| Ischemic brain injury is a common injury type of cerebrovascular disease in clinic.The pathological process of ischemic brain injury is complex and involves many factors.The excitotoxicity caused by excessive glutamate is one of the important pathological bases of ischemic injury.Glutamate is the main excitatory neurotransmitter in the central nervous system,which is essential to maintain normal brain function.However,excessive concentration of glutamate has strong neurotoxicity.Therefore,it is an important strategy to alleviate injury through reduction of the extracellular high concentration glutamate during cerebral ischemia.There is no glutamate metabolic enzyme in the central nervous system extracellular.The transport of glutamate by astrocytes is the main way to eliminate extracellular glutamate and limit excitotoxicity.About 90%of extracellular glutamate enters the cell through the astrocyte glutamate transporter-1(GLT-1),and then most of it is converted into glutamine through glutamine synthetase(GS)and enters the process of reuse.Therefore,GLT-1 and GS are key regulatory links of glutamate transport in the central nervous system.Ginsenoside Rb1(Ginsenoside Rb1)is one of the main active ingredients of Panax ginseng C.A.Meyer.It can penetrate the blood brain barrier,has neuroprotective effects,and can improve epilepsy,Alzheimer’s disease,Parkinson’s disease and other neurological diseases.Our previous research found that ginsenoside Rbl has a certain regulatory effect on glutamate transporter GLT-1 and glutamine synthetase GS after cerebral ischemia,but its action pathway and mechanism are still unclear.The purpose of this study is to explore the regulatory effect of ginsenoside Rb1 on glutamate transport after cerebral ischemia,to provide scientific basis for explaining the mechanism of ginsenoside Rbl in nerve injury after cerebral ischemia,and to provide thinking for the excavation and development of traditional Chinese medicine targeting the mechanism of glutamate injury.Part 1 Protective effect of ginsenoside Rbl on cerebral ischemia injury in mice and its effect on glutamate transport systemObjective:To observe the brain protective effect of ginsenoside Rbl and the effect of drug on glutamate transport in mice with cerebral ischemia reperfusion injury.Methods:LC-MS/MS was used to observe the distribution of ginsenoside Rbl in the brain of mice after intraperitoneal injection.The effect of ginsenoside Rbl was observed on the cerebral ischemia reperfusion injury mice.The animals were randomly divided into normal group(Control),sham operation group(Sham),cerebral ischemiareperfusion group(CIR),ginsenoside Rb1 20 mg/kg,40 mg/kg groups.The model of cerebral ischemia injury mice was prepared by middle cerebral artery occlusion(MCAO).Postoperative intraperitoneal injection for 7 days,once a day.Set 24 h,72 h and 168 h of reperfusion as observation periods to record the overall state,behavior score and brain damage of animals;Glu content and GS enzyme activity in ischemic side brain were detected;The mRNA expression and protein level of GLT-1 and GS in ischemic side brain were detected;The expression of protein kinase Akt(p-Akt),PKA in ischemic side brain related to GLT-1 and GS regulation were detected.Results:LC-MS/MS results showed that the mice were intraperitoneally injected with ginsenoside Rb1,once a day,20 mg/kg body weight each time.The drug content in the mice brain was 11.37±3.05 ng/mg on the first day after administration,and 14.18±3.25 ng/mg on the seventh day after administration,indicating that intraperitoneal injection of ginsenoside Rb1 can achieve effective distribution of brain.The results of cerebral ischemia injury showed that ginsenoside Rbl significantly improved the overall activity of cerebral ischemia mice,reduced the mortality,increased the weight of surviving animals,improved the neurological behavior score,reduced brain edema and reduced the area of ischemic cerebral infarction,indicating that ginsenoside Rb1 has a definite protective effect on brain of mice with ischemiareperfusion.Neurotransmitter assay showed that Glu content in the ischemic side brain of CIR group increased significantly on the first day after ischemia(P<0.01).Until the 7th day after ischemia,the Glu content in the ischemic side brain of CIR mice was still higher than that of Sham mice(P<0.01).Compared with CIR group,20 mg/kg and 40 mg/kg ginsenoside Rb1 treatment significantly reduced the Glu content in the ischemic side mice brain.At the three time points tested,i.e.the first,third and seventh day after ischemia,the Glu content in the ischemic side mice brain of both two ginsenoside Rbl dose groups were significantly lower than that of sham group(P<0.05 or P<0.01).The determination of GLT-1 and GS showed that the mRNA and protein levels of GLT-1 in the CIR mice ischemic side brain were significantly