treated for 0, 5, 15, 30, 60 or 120 min after 100 μmol/L PD98059 incubation for 1 h. PD98059 prevented the lead-induced increase of p-ERK protein. The experiment was conducted 3 times and a representative gel is shown

Fig. 7

p-ERK and β-actin simultaneous SDS-PAGE gel electrophoresis for bFGF pretreated group 0~5: 1.0 μmol/L Pb acetate treated for 0, 5, 15, 30, 60 or 120 min after 40 ng/L bFGF incubation for 1 h. bFGF could restrain the increase of lead-induced p-ERK protein. The experiment was conducted 3 times and a representative gel is shown