| Glutamate is one of the primary neurotransmitters responsible for excitatory signaling transmission in the central nervous system.A large body of data suggested that the excessive extracellular accumulations of exciting amino acids,particularly such as glutamate,lead to neuronal death after brain ischemia.The excessive release of glutamate after cerebral ischemia and reperfusion with insufficient clearance by glutamate transporters leads to accumulation of glutamate in synaptic cleft,which acts on ionotropic and metabotropic glutamate receptors to initiate excitotoxicity including calcium and sodium overload,radicals generation and apoptosis,etc.Glutamate transporters are distributed in both glial cells and transporting glutamate from extracellular space to the intracellular is the only mechanisms for eliminating the redundant glutamate.Glial glutamate transporter-1(GLT-1)plays the most important part in removing the extracellular glutamate.A variety of evidence showed that upregulation of GLT-1 expression and its uptake activity for glutamate either by cerebral ischemic preconditioning or drugs could play neuronal protective effect against ischemic insult.Rothstein et al.reported that β-lactam antibiotics,such as ceftriaxone strongly and selectively promote GLT-1 and active splice variant GLT-1b expression in vivo and markedly activated the GLT-1 promoter in human fetal astrocytes or COS7 cell lines.However,many new problems have arisen to use ceftriaxone for prevention and treatment of brain ischemia in clinic,such as the excessive doses,dysbacteriosis and bacterial resistance.Our recent study has shown that sulbactam,an atypical β-lactam antibiotics,could prevent ischemic injury of pyramidal neurons in the CA1 hippocampal region by up-regulating GLT-1 in a rat global cerebral ischemic model.These findings provided a beneficial basis and reference for clinical study using sulbactam to prevent and therapy cerebral ischemic injury,because sulbactam has little anti-bacterial capacity and side effect such as dysbacteriosis and bacterial resistance.However,in vivo it is too difficult to clarify the direct effect of sulbactam on astrocytic GLT-1 expressions.Therefore,it is necessary to study in vitro the direct effect of sulbactam on the expression of GLT-1 in astrocytes using pure astrocytes culture.Additionally,It is important to further study the signaling pathway in upregulation of GLT-1 after the treatment of sulbactam for better understanding the role of sulbactam and promoting its clinical application as an anti-cerebral ischemia medicine.p38 mitogen-activated protein kinase(p38 MAPK)is an important intracellular signal transduction system and participates in a series of physiological and pathological processes including cell death and survival,like a double-edged sword Although some reports showed that activation of p38 MAPK might facilitate neuronal death after brain ischemic insult,the beneficial or protective effect of moderate p38 MAPK activation has been well demonstrated in brain or other organs ischemic models,especially in ischemic preconditioning in the organs being preconditioned or remote organs from the preconditioned organs.Our recent study suggested in rats that the upregulation of GLT-1 expression and neuronal protection induced by cerebral ischemic preconditioning were blocked by inhibition of the p38 MAPK activation with SB 203580,the specific inhibitor of p38 MAPK.These findings suggest that activation of p38 MAPK might mediates upregulation of GLT-1 expression.Therefore,the present study was undertaken to study the role of p38 MAPK signal pathway in the sulbactam-induced upregulation of GLT-1 expression during the process of anti-ischemic effect of sulbactam.Part 1 The effect of sulbactam on neuronal survival in neuron-astrocyteco-cultures and GLT-1 expression in pure astrocyte cultures afterOGDObjectives:1 The effect of sulbactam on neuronal survival after OGD in neuron-astrocyte co-cultures is investigated in order to provide evidence to elucidate the protective effect of sulbactam against OGD.2 The effect of sulbactam on GLT-1 expression after OGD in pure astrocyte cultures is also investigated in order to illustrate the direct upregulation of GLT-1 by sulbactam incubation against OGD so as to further elucidate one of the astrocytic mechanism for the protective effect of sulbactam against OGD.Methods:GLT-1 primarily expresses in astrocytes.To directly demonstrate the effect of sulbactam on astrocytic GLT-1 expression,all parts in the present study were performed using neuron-astrocyte co-cultures for the experiment of neuronal protection and using pure astrocyte cultures for the experiment of the expression regulation from 0-1 day old newborn Wistar rats.Steady primary neuron-astrocyte co-cultures for 10 days and pure astrocyte cultures at 3 or 4 generations were used and divided into the following 4 groups.