Regulatory Effect Of Panax Notoginseng Saponins On Glutamate Metabolic Pathway In Astrocytes After Cerebral Ischemi

In the central nervous system,glutamic acid is the major excitatory neurotransmitter responsible for excitatory signaling transmission.A large body of research suggested that the excessive extracellular accumulations of glutamic acid lead to neuronal death.Accumulating evidences support that glia cells,particularly astrocytes play an important role in glutamate metabolism and homeostasis.Following synaptic release,glutamate is mainly taken up by the astrocytic glutamate transporter GLT-1,and then converted into glutamine by glutamine synthetase(GS).Subsequently,the glutamine in astrocytes is transported back to the neuron and converted to glutamate for reuse,which is named glutamate-glutamine cycle.Interfering the glutamate-glutamine cycle or its key components such as GLT-1 and GS has been reported to influence the glutamate levels and glutamate-induced excitotoxicity.Panax Notoginseng Saponins(PNS)is an active ingredient extracted from Chinese medicine Panax notoginseng.It can effectively inhibit the development of cerebral ischemia,and protect neuron after brain ischemia.Thus,based on the researches on glutamate transports and metabolism related enzymes,the main study is to elicit the molecular mechanism of PNS-induces neuroprotection after cerebral ischemia.Part 1 The effect of PNS on dysfunction of Glu metabolism and transport in cortex induced by cerebral ischemia/reperfusionObjective:The neuroprotective effect of PNS and its influence on Glu metabolism pathway on mice of cerebral ischemic reperfusion injuryMethod:Cerebral ischemia/reperfusion was induced by middle cerebral artery occlusion(MCAO)in mice which were randomly divided into 5 groups:Sham,cerebral ischemia/reperfusion(CIR),Nimodipine(Nim)13.65 mg/kg,PNS 60 mg/kg and PNS 120 mg/kg.Experimental mice administered intragastric once a day post-ischemia.The reperfusion period of 24 h,72 h and 168 h were set as the observation time,1)The general state,body weight,neurological function score,and forelimp grip of cerebral ischemia/reperfusion mice were observed.2)With Dry/wet specific gravity method,brain water content was detected;TTC staining was used to measure cerebral infarct size;Nissl staining was used to detect neuronal changes.3)Biochemical assay was used to detect Glu concentration;Na+-K+-ATPase and PAG activity;Western blot and RT-PCR were used to detect the expression levels of GLT-1,GS protein and mRNA.Results:1)In the experiment,mice in the Sham group had normal status of food and drinking intake,smooth movements,stable weight gain,and no death.The mice in CIR group were dull,contracted,and turned to the affected side.Body weight was significantly decreased compared with sham group(P<0.01),and the mortality rate of mice was 50%at 168 h after reperfusion.After PNS treatment,the mental state of the mice was improved,and the weight of mice was gradually stabilized,the mortality of mice was decreased;the weight of the mice in PNS(120 mg/kg)group showed a significant increase(P<0.05)after 144 h reperfusion.The result of neurological function score showed that the CIR mice had obvious symptoms of neurological deficits;with the extension of time,the recovery of neurological deficits was observed in all groups,the neurological function scores of mice in the PNS(120 mg/kg)group was lower at 96 h after reperfusion(P<0.05).Forelimb grip results showed that the forearm holding power of CIR mice was significantly reduced(P<0.01),and treatment with Nim and PNS(120 mg/kg)for 168h,the forelimb grip of the mice was obviously restored(P<0.05).2)At 24 h and 72 h after reperfusion,the brain water content of mice in CIR group was significantly increased(P<0.01);PNS(120 mg/kg)obviously reduced brain water content after 24 h reperfusion(P<0.01);Nim and PNS(60 mg/kg,120 mg/kg)significantly decreased brain water content after 72 h reperfusion(P<0.05).Compared with Sham group,the cerebral infarction area in CIR group mice was obviously increased(P<0.01);at 24 h and 72 h after reperfusion,Nim and PNS(120 mg/kg)significantly reduced the cerebral infarct size of mice(P<0.01);The result of Nissl staining showed that the neurons in the cortical infact area of CIR mice were disorderly,swollen and structure blurred;after PNS treatment,the number of normal neurons in cortex was increased,and the swelling of the neurons in cerebral ischemic mice were improved.3)Compared with Sham group,Glu concentration was significantly increased(P<0.01),and Na+-K+-ATPase activity was obviously decreased in mice cortex of CIR group;After Nim and PNS(60 mg/kg,120 mg/kg)treatment,the Glu concentration was significantly decreased(P<0.05),and Na+-K+-ATPase activity was obviously increased for 72 h and 168 h.Compared with Sham group,the mRNA and protein expressions of GLT-1 and GS in the ischemic cortex of mice in CIR group were significantly down-regulated(P<0.01);Nim and PNS(60 mg/kg,120 mg/kg)treatment for 72 h and 168 h,the expression of GLT-1 mRNA and protein in the cortex were increased significantly(P<0.05);after 24 h of PNS(120 mg/kg)treatment,only GLT-1 protein level was increased obviously(P<0.05);the GS mRNA expression and protein levels in the cortex of the mice in Nim and PNS(60 mg/kg,120 mg/kg)group at 168 h after ischemia were