| Cerebral ischemic diseases seriously threaten human health because of its high mortality and disability.With the accelerated pace of life and growing social aging problem,the incidence rate of cerebral ischemia is increasing.Mobilization of endogenous protective mechanism against ischemic stimulation is an important strategy for the prevention and treatment of such diseases.Glutamate is the most important excitatory neurotransmitter in the center neural system and plays an essential role in the maintenance of normal brain function.However,ischemia and hypoxia may cause a great deal of glutamate release to the synaptic gap during ischemic stimulation.Studies showed that glutamate excitotoxicity was a considerable mechanism of ischemic neuronal damage.Therefore,maintenance of the lower glutamate concentration in the synaptic gap is a vital way to reduce neuronal damage caused by cerebral ischemia.The stability of the glutamate concentration in the brain is mainly dependent on the high-affinity transport system--excitatory amino acid transporters(EAATs)with 5 members namely EAAT 1-5.EAAT2 mainly distributes on the cell membrane of astrocytes,and then also known as glial glutamate transporter-1(GLT-1).Many studies have shown that GLT-1 plays a principal role in removing the released glutamate from the extracellular space and maintaining the extracellular glutamate below neurotoxic level in the brain.The mRNA and protein of GLT-1 were decreased during the process of delayed neuronal death(DND)in the CA1 hippocampus in rats with global cerebral ischemia.Our previous studies have suggested that the up-regulation of GLT-1 played a vital role in the acquisition of brain ischemic tolerance induced by cerebral ischemia preconditioning(CIP).Therefore,modulation in the expression and function of GLT-1 might provide a new way for the prevention and treatment of cerebral ischemic disease.Rothstein et al.reported that β-lactam antibiotics could promote the expression of the GLT-1 protein and enhance the ability of astrocytes to clear the glutamate from synaptic gap,which was of great associated with the study of cerebral ischemic tolerance induction.Pretreatment with ceftriaxone sodium could up-regulate the expression of GLT-1 in CA1 hippocampus of rats and reduce the neuron damage caused by cerebral ischemia.This is particularly significant for the prevention and treatment of ischemic brain injury.However,in view of a large number of side effects caused by the extensive use of antibiotics,usage of ceftriaxone sodium for the prevention and treatment of ischemic brain damage has been limited greatly.Therefore,looking for alternatives with small side effects is of great significance for the translation of the above research results to clinical application.Sulbactam is structurally similar to ceftriaxone sodium and has a β-lactam ring,but its antibacterial activity is little and generally combined with other β-lactam antibiotics to enhance the antibacterial effect of antibiotics.Our previous studies showed that preventive administration of sulbactam could up-regulate the expression of the GLT-1 protein in CA1 hippocampus,and enhance the viability of neurons in the subsequent global cerebral ischemia.However,the mechanism about the up-regulation of GLT-1 during the process of sulbactam pretreatment is not yet fully known.Mitogen-activated protein kinases(MAPKs)belong to the serine/threonine protein kinase family and mainly consist of the following four members including ERK,JNK,p38 MAPK and BMK.The p38 MAPK is an important intracellular signal transduction system and participates in a series of physiological and pathological processes,including cell death and survival.In middle cerebral artery occlusion model,hypoxic preconditioning could promote the expression of p38 MAPK in cells of the infarcted area,and enhance the tolerance of the neurons to ischemia,and especially,ischemic tolerance of the neurons is inhibited by inhibition of p38 MAPK.Our previous studies showed that CIP could up-regulate the expression of GLT-1 via p38 MAPK signaling pathway in the induction of cerebral ischemic tolerance.Accordingly,we hypothesized that the p38 MAPK signaling pathway may participates in the sulbactam-induced GLT-1 up-regulation and neuronal protection.To prove this hypothesis,the present study was undertaken to(1)investigate: the effect of sulbactam on the expression of phosphorylated p38 MAPK(p-p38 MAPK)and GLT-(2)1 protein in the CA1 hippocampus;the effect of p38 MAPK inhibition on the sulbactam-induced GLT-1 up-regulation and cerebral ischemic tolerance using the rat global cerebral ischemia model.Part 1 The effect of sulbactam on the expression of p-p38 MAPK and GLT-1 in CA1 the hippocampus of ratsObjective: To compare the temporal relationship of sulbactam-induced p-p38 MAPK and GLT-1 expression upregulation in the CA1 hippocampus of rats.Methods:After a stainless steel cannula was implanted in the right lateral ventricle,60 healthy male Wistar rats were randomly divided into the following four groups(n = 5 in each group):1 Vehicle control(NS+sham)group(n=5): The rats was administrated with normal saline(NS)of 10 μl via the cannula first,and then the sham operation for global cerebral ischemia was performed.2 Sulbactam + sham group(n=5): Sulbactam solution(10 μl,360 nmol)was