(1)Control group: The neuron-astrocyte co-cultures and pure astrocyte cultures were maintained in normal medium for 48 h+2 h+24 h,which were corresponded to the times for sulbactam incubation,OGD and recovered cultures after re-oxygenation,respectively,in the following groups.(2)OGD group: Firstly,the neuron-astrocyte co-cultures and pure astrocyte cultures were kept under normal medium for 48 h.Then the cultures were endured 2 h OGD and then were cultured for 24 h under normal condition.(3)Sulbactam+OGD group: Firstly,the neuron-astrocyte co-cultures and pure astrocyte cultures were maintained for 48 h under the presence of sulbactam(dissolved in normal saline(NS),and the same in the following)in the final concentrations of 5 μM,25 μM and 125 μM in the cultures.Then the cultures were endured 2 h OGD.Whereafter,the medium for the cultures was replaced with fresh normal medium free of sulbactam and continuously cultured for 24 h under normal condition after re-oxygenation.In addition,a NS+OGD group was designed as the sulbactam?s vehicle control group,in which only NS was administrated instead of sulbactam.(4)Sulbactam control group: This group was designed only as control of neuronal survival and viability for the neuron-astrocyte co-cultures.The co-cultures were administrated with sulbactam in the final concentration of 125 μM in the cultures and cultured for 48 h under the presence of sulbactam.Then the medium was replaced with fresh normal medium free of sulbactam and continuously cultured for 2+24 h.The neuronal survival condition was was performed with HO/PI staining(HOPI)and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)methods for neuron-astrocyte co-cultures at 24 h after the reoxygenation from OGD.The evaluation was presented by percent of cell death observed by HO/PI staining and cell viability observed by MTT to reflect the neuronal survival condition in neuron-astrocyte co-cultures.The GLT-1 expression in the pure astrocyte cultures was assayed by immunocytochemistry and western blot analysis at 12 h and 24 h after the re-oxygenation from OGD.Results:Sulbactam protects neurons and upregulates astrocytic GLT-1 expression against OGD 1 Neuronal survival and viabilityHO/PI staining showed that sulbactam reduced the percentage of neuronal death induced by OGD in a dependent manner in hippocampal neuron-astrocyte co-cultures.It could be found that there were a lot of living neurons stained in dark blue with normal shape(big and round)of cellular nucleus,and the percentage of neuronal death was only 9.2±0.5% in control group.OGD induced many neuronal death which were stained in red(PI positive)or bright white blue(intensive HO positive)with small nuclei because of pyknotic,and increased the percent of neuronal death to 71.6±2.4%.Sulbactam treatment in the sulbactam+OGD group effectively prevented the neuronal death induced by OGD in a dose dependent manner,which was represented with the increased numbers of living neurons and then decreased the percentage of neuronal death.The administration of sulbactam at 125 μM had the best drug efficacy,which reduced the percentage of neuronal death to 15.6±1.0%.Administration of vehicle instead of sulbactam had no effect on the OGD induced cell death.In addition,the largest dose sulbactam(125 μM)had no effect on the percentage of neuronal death in the sulbactam control group compared with control group.MTT assay showed that OGD significantly decreased the cell viability and sulbactam incubation dose dependently increased the cell viability in neuron-astrocyte co-cultures.Sulbactam incubation had no effect on the cell viability of normal neuron-astrocyte co-cultures in the sulbactam control group compared with control group.These results indicated that sulbactam could protect the neurons against OGD in a dose dependent manner in neuron-astrocyte co-cultures.2 GLT-1 expressionThe GLT-1 expression was evaluated at two time points of 12 h and 24 h after re-oxygenation from OGD.Immunocytochemistry assay showed that there was a basic amount of distribution of brown GLT-1 immunoparticles in astrocytes in control group.Compared with control group,the GLT-1 expression was significantly downregulated in OGD group.Compared with OGD group,sulbactam incubation significantly upregulated the GLT-1 expression in sulbactam+OGD group in a dose dependent manner at both 12 h and 24 h after OGD.In the largest dose(125 μM),the sulbactam incubation not only prevented the downregulation of GLT-1 expression induced by OGD,but made the expression exceeded and even reached approximately 2 to 3 folds higher than the control level.The vehicle for sulbactam had no effect on GLT-1 protein expression levels in OGD-treated astrocytes.Western blot analysis showed similar results in the expression