| Objective: Mitogen-activated protein kinases (MAPKs) is a family of widespread protein kinases containing the residue of serine and threonine in cell, which participate in many physiological processes including cell growth, development, division and apoptosis. p38 MAPK is a member of MAPKs family. Many studies have proved that the relation ship between p38 MAPK and brain ischemic tolerance as well as brain ischemic injury is intimate. Our previous study has proved the cerebral ischemic preconditioning (CIP) via up-regulating of GLT-1 protein induced brain ischemic tolerance. However, the mechanism of which is not clear until now. By our knowledge, no literature is about whether p38 MAPK participates in the up-regulating of GLT-1 protein during brain ischemic tolerance induced by CIP. Therefore, the present study is aimed to observe whether p38 MAPK participates in the above process. The result will give new idea to prevent and cure brain ischemia.Method: Three hundred adult male Wistar rats (280-320 g) were used and randomly divided into 3 parts as follows.1 The effects of CIP on p-p38 MAPK protein and GLT-1 proteinExcept for the control group, rats of the other groups were permanently occluded bilateral vertebral arteries and recovered for 2 days before brain ischemia or sham operation, the details are as follows:①control group (n=5): no treatment;②sham group (n=45): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow;③CIP group (n=45): the BCCA were clamped for 3 min then reperfused with the blood flow;④ischemic insult (II) group (n=45): the BCCA were clamped for 8 min then reperfused with the blood flow;⑤CIP+II group (n=45): a CIP for 3 min was preformed then reperfused, 2 day-interval, a lethal ischemic insult for 8 min was given then reperfused with the blood flow. Except for the control group, the other groups were further divided into 9 time points: immediate time point (0min), 30min, 15min, 3h, 6h, 1d, 2d, 4d and 7d after the sham operation or the last operations (n=5 in each time point). At the determined time point, the rats were sacrificed by decapitation. Thionine staining was used for neuronal evaluation. Western blot was used for observing the expression of p-p38 MAPK protein and GLT-1 protein.2 The effect of SB203580, an inhibitor of p38 MAPK, on the induction of brain ischemic toleranceThe grouping was as follows:①sham group (n=5);②CIP group (n=5);③II group (n=5);④CIP+II group (n=5);⑤SB203580+sham group (n=15): The rats were injected with 20 microliters SB203580 solution into the right lateral cerebral ventricle at 30 min before sham operation, the others was the same as sham group;⑥3% DMSO+CIP+II group (n=5): The rats were injected with 20 3% DMSO solution into the right lateral cerebral ventricle at 30 min before CIP for 3min, the others was the same as CIP+II group;⑦SB203580+CIP+II group (n=15): The rats were injected with 20 microliters SB203580 solution into the right lateral cerebral ventricle at 30 min before CIP for 3min, the others was the same as CIP+II group. According to the doses of SB203580 used,⑤group and⑦group were further divided into 1.25 nmol, 2.5 nmol and 5 nmol three subgroups (n=5 in each dose of SB203580).All the rats were sacrificed at 7 d after the global brain ischemia or the sham operation for neuropathological evaluation by thionin staining. 3 The effect of SB203580, an inhibitor of p38 MAPK, on the expression of GLT-1 during the inducing of brain ischemic tolerance by CIPThe grouping was as follows:①3% DMSO+CIP+II group (n=30);②SB203580+CIP+II group (n=60). According to the design, the rats were injected with 20 microliters 3% DMSO solution or SB203580 solution into the right lateral cerebral ventricle at 30 min before CIP for 3min, the others was the same as CIP+II group. According to the doses of SB203580 used, SB203580+CIP+II group was further divided into 2.5 nmol and 5 nmol two subgroups (n=30 in each subgroup).Each group was further divided into 6 time points: immediate time point (0min), 3h, 6h, 1d, 4d and 7d after the sham operation or the last operations (n=5 in each time point). Western blot was used for observing the effect of SB203580 on the expression of GLT-1 during the inducing of brain ischemic tolerance by CIP.Results:1 The expression of p-p38 MAPK protein and GLT-1 protein in the induction of brain ischemic tolerance, and the time course of the two protein were observed.Western blotting analysis showed that the basal expression of GLT-1 in CA1 hippocampal subfield of control group was in a certain amount, but the basal level of p-p38MAPK protein was very low.In the sham group, the expression of GLT-1 was up-regulated significantly at each time point compared with the control group (P<0.01). The peak value of GLT-1 was at 3h time point. The expression of p-p38MAPK protein was up-regulated significantly at each time point compared with the control group (P<0.01). The peak value of p-p38MAPK was at 15min time point.After CIP for 3min, the expression of GLT-1 was up-regulated significantly at each time point compared with the control group. Compared with 0min time point of the same group, the expression of GLT-1 was up-regulated from 3h, and the peak values were at 6h and 1d (P<0.01). The expression of p-p38MAPK protein in CIP group was up-regulated significantly at each time point compared with the control group (P<0.01). Compared with 0min time point of the same group, the expression of p-p38MAPK was up-regulated from 15min, and the peak values were at 3h time point. The