administrated via the cannula first,and then the sham operation for global cerebral ischemia was performed.3 Global cerebral ischemia(NS+ischemia)group(n=5): NS(10 μl)was administrated via the cannula first and then performed 8 minutes of global cerebral ischemia.4 Sulbactam+cerebral ischemia group(n=5): Sulbactam solution(10 μl,360 nmol)was administrated via the cannula first and then performed 8 minutes of global cerebral ischemia.Rats in each group were sacrificed at 6 h,12 h and 48 h after the administration.The expression of p-p38 MAPK and GLT-1 protein in the CA1 hippocampus of rats were observed by methods of immunohistochemistry and western blot and the time sequence of p-p38 MAPK and GLT-1 protein expression was compared.Results:Immunohistochemistry assay showed that there was basic expression of p-p38 MAPK and GLT-1 showing light brown particles in the CA1 hippocampus of the rats,and no significant change in the expression of p-p38 MAPK and GLT-1 among each time point in the vehicle control(NS+sham)group.Compared with the vehicle control group,sulbactam administration in sulbactam+sham group significantly up-regulated the expression of p-p38 MAPK and GLT-1(P<0.05).The immunoparticles of p-p38 MAPK were mainly distributed in the nucleus and the GLT-1 immunoparticles distributed tightly around the pyramidal neurons in the CA1 hippocampus.The time sequence of up-regulation of p-p38 MAPK and GLT-1 was different.Compared with the vehicle control(NS+sham)group,the expression p-p38 MAPK reached the peak at 6 h,and began to fall down at 12 h,while the expression of GLT-1 began to increase at 12 h and peaked at 48 h after administration of sulbactam in sulbactam+sham group.The global cerebral ischemia(NS+ischemia)significantly enhanced p-p38 MAPK expression with a peak at 12 h(P<0.05),while the expression of GLT-1 had no significant change at any time points after the ischemic stimulation compared with the vehicle control(NS+sham)group.The administration of sulbactam in the sulbactam+ischemia group significantly upregulated the expressions of p-p38 MAPK and GLT-1 compared with the vehicle control(NS+sham)group.The expression of p-p38 MAPK peaked at 6 h,then fell down to level of vehicle control(NS+sham)group at 12 h.The expression of GLT-1 started to upregulate at 12 h after administration of sulbactam and peaked at 48 h(P<0.05).In addition,compared with the global cerebral ischemia group,sulbactam inhibited the over-expression of p-p38 MAPK in CA1 hippocampus caused by global cerebral ischemia.The above results indicated that the up-regulation of GLT-1 expression induced by sulbactam was later than that of p-p38 MAPK in either normal or global cerebral ischemic rats.Western blot analysis showed a similar trend in the expression of p-p38 MAPK and GLT-1 protein with the immunohistochemistry.The expression of p-p38 MAPK and GLT-1 protein was up-regulated(P<0.05)after administration of sulbactam in normal and global cerebral ischemic rats,and the up-regulation of GLT-1 expression was later than that of p-p38 MAPK in time sequence.Summary:The expression of p-p38 MAPK and GLT-1 in the hippocampus CA1 region was significantly increased after pretreatment with sulbactam in both normal and global cerebral ischemic rats.The up-regulation of GLT-1 was later than that of p-p38 MAPK in time sequence.The results suggested that p38 MAPK signal pathway might be the upstream mechanism for the sulbactam-induced up-regulation of GLT-1 expression.Part 2 The effect of inhibition of p38 MAPK pathway on the up-regulation of GLT-1 protein in CA1 hippocampus of rats induced by sulbactamObjective: To clarify the role of p38 MAPK signal pathway in the up-regulation of GLT-1 expression induced by sulbactam pretreatment,we the effect of inhibition of p38 MAPK pathway on up-regulation of GLT-1 protein in CA1 hippocampus of rats induced by sulbactam was further observed.Methods:After a stainless steel cannula was implanted in the right lateral ventricle 35 healthy male Wistar rats were randomly divided into the following groups(n=5 in each group):1 NS+sham group(n=5): The same with the first part;2 Sulbactam+sham group(n=5): The same with the first part;3 SB203580+sulbactam+sham group: SB203580(10 μl,5 nmol)was administrated via the cannula 30 min before sulbactam(10 ul,360 nmol)and then performed the sham operation for global cerebral ischemia immediately according to the operation of the sulbactam+sham group of part one;4 SB203580+sham group(n=5): SB203580 solution(10 μl,5 nmol)administrated via the cannula first and then performed the sham operation for global cerebral ischemia immediately.5 NS + ischemia group(n=5): The same with the first part;6 Sulbactam+ischemia group(n=5): The same with the first part;7 SB203580+sulbactam+ischemia group(n=5): SB203580(10 μl,5 nmol)was administrated via the cannula 30 min before sulbactam(10 ul,360 nmol)and then performed 8min of global cerebral ischemia immediately according to the operation of sulbactam + ischemia group of part one;Rats in each group were sacrificed at 48 h after the administration of sulbactam solution.The expression of GLT-1 in CA1 hippocampus was observed by methods of immunohistochemistry and western blot.Results:Immunohistochemistry assay showed that compared with NS+sham group,the GLT-1 expression in CA1 hippocampus was significantly