significantly increased(P<0.01).Part 2 The effect of PNS and its components R1,Rb1,Rg1 on dysfunction of Glu metabolism and transport in astrocytes after OGD/RObjective:the protective effects of PNS and its components R1,Rb1,Rg1 on astrocytes after OGD/R,and their influence on Glu metabolism pathway.Method:In this section,cortical astrocytes were cultured in vitro.1)Morphological changes of astrocytes were observed by inverted microscope;GFAP-labeled astrocytes were identified by immunocytocfluorescence staining;the cell growth curve was recorded.2)An in vitro ischemic stroke model(OGD/R)was established,astrocytes were divided into 6 groups:Control,OGD/R,PNS,R1,Rb1,Rg1.CCK-8 asssy was used to detect the cell viability,and the release of extracellular LDH was measured.3)The astrocytes Na+-K+-ATPase activity and the Glu intake rate were detected by biochemical method;Western Blot,RT-PCR and immunocytocfluorescence assay were used to detect GLT-1,GS mRNA expression and protein levels.Results:1)Observed under an inverted microscope,astrocytes showed unregulated shapes,big cell body,abundant cytoplasm,many longer apophyses.The cultured astrocytes were identified by GFAP immunofluorescence staining,the cells were positive fluorescence staining.2)Compared with the Control group,cell viability was significantly decreased(P<0.01),LDH release was obviously increased(P<0.01)after OGD/R injury,and astrocytes protrusion became short,shrinking,and even detectment;Compared with OGD/R group,PNS(10,20,and 50 μg/mL),Rbl(2,5,and 10 μmol/L),and Rg1(10 μmol/L)markly increased cell viability(P<0.05),and significantly reduced LDH released(P<0.01).R1(10,20,and 50μmol/L)significantly reduced LDH released(P<0.01).Base on the above research results,we selected the most adequate concentration for next experiments:PNS(20 μg/mL),R1(50μmol/L),Rbl(5 μmol/L),and Rg1(10 μmol/L).3)Compared with the Control group,the uptake rate of Glu decreased significantly(P<0.01),Na+-K+-ATPase activity decreased(P<0.01),and expression of GLT-1 and GS mRNA and protein were obviously down-regulated,and the fluorescence intensity of GLT-1 and GS were reduced after OGD/R injury;after treatment with PNS,Rbl and Rg1,Glu uptake was markly enhanced(P<0.05),and Na+-K+-ATPase activity was obviously increased(P<0.05);The expression of GLT-1 and GS mRNA and protein were significantly up-regulated(P<0.05)with PNS,Rbl and Rg1 treatment.After R1 treatment,only the expression of GLT-1 protein was markly up-regulated(P<0.05).Part 3 The effect of PNS and its components R1,Rb1,Rg1 on neuronal survival in neuron-astrocyte co-cultures induced by OGD/RObjective:in order to convincingly elucidate the role of Glu-Gln cycle on PNS and its compoments R1,Rb1,Rg1 induced neuronal protection agsinst OGD/R,the effects of GLT-1 inhibition with DHK,GS inhibition with MSO on PNS and its compoments R1,Rb1,Rg1 induced neuronal protection agsinst OGD/R were investigatated in neuron-astrocyte co-cultures.Method:In this section,the transwell system was used for neuron-astrocyte co-culture.1)Mouse cortical neurons were cultured in vitro,the neuron morphology was observed using an inverted microscope;MAP-2 labeled neurons were identified by immunofluorescence staining.2)The model of neuron-astrocyte co-coutures by OGD/R injured was established,and the co-cultured cells were divided into:Control,DHK,MSO,OGD/R,PNS(20μg/mL),R1(50μmol/L),Rb1(5μmol/L),Rg1(10μmol/L),DHK+(PNS,R1,Rb1,Rg1),and MSO+(PNS,R1,Rb1,Rg1).Before OGD/R,the function and activities of GLT-1 and GS were inhibited separately by adding DHK(1.0 mmol/L)or MSO(3.0 mmol/L)for 30 min,CCK-8 assay was used to detect the cell activity after OGD/R;the release of extracellular LDH and the concentration of Glu in extracellular fluid were measured by biochemical method.Results:1)Observed under an inverted microscope,neurons were distributed evenly,tightly adherent,and grew well.Never cell axons and neurite were connected to each other to form the "cell-net",showing the typical morphology of neurons.The cultured neurons were identified by MAP-2 immunofluorescence staining,the cells showed positive fluorescence staining.2)In the system of neuron-astrocyte co-cultures,compared with Control group,neuron viability was significantly decreased(P<0.01),LDH release was obviously increased(P<0.01),and extracellular Glu concentration was markly increased after OGD/R injury(P<0.01);PNS,Rb1 and Rg1 effectively evenated neuron viability(P<0.05),reduced LDH release(P<0.05)and extracellular Glu concentration(P<0.05)in neuron-astrocyte co-cultures.Administration of DHK prior to PNS,Rb1,Rg1 incubation inhibited the neuronal protection,which was represented with decreasing neuron viability(P<0.05),increasing LDH release(P<0.01)and extracellulur Glu concentration(P<0.05);Administration of MSO prior to PNS,Rbl,Rgl incubation inhibited the neuronal protection,which was represented with decreasing neuron viability(P<0.05),increasing LDH release(P<0.01)and extracellulur Glu concentration(P<0.05);the vehicle for DHK or MSO had no effect on cell viability,LDH release and Glu concerntration in the neuron-astrocyte co-cultures without OGD/R.Conclusion:PNS ameliorate cerebral ischemia-induced excitotoxicity via increasing expression GLT-1 and GS,improving Glu metabolism pathway.Rbl and Rg1 are effective monomer components for inhibiting excitotoxicity.