increased(P<0.05 or P<0.01),while the mRNA and protein levels of GS in the ischemic side brain were significantly decreased(P<0.05 or P<0.01);After treatment with ginsenoside Rbl,the mRNA and protein levels of GLT-1 and GS were significantly increased,and the effect of ginsenoside Rb1 at 40 mg/kg was more significant.Immunofluorescence staining showed that intraperitoneal injection of ginsenoside Rbl could promote the expression of GLT-1 and GS in ischemic regions,especially in hippocampus.The results of intracellular signal pathway showed that compared with Sham group,the level of Akt protein in the CIR group ischemic side brain did not change significantly,but the phosphorylated Akt(p-Akt)increased significantly(P<0.05)and remained until the 7th day after ischemia.Contrary to Akt protein changes,PKA protein level in ischemic side brain of CIR group was significantly lower than that of Sham group,and its protein level was still significantly lower than that of Sham group on the 7th day after ischemia.Compared with CIR group,after 20 mg/kg and 40 mg/kg ginsenoside Rb1 treatment,Akt and PKA protein levels in ischemic side brain were significantly increased,and 40 mg/kg ginsenoside Rb1 had more obvious effects on pAkt and PKA.Summary:Ginsenoside Rbl can be effectively distributed to mouse brain by intraperitoneal injection;Ginsenoside Rbl has obvious protective effect on ischemic brain injury;Ginsenoside Rb1 can reduce the content of Glu in ischemic brain,promote the expression of GLT-1 and GS in ischemic region,and regulate the related signal pathways Akt and PKA.Part 2 Protective effect of ginsenoside Rbl on astrocytes injured by OGD/R and its effect on glutamate transport systemObjective:Using primary cultured astrocytes from mouse cerebral cortex,to observe the protective effect of ginsenoside Rb1 on astrocytes injured by glucose deficiency and hypoxia,study the effects of drugs on glutamate transport system of astrocytes,and explore the changes of signal pathways related to glutamate transport regulation.Methods:The primary cultured astrocytes were prepared from the cerebral cortex of newborn mice,and their purity was identified by glial fibrillary acidic protein(GFAP).Oxygen glucose deprivation/reoxygenation(OGD/R)cell injury model was prepared according to oxygen glucose deprivation for 6 h,and then reoxygenation for 24 h.The effects of ginsenoside Rbl intervention on astrocytes were observed.CCK-8,lactate dehydrogenase(LDH)and cell morphology were used to observe the cell damage;The changes of Glu uptake rate and GS enzyme activity were detected by biochemical method;The mRNA expression and protein level of GLT-1 and GS were detected by Western Blot and RT-PCR respectively;Detection the content of β-catenin and GSK-3β in cells by ELISA;Detection of cellular NF-κB binding activity to GLT-1 promoter by CHIP method;Western Blot,RT-PCR,immunocytochemistry and other methods were used to detect the mRNA expression and protein level changes of cell transcription factor NF-κB,CREB and β-catenin,and the changes of PI3K/Akt,p38 MAPK,ERK and cAMP/PKA signaling pathways were also detected.Results:In this study,the primary cultured cells adhered to the wall and grew with rich cytoplasm,and the cell body emitted many long branching fibers.The cells were identified as high-purity astrocytes by GFAP.Compared with the Control group,the OGD/R treated cells had shorter fibers and rounder cells,lower cell activity(P<0.01),and increased LDH release(P<0.01).After the ginsenoside Rb1 intervention of 2 μmol/L,5 μmol/L,10 μmol/L on OGD/R cells,the cell morphology tends to be normal,the cell activity increases,and the LDH release decreases significantly,of which 5μmol/L ginsenoside Rb1 was the most significant effect(P<0.01).The results of glutamate uptake showed that compared with the Control group,after OGD/R injury,the astrocytes’ uptake rate of extracellular Glu decreased significantly(P<0.01).After treatment with ginsenoside Rbl,the astrocytes’ uptake of Glu increased significantly(P<0.01),and the effect of increased concentration is enhanced,of which 5 μmol/L Glu uptake rate of cells was higher than that of normal cells.The results of enzyme activity and ATP determination showed that after OGD/R injury,the activities of glutamine synthetase and Na+-K+-ATPase in astrocytes decreased significantly,and the content of ATP in cells decreased significantly.The OGD/R cells treated with ginsenoside Rbl showed a significant increase in GS enzyme activity,Na+-K+-ATPase activity and ATP content in cells,of which 5 μmol/L ginsenoside Rb1 was more significant effect(P<0.01).GLT-1 detection results showed that the mRNA and protein of GLT-1 were stably expressed in astrocytes under primary culture conditions,and ginsenoside Rb1 treatment had no significant effect on the expression of GLT-1 in normal cells.After OGD/R treatment,the mRNA and protein