enhanced after administration of sulbactam in either normal(sulbactam+sham group)or ischemic(sulbactam+ischemia)rats(P<0.05).Compared with sulbactam+sham and sulbactam+ischemia group,pretreatment with SB203580 significantly attenuated the sulbactam-induced upregulation in GLT-1 expression in the CA1 hippocampus either in SB203580+sulbactam+sham or SB203580+sulbactam+ischemia group(P<0.05).Compared with vehicle control(NS+sham)group,SB203580 alone had no significant change in the positive staining of GLT-1 in CA1 hippocampus region of ratsWestern blot analysis showed a similar results that the upregulation of expression of GLT-1 protein induced by sulbactam in CA1 hippocampus was significantly inhibited after pretreatment with SB203580 either in SB203580+sulbactam+sham or SB203580+sulbactam+ischemia group(P<0.05).Summary:Results above showed that the inhibition of p38 MAPK signaling pathway by SB203580 could inhibit the expression of GLT-1 protein in CA1 hippocampus induced by sulbactam in rats.Part 3 The effect of the inhibition of p38 MAPK signal pathway on neuronal protective effect induced by sulbactam.Objective: The first and second part of this study showed that P38 MAPK participated in the up-regulation of GLT-1 expression in CA1 hippocampus of rats induced by sulbactam.To further demonstrate the role of p38 MAPK signal pathway in the sulbactam-induced neuronal protective effect via GLT-1 upregulation,we further observed the effect of the inhibition of p38 MAPK signal pathway on the neuronal protective effect induced by sulbactam.Methods:After a stainless steel cannula was implanted in the right lateral ventricle,30 healthy male Wistar rats were randomly divided into the following groups(n=5 in each group):1 Sham group(NS+sham): The rats were administrated with normal saline(NS)of 10 μl via the cannula first,and then the sham operation for global cerebral ischemia was performed.2 Global cerebral ischemia group(NS+ischemia): NS(10 μl)was administrated via the cannula first and then performed 8 minutes of global cerebral ischemia 2 days later.3 Sulbactam+ischemic group: Sulbactam solution(10 μl,360 nmol)was administrated via the cannula first and then performed 8 minutes of global cerebral ischemia 2 days later.4 SB203580+sulbactam+ischemic group: SB203580(10μl,5nmol)was administrated via the cannula 30 min before sulbactam(10ul,360nmol)and then performed 8min of global cerebral ischemia2 days later according to the operation of sulbactam + ischemia group of part one;5 SB203580+sham group: SB203580 solution(10 μl,5 nmol)was administrated via the cannula first and then performed sham operation for global cerebral ischemia 2 days later.Rats in each group were sacrificed at 7 days after the sham operation or global brain ischemia in each group.Neuropathological evaluation under thionin stain was performed and the evaluation was presented by histological grade(HG)and neuronal density(ND)to reflect the content of DND of pyramidal neurons in the CA1 hippocampus.Results:The result of thionin stain and neuro-pathological evaluation showed that there were about 3-4 layers of neurons in the CA1 hippocampus of the sham group,and the pyramidal neurons arranged neatly and densely with contours clearly and intactly.There were large numbers of Nissl body in the cell,and the nucleus was full,the nucleolus was clearly visible.The HG of rats in the sham group was grade 0,and ND was 198.6±7.89(cells / mm,the same below).Global cerebral ischemia for 8 min induced obvious DND which resulted in large area of neural absence in the CA1 subfield represented with the significant increase in HG to grade Ⅱ-Ⅲ and decrease in ND to 51.8±7.40(P<0.05).Pre-treamented with sulbactam(sulbactam+ischemia)effectively prevented the DND normally induced by global brain ischemia,which was represented with the increased survival of pyramidal neurons(P<0.05).The HG decreased to gradeⅠ-Ⅱ and ND increased to 188.6±12.76.These results suggested that sulbactam played a neural protective role when suffering ischemia.Compared with the sulbactam + ischemia group,pretreatment with SB203580(SB203580+sulbactam+ischemia group)blocks the neuroprotective effect mediated by sulbactam,which resulted in the significant increase in HG to grade Ⅱ-Ⅲ and decrease in ND to 52.2±6.61(P<0.05).Compared with the sham group,SB203580 alone showed no significant change on neurons in CA1 hippocampus.Summary:Sulbactam pretreatment played a neural protective role during ischemia-reperfusion.SB203580,a specific inhibitor of p38 MAPK pathway,could inhibit the neural protective induced by sulbactam.Conclusions:1 The administration of sulbactam upregulated the expression of p-p38 MAPK and GLT-1 protein in the CA1 hippocampus of normal and global cerebral ischemia rats,and the up-regulation of GLT-1 was later than that of p-p38 MAPK.2 The inhibition of p38 MAPK signaling pathway by SB203580 inhibited the up-regulation of GLT-1 protein in the CA1 hippocampus induced by sulbactam.3 The inhibition of p38 MAPK signaling pathway by SB203580 inhibited the ischemic tolerance of the CA1 hippocampal pyramidal neurons induced by sulbactam.4 Results above indicated that P38 MAPK participated in the sulbactam-induced brain ischemic tolerance mediated by GLT-1 up-regulation in rats... |
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