of GLT-1 in the OGD-treated astrocytes at the time point of either 12 h or 24 h.The results suggested the association of the sustainability of GLT-1 in high level expression in astrocytes with the neuronal protection of sulbactam against OGD.Part 2 The comparison between the time course of GLT-1 andphosphorylated p38 MAPK expressions after sulbactamtreatment in normal astrocytesObjectives:1 The dose characteristic of the expression of GLT-1,p-p38 MAPK and p38 by sulbactam incubation in normal pure astrocyte cultures is investigated in order to provide evidence to elucidate the effect of sulbactam on expressions of these proteins and the dose characteristics of these expressions.2 The time characteristic of the expression of GLT-1,p-p38 MAPK and p38 by sulbactam incubation in normal pure astrocyte cultures is investigated in order to provide evidence to elucidate the effect of sulbactam on the time characteristics of these expressions.3 The time course of GLT-1 and phosphorylated p38 MAPK expressions after sulbactam incubation in normal astrocytes was compared in order to provide evidence to estimate wether p38 MAPK pathway was the upstream of the GLT-1 expressions.Methods:1 The pure astrocyte cultures were used for this part.First,to determine the upregulating effect of sulbactam on these proteins,dose dependency of the expression of these proteins to sulbactam was observed.Sulbactam in a final concentration of 5 μM,25 μM and 125 μM,respectively was added to the cultures.The astrocytes were harvested at 48 h for GLT-1 expression,3 h for phosphorylated p38 MAPK and 24 h for total p38 MAPK after the culture in the presence of sulbactam.The reason for selection of these time points is due to relative high expression of these proteins after sulbactam incubation in our preliminary experiment.2 To determine the time course of the effect of sulbactam on these proteins,the pure astrocytic cultures were administrated with sulbactam in the final concentration of 125 μM and the astrocytes were harvested at 1 h,3 h,6 h,12 h,24 h,48 h and 72 h after the culture in the presence of sulbactam.Vehicle control groups were designed as well,in which only NS was administrated instead of sulbactam.Results: 1 GLT-1 expressionImmunocytochemistry assay showed that brown GLT-1 immunoparticles were extensively distributed in the cultured normal astrocytes.In vehicle control group,there was basic amount of expression.After incubation with sulbactam for 48 h in concentration of 5 μM,25 μM,125 μM,the immunostaining significantly increased and showed a dose-dependency.Western blot analysis showed a similar dose dependency in the upregulation of GLT-1 after incubation of sulbactam.Then,we observed the time course of the upregulation of GLT-1 after sulbactam incubation in concentration of 125 μM.It was found with immunocytochemistry assay and western blot analysis that the GLT-1 upregulation started at 12 h or 24 h,reached the peak at 48 h and maintained high level till 72 h after sulbactam incubation.2 p38 MAPK expressionImmunocytochemistry assay showed that phosphorylated-p38 MAPK expression were mainly located in nucleus and also in the cytoplasm near the nucleus in small amount.There were small amount of the phosphorylated-p38 MAPK immunoparticles in the vehicle control group in the cultured astrocytes.Compared with the vehicle control group,the expression of phosphorylated-p38 MAPK was upregulated after incubation of sulbactam for 3 h in a dose of 5 μM,25 μM,125 μM and showed a clear dose-dependency,and the upregulation was especially significant in the nucleus.Western blot analysis showed a similar effect and dose-dependency in the upregulation of phosphorylated-p38 MAPK expression after incubation of sulbactam for 3 h.The expression of p38 MAPK did not show changes after sulbactam incubation for 24 h in a dose of 5 μM,25 μM,125 μM by immunocytochemistry and western blot assay.Then,we observed the time course of the upregulation of phosphorylated-p38 MAPK after sulbactam incubation in concentration of 125 μM.It was showed that the upregulation of phosphorylated-p38 MAPK expression started at 1 h,reached the peak at 3 h to 6 h and fallen to the baseline at 24 h after the sulbactam incubation with immunocytochemistry assay and western blot analysis.The expression of p38 MAPK also did not show changes after incubation of 125 μM sulbactam at every time point by immunocytochemistry and western blot assay.3 Comparion of the time course between GLT-1and p-p38 MAPK expressionSpecially importantly,it was notable that the time course of the phosphorylated-p38 MAPK expression was obviously earlier than that of GLT-1 expression under sulbactam incubation by comparison of the time course between the GLT-1 and phosphorylated-p38 MAPK expressions.This result suggested a possibility that the phosphorylation of