expressions of GLT-1 were significantly increased.After being treated with ginsenoside Rbl,OGD/R cells showed a significant increase in GLT-1 expression,of which the GLT-1 protein amount in 5 μmol/L ginsenoside Rbl group cells was nearly twice that of normal cells.GS detection results showed that the expression of glutamine synthetase in normal astrocytes was rich,and ginsenoside Rbl treatment had no significant effect on the expression of GS in normal astrocytes.After OGD/R treatment,the mRNA and protein expression of GS in astrocytes decreased significantly.After OGD/R cells were treated with ginsenoside Rbl,the mRNA expression of GS was significantly increased,of which 5μmol/L ginsenoside Rbl was more significant effect,but its expression was still significantly lower than that of normal cells.The results of signal pathway showed that the expression of p-PI3K and p-Akt,pERK and p-p38 in astrocytes increased after OGD/R injury;At the same time,the expression of PKA and p-CREB decreased,total GSK-3β expression increased,but pGSK-3β expression decreased.Via the treatment of OGD/R cells with 5 μmol/L ginsenoside Rbl can increase the expression of p-PI3K and p-Akt,and increase the expression of p-ERK and p-p38.The expression level of p-GSK-3β after drug treatment was also increased.Chromatin immunoprecipitation results showed that ginsenoside Rbl treatment of OGD/R cells could make NF-κB(p65)on GLT-1 promoter binding increased significantly.Summary:Ginsenoside Rb1 has a clear protective effect on astrocytes injured by OGD/R.Ginsenoside Rb1 can significantly increase the uptake of glutamate by OGD/R astrocytes,and the drug can significantly increase the expression of GLT-1 and GS related to the transport system.Ginsenoside Rb1 can significantly promote the expression of astrocyte related signal pathways under OGD/R conditions,and the binding of GLT-1 promoter with p65 is significantly enhanced.Part 3 Study on the Key Signaling Pathway of Ginsenoside Rbl on the Regulation of Glutamate Transport in AstrocytesObjective:Based on the effect of ginsenoside Rbl on GLT-1/GS regulatory pathway,by specifically inhibiting or blocking the signal pathway,to study the key link of ginsenoside Rbl on the regulation of glutamate transport in astrocytes,and preliminarily clarify the mechanism of ginsenoside Rbl on the regulation of glutamate transport in astrocytes.Methods:The primary cultured astrocytes of mouse cerebral cortex were used to establish the cell injury model by OGD/R method.PDTC was used to inhibit NF-κB,KG-501 inhibits CREB-CBP complex,LY294002 inhibits PI3K activity,SB203580 inhibits p38 MAPK,PD98059 inhibits ERK,and H89 inhibits cAMP/PKA activity,the expression change of glutamate transporter was detected;With XAV939 inhibition ofβ-catenin activity,LY294002 inhibition of PI3K activity,SB203580 inhibition of p38 MAPK,PD98059 inhibition of ERK and H89 inhibition of PKA activity,to detect the expression change of glutamine synthetase and study the key link of ginsenoside Rb1 regulating glutamate transport.Result:When transcription factor NF-κB entry into the nucleus was inhibited,the expression of glutamate transporter GLT-1 in cells of all groups decreased,and the expression of GLT-1 in OGD/R+ginsenoside Rb1 cells was significantly different before and after inhibition(P<0.01);When the transcription factor CREB was inhibited,the facilitating effect of ginsenoside Rb1 on GLT-1 expression was weakened.When PI3K and PKA activities were inhibited,the expression of GLT-1 in astrocytes decreased,and the up regulation of ginsenoside Rbl on GLT-1 expression was also inhibited.Combined with the changes of GLT-1 and PI3K/PKA signal pathway in astrocytes before and after OGD/R injury,it suggests that PI3K/Akt/NF-κB plays a leading role in the regulation of GLT-1 by ginsenoside Rb1.When transcription factor β-catenin was inhibited by XAV939,the expression of glutamine synthetase protein in cells of each group was significantly decreased,and the GS promoting effect of ginsenoside Rbl on OGD/R cells was significantly inhibited,indicating that β-catenin participates in GS expression,and the effect of ginsenoside Rbl on GS requires the help of transcription factors β-catenin effect;When PI3K activity and PKA activity were inhibited respectively,the GS effect of ginsenoside Rb1 on OGD/R cell was inhibited.In combination the GS changes and PI3K/PKA/GSK-3βpathway changes of astrocytes before and after OGD/R injury,and phosphorylated GSK-3β(p-GSK-3β)suppressed changes,PKA/GSK-3β/β-catenin plays a leading role in the regulation of GS by ginsenoside Rb1.Summary:Ginsenoside Rbl regulates the key proteins GLT-1 and GS of glutamate transport through nuclear transcription factor NF-κB and β-catenin respectively.And the ginsenoside Rb1 regulation of GLT-1 and GS respectively through PI3K/Akt/NF-κB and PKA/GSK-3β/β-catenin signal pathway. |