high level of p-p38MAPK was last to 1d. The expression of p-p38MAPK decreased from 2d. Cerebral ischemic preconditioning induced not synchronous up-regulating of p-p38MAPK and GLT-1. The up-regulation of p-p38MAPK was earlier than the up-regulation of GLT-1.In the ischemic insult group, compared with 0min time point, the GLT-1 protein decreased from 3h time point and gradually decreased with time (P<0.01). The level of GLT-1 protein at 4d or 7d time point was very low. No difference of the GLT-1 expression was observed at 15min or 30min time point compared with 0min time point (P>0.05). The expression of p-p38 MAPK protein in hippocampal CA1 subfield was up-regulated significantly after global brain ischemia for 8min, which appeared double peaks. Compared with 0min time point, the first peak was evoked at 15min after ischemic insult and recovered at 3h. Form 6h time point, the expression of p-p38 MAPK increased again, and the second higher peak was appeared at 4d (P<0.01). The data indicated that the change of p-p38 MAPK was earlier than that of GLT-1 after ischemic insult for 8min. The expression of p-p38 MAPK protein increased significantly, and the expression of GLT-1 protein decreased significantly.In the CIP+II group, the expression of GLT-1 was up-regulated significantly at each time point compared with the control group (P<0.01). Compared with 0min time point of the same group, the expression of GLT-1 was up-regulated at 6h time point. The level of the expression of p-p38 MAPK protein at early stage was high. Compared with 0min time point, the expression of p-p38 MAPK down-regulated significantly form 6h (P<0.01). In a word, the CIP for 3min 2 days before the 8min global brain ischemia depressed the excessive up-regulation of p-p38 MAPK and the down-regulation of GLT-1 induced by ischemic insult for 8min.2 The specific inhibitor of p38 MAPK, SB203580, dose-dependently depressed the brain ischemic tolerance induced by CIP.Neuropathological evaluation by thionin staining showed that in the sham group, pyramidal neurons in the hippocampal CA1 subfield were arranged in order with 2 to 3 cell layers, the outline of the neurons was intact, nucleus was full and nucleolus was clear. The HG was 0 grade and ND was 203±5.32 mm-1.In the CIP group, no significant neuronal damage was observed at each time point. At 7d time point, neither the HG grade nor the ND value was different from that of the sham group (P>0.05).Pyramidal neurons in hippocampal CA1 subfield were damaged significantly and appeared almost all dead after the lethal ischemic insult for 8 min. The ND was 17±8.06 mm-1. Compared with the sham group, the HG grade of the II group increased obviously (P<0.01) and the ND decreased obviously (P<0.01).In the CIP+II group, almost complete neurons survived except for few neurons damaged and appeared nucleus pycnosis. Compared with the II group, the HG grade decreased obviously (P<0.01) and the ND, 180±11.67 mm-1, increased obviously (P<0.01).In SB203580+sham group, compared with sham group, neither HG nor ND in each dose of SB203580 (1.25nmol, 2.5nmol, 5nmol) was different from that of sham group.In 3%DMSO+CIP+II group, no neuronal damage was observed. Compared with CIP+II group, neither HG nor ND was different from that of CIP+II group.In SB203580+CIP+II group, the damage of pyramidal neuron deteriorated with the dose of SB203580. Some neuronal damaged was observed in 1.25nmol SB203580+CIP+II subgroup, and ND value decreased significantly compared with that of 3%DMSO+CIP+II group. Many neuronal damaged was observed in 2.5nmol SB203580+CIP+II subgroup. Compared with DMSO+CIP+II group and 1.25nmol SB203580+CIP+II subgroup, the HG grade increased obviously (P<0.01) and the ND decreased obviously (P<0.01). In 5nmol SB203580+CIP+II subgroup, almost all pyramidal neurons in the CA1 subfield died. Compared with 2.5nmol SB203580+CIP+II subgroup, the HG grade increased obviously (P<0.01) and the ND decreased obviously (P<0.01). All the above indicated that the specific inhibitor of p38 MAPK, SB203580, dose-dependently depressed the brain ischemic tolerance induced by CIP.3 The effect of the specific inhibitor of p38 MAPK, SB203580, on the expression of GLT-1 protein during the induction of brain ischemic tolerance induced by CIP.At the all time points (0min, 3h, 6h, 1d, 4d and 7d) of 3%DMSO+CIP+II group, all the levels of GLT-1 expression were high. Both 2.5nmol SB203580 and 5nmol SB203580 induced significant down-regulation of GLT-1 expression at each time point (P<0.01).Summary:1 Cerebral ischemic preconditioning induced up-regulating of p-p38MAPK protein and GLT-1 protein nonsynchronously. The up-regulation of p-p38MAPK was earlier than the up-regulation of GLT-1.2 The change of p-p38 MAPK was earlier than that of GLT-1 after ischemic insult for 8min. The expression of p-p38 MAPK protein increased significantly, and the expression of GLT-1 protein decreased significantly.3 The CIP for 3min 2 days before the 8min global brain ischemia depressed the excessive up-regulation of p-p38 MAPK and the down-regulation of GLT-1 induced by ischemic insult for 8min.4 The specific inhibitor of p38 MAPK, SB203580, dose-dependently depressed the brain ischemic tolerance induced by CIP.5 SB203580 inhibited the up-regulating of GLT-1 protein during the induction of brain ischemic tolerance induced by CIP.Conclusion:CIP via p38 MAPK up-regulating the expression of GLT-1 induced brain ischemic tolerance.
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