Thrombin-induced neuronal protection: role of the mitogen activated protein kinase/ribosomal protein S6 kinase pathway

Abstract

Our previous studies have found that intracerebral pretreatment with a low dose of thrombin (thrombin preconditioning, TPC) reduces infarct volume and attenuates brain edema after focal cerebral ischemia. In this study, we examined whether TPC protects against the neuronal death induced by oxygen glucose deprivation (OGD), and whether the protection is through thrombin receptors and the p44/42 mitogen activated protein kinases (MAPK)/ribosomal protein S6 kinases (p70 S6K) pathway. Expression of protease-activated receptors (PARs) mRNA was detected in cultured primary rat neurons and thrombin upregulated PAR-1 and PAR-4 mRNA expression. TPC reduced OGD-induced neuronal death (e.g. dead cells: 52.5 ± 5.4% vs. 72.3 ± 7.2% in the control group, n=6, p<0.01). Agonists of PAR-1 and PAR-4 mimicked the effects of thrombin and reduced OGD-induced neuronal death. Pretreatment with thrombin or PAR agonists induced the upregulation of activated p44/42 MAPK and p70S6K (Thr 421/Ser 424). PD98059, an inhibitor of p44/42 MAPK kinase, blocked thrombin-induced upregulation of activated p44/42 MAPK and p70S6K. It also reduced TPC-induced neuronal protection (e.g. dead cells: 68.2 ± 5.2% vs. 56.9 ± 4.6% in vehicle+TPC group, n=6, p<0.05). These results suggest that TPC-induced ischemic tolerance is through activation of thrombin receptors and the p44/42 MAPK/p70S6K pathway.

Figure 1

Primary cultured neurons were pretreated with different doses of thrombin for 24 hours and then exposed to oxygen glucose deprivation (OGD) for 2 hours. The levels of lactate dehydrogenase (LDH) released into the medium (A) and the percent of dead cells by live/dead cell staining (B and C) were measured after 22 hours of reoxygenation. Values are expressed as means ± SD. *p< 0.05, # p< 0.01 vs. control (0 U/ml thrombin). In (C), neurons stained red indicate dead neurons and those green are live. TPC = thrombin preconditioning with 1U/ml. Scale bar = 20 μM.

Figure 2

PAR-1 and PAR-4 mRNA levels in cultured neurons 24 hours after thrombin (1 U/ml) stimulation. (A) Lanes 1–3: vehicle control-treated neurons; Lanes: thrombin-treated neurons. (B) Bar graphs showing neuronal PAR-1 and PAR-4 mRNA levels. Values are means ± S.D., #p<0.01 vs. control.

Figure 3

Neurons pretreated with agonist peptides of PAR-1 and PAR-4 mimicked the effects of thrombin. Neurons were pretreated with vehicle, thrombin (1 U/ml) or agonists of PAR-1 and PAR-4 (50 nM) for 24 hours and then exposed to OGD for 2 hours. Levels of LDH release in the culture medium (A) and percent of dead cells (B) were measured after 22 hours of reoxygenation. Values are expressed as means ± SD. #p< 0.01, compared with control group.

Figure 4

Immunoreactivity of activated p44/42 MAPK and p70 S6K (Thr421/Ser424) in neurons 24 hours after vehicle or thrombin (1 U/ml) treatment. Scale bar=10 μm.

Figure 5

Activated p44/42 MAPK and p70S6K (Thr421/Ser424) protein levels in neurons after vehicle (lanes 1–3), thrombin (1 U/ml, lanes 4–6) or thrombin (1U/ml) plus PD098059 (20 μM, lanes 7–9) treatment for 24 hours. Values are mean ± SD, *p< 0.05 vs. the other groups.

Figure 6

Western blot showing activated p44/42 MAPK and p70S6K (Thr421/Ser424) protein levels in neurons 24 hours after vehicle (lanes 1–3), PAR-1 agonist (50nM, lanes 4–6) or PAR-4 (50nM, lanes 7–9) agonist treatment. Values are mean ± SD, *p< 0.05, #p < 0.01 vs. vehicle-treated group.

Figure 7

Neurons were pretreated with PD098059 (20 μM), an inhibitor of p44/42 MAPK kinase, or vehicle 60 minutes before thrombin (1 U/ml) treatment. Neurons were then exposed to OGD for 2 hours and LDH levels (A) and number of dead cells (B) were measured after 22 hours of reoxygenation. Values are mean±SD, *p<0.05, vs. the other groups.