p38 MAPK might be the upstream signal mechanism for GLT-1 upregulation induced by sulbactam in astrocytes.Part 3 The effect of p38 MAPK inhibition on sulbactam-induced GLT-1expression in normal and OGD-treated astrocytesObjectives: In order to convincingly elucidate the role of p38 MAPK activation on sulbactam induced GLT-1 up-regulation in normal and OGD state,the effect of p38 MAPK inhibition with SB203580 on GLT-1 up-regulation of sulbactam were investigated in normal and OGD-treated astrocytes.Methods: 1 The GLT-1 expression in normal astrocytesThis part was performed using pure astrocyte cultures and consisted of the following 4 groups:(1)Control group: Astrocytic cultures were maintained in normal medium for 1 h+48 h,which were corresponded to the times for SB203580 and sulbactam incubations in the following groups.(2)Sulbactam group: Astrocytic cultures were maintained in normal medium for 1 h and then were administrated with sulbactam in concentration of 125 μM and incubated for 48 h in the presence of sulbactam.(3)SB203580+sulbactam group: Firstly,astrocyte cultures were administrated with SB203580(dissolved in DMSO,and the same in the following).After incubation in the presence of SB203580 for 1 h,the cultures were administrated with sulbactam in a concentration of 125 μM,and then were incubated for 48 h in the presence of SB203580 and sulbactam in the medium.According to the concentration of SB203580,this group was further divided into 2.5 μM,5 μM and 10 μM subgroups.In addition,a DMSO+sulbactam group was designed as the SB203580?s vehicle control group,in which only DMSO was administrated instead of SB203580.(4)SB203580 control group: The culture was administered with SB203580 in a concentration of 10 μM and maintained for 1h+48 h.After the above treatments in each group,the astrocytes were harvested and the GLT-1 expression levels in the astrocytes were evaluated with immunocytochemistry and western blot analysis.2 The GLT-1 expression in OGD-treated astrocytesThis part was performed using pure astrocyte cultures as well and consisted of the control,OGD,sulbactam+OGD,and SB203580+sulbactam+ OGD groups.The protocols for control,OGD,sulbactam+OGD groups were the same as those in the corresponding groups in Part 1,except only one dose of sulbactam in 125 μM was added.The protocols for SB203580+ sulbactam+OGD group consisted of the following procedures: Firstly,astrocyte cultures were administrated with SB203580.After incubation in the presence of SB203580 for 1 h,the cultures were administrated with sulbactam in a concentration of 125 μM,and then were incubated for 48 h in the presence of SB203580 and sulbactam in the medium.Then the cultures were endured 2 h OGD,followed by a subsequent recovered culture for 24 h under normal condition and medium free of SB203580 and sulbactam.According to the concentration of SB203580,this group was further divided into 2.5 μM,5 μM and 10 μM subgroups.In addition,a DMSO+sulbactam+OGD group was designed as the SB203580?s vehicle control group,in which only DMSO was administrated instead of SB203580.After the above treatments in each group,the astrocytes were harvested at 12 h or 24 h after the OGD and the GLT-1 expression levels in the astrocytes were evaluated with immunocytochemistry and western blot analysis.Results: 1 Normal astrocytesImmunocytochemistray and western blot analysis showed that the GLT-1 upregulation induced by sulbactam incubation was dose dependently inhibited by the specific p38 MAPK inhibitor,SB 203580,which inhibits the phosphorylation of p38 MAPK.It was shown that in control group,there was a basic amount of expression of GLT-1.Compared with control group,GLT-1 was upregulated by 125 μM sulbactam in sulbactam group.Compared with sulbactam group,SB203580 in doses of 2.5 μM,5 μM,10 μM was dose dependently inhibited GLT-1 upregulation induced by sulbactam.The largest dose of 10 μM SB 203580 presented the most inhibition on GLT-1 upregulation.Vehicle(DMSO)for SB 203580 had no inhibited effect on GLT-1 upregulation induced by sulbactam.Compared with control group,the basic expression of GLT-1 was not significantly inhibited by the administration of SB 203580 in SB 203580 control group,even in the largest dose of 10 μM.2 OGD-treated astrocytesImmunocytochemistry and western blot analysis showed that the GLT-1 upregulation induced by sulbactam in OGD-treated astrocytes was dose dependently inhibited by SB 203580 as well.Compared with sulbactam+OGD group at 12 h and 24 h,the administration of SB 203580 inhibited GLT-1 upregulation induced by sulbactam in astrocytes either at 12 h or 24 h after OGD in the group of SB203580+sulbactam+OGD in a dose dependent manner,in which the most inhibiting effect was observed in the dose of 10 μM of SB 203580.Vehicle(DMSO)for SB 203580 had no inhibited effect on GLT-1 upregulation induced by sulbactam in the OGD-treated astrocytes as well.It is showed that the GLT-1 upregulation induced by sulbactam was dose dependently inhibited by SB203580 in both normal and OGD-treated astrocytes.The above findings indicated that p-p38 MAPK pathway participated in the GLT-1 upregulation induced by sulbactam in both normal and OGD-treated astrocytes.Part 4 The effect of p38 MAPK inhibition on the neuronal protectiveeffect of sulbactam against OGD in neuron-astrocyte co-culturesObjectives: In order to convincingly elucidate the role of p38 MAPK activation on sulbactam-induced neuronal protection against OGD,the effect of p38 MAPK inhibition with SB203580 on sulbactam-induced neuronal protection against OGD was investigated in neuron-astrocyte co-cultures.Methods:This part was performed using neuron-astrocyte co-cultures for 10 days and consisted of five groups: control,SB203580 control,OGD,sulbactam+OGD,and SB203580+sulbactam+OGD groups.The protocols of control,OGD,sulbactam+OGD,SB203580+sulbactam+OGD were the same as those in the corresponding group in the part 3.2.The cultures in SB203580 control group were administered with SB203580 in a concentration of 10 μM and maintained for 1 h+48 h.Cells were harvested at 24 h after OGD or corresponding time point(SB203580 control group and control group).The neuronal survival condition was evaluated with HOPI and MTT methods.Results:Consistent with the results in the part 1.1,it was shown in Fig 8 by HO/PI staining that OGD for 2 h induced many neuronal death(red or bright white blue staining)and increased the percent of neuronal death and 125 μM sulbactam incubation in the sulbactam+OGD group effectively prevented the neuronal death induced by OGD in the neuron-astrocyte co-cultures.Administration of SB203580 prior to sulbactam incubation inhibited the neuronal protection of sulbactam incubation against OGD in a dose dependent manner,in which the neuronal death occurred again and the percentage of neuronal death was again increased after the administration of SB203580,especially in the dose of 10 μM in the SB203580+ sulbactam+OGD group compared with sulbactam+OGD group.The vehicle(DMSO)for SB203580 had no effect on the neuronal protection of sulbactam incubation against OGD.In addition,administration of SB203580 only even in the largest dose of 10 μM did not increase dead cell in the SB203580 control group compared with control group.MTT assays showed that the OGD significantly decreased the cell viability and 125 μM sulbactam incubation prevented the decrease in the cell viability induced by OGD in the neuron-astrocyte co-cultures.Compared with sulbactam+OGD group,administration of SB203580 in a dose of 2.5 μM,5 μM,10 μM,respectively,inhibited the increase in cell viability induced by sulbactam incubation against OGD in SB203580+sulbactam+OGD in a dose dependent manner.The vehicle for SB203580 had also no effect on the increase of cell viability induced by sulbactam incubation.SB203580 only even in the largest dose of 10 μM had no effect on the cell viability in the SB203580 control group compared with control group as well.The above results indicates that SB203580 could dose dependently inhibit the sulbactam-indued protection on hippocampal neurons against OGD in the neuron-astrocyte co-cultures.The p-p38 MAPK pathway parcitipated the sulbactam-indued protection on hippocampal neurons against OGD in the neuron-astrocyte co-cultures.Conclusions:1 The sulbactam pretreatment played neuronal protective effect against OGD in the neuron-astrocyte co-cultures and upregulated GLT-1 expression in astrocytes.2 The GLT-1 and phosphorylated-p38 MAPK was both upregulated by incubation of sulbactam in a clear dose and time dependent manner in normal astrocytesIt was notable that the time course of the phosphorylated-p38 MAPK expression was obviously earlier than that of GLT-1 expression under sulbactam incubation,which suggested a possibility that the phosphorylation of p38 MAPK might be the upstream signal mechanism for GLT-1 upregulation induced by sulbactam in astrocytes.3 The GLT-1 upregulation induced by sulbactam was dose dependently inhibited by SB203580 in both normal and OGD-treated astrocytes,which indicated that p-p38 MAPK pathway participated in the GLT-1 upregulation induced by sulbactam in both normal and OGD-treated astrocytes.4 SB203580 could dose dependently inhibit the sulbactam-indued protection on hippocampal neurons against OGD in the neuron-astrocyte co-cultures,which indicated that p-p38 MAPK pathway parcitipated in the sulbactam-indued protection on hippocampal neurons against OGD in the neuron-astrocyte co-cultures.5 The above findings indicated that p-p38 MAPK pathway parcitipated in the sulbactam-indued neuronal protection via upregualtion of GLT-1. |
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