| The incidence of acute ischemic stroke is increasing year by year with a trend of getting younger,and has become the leading cause of death/disability in Chinese adults.The standard treatment for acute ischemic stroke is endovascular mechanical thrombectomy and intravenous thrombolysis in the time window to restore blood perfusion by dredging blocked vessels,so as to save brain tissue in the ischemic penumbra,thereby improving the clinical symptoms and prognosis of stroke patients.However,reperfusion may be followed by inflammation,oxidative stress,and calcium overload,which may further aggravate the brain tissue injury,known as cerebral ischemia-reperfusion injury(CIRI).Apoptosis plays an important role in the development of CIRI,and determines the size of cerebral infarction and the severity of clinical symptoms.Inhibition of apoptosis can improve the outcome and prognosis of stroke patients.The calcium-sensing receptor(CAsr),a member of the G protein coupled receptor superfamily,is expressed in central nervous system neurons and can be activated by extracellular calcium,and participates in ischemia reperfusion injury by inducing apoptosis.Astragaloside IV,the main active ingredient of astragalus membranaceus,which is commonly used in the treatment of ischemic cerebrovascular diseases,has the properties of scavenging free radicals,anti-inflammatory,anti-apoptosis,regulating energy metabolism,and alleviating CIRI by inhibiting apoptosis.However,it is not clear whether the anti-CIRI effect of Astragaloside IV is related to CaSR.Therefore,with CaSR as the breakthrough point,the middle cerebral artery occlusion/reperfusion(MCAO/R)model of SD rats and the oxygen and glucose deprivation/reoxygenation(OGD/R)model of PC12 cells were established to simulate the process of CIRI.To investigate whether the protective effect of Astragaloside IV on CIRI is related to CaSR and to explore its specific mechanism,CaSR inhibitor and activator was used.Part one effect of calcium-sensing receptor on Astragaloside IV attenuating cerebral ischemia-reperfusion injuryObjective:To observe the neuroprotective effect of Astragaloside IV and CaSR inhibitor(NPS-2143)on CIRI in vivo and in vitro.Methods:The MCAO/R model of rats was established by modified suture method to simulate CIRI,and the OGD/R model of PC12 cells was established to simulate the damage of CIRI on neurons.In vivo experiments:clean,healthy,male SD rats were randomly divided into Sham operation group(Sham),model group(MCAO/R),Astragaloside IV group(AST-IV)and CaSR inhibitor group(NPS-2143).Except for the sham group,all the other groups were subjected to ischemia for 2 h and reperfusion for 24 h.AST-IV group and NPS-2143 group were given Astragaloside IV 20mg·kg-1 and NPS-2143 10?mol·kg-1 intraperitoneally at the same time of reperfusion.According to the results of Zea Longa scoring,the rats with successful modling were selected for experimental study.The neuroprotective effects of Astragaloside IV on MCAO/R rats were observed by TTC staining,H&E staining and Nissl staining,and CaSR expression was detected by Western blot.In vitro experiment:PC12 cells were randomly divided into normal control group(Control),model group(OGD/R),Astragaloside IV group(AST-IV)and CaSR inhibitor group(NPS-2143).Except for the Control group,all the other groups were subjected to oxy-glucose deprivation for 2 h and reoxygenation for 24 h.AST-IV group and NPS-2143 group were treated with the optimal concentration which was determined by preliminary experiments(100?mol/L)Astragaloside IV and 25?mol/L NPS-2143 at the time of reoxygenation.The cell growth state weas observed by inverted microscope,and CCK-8 was used to detect the effects of Astragaloside IV and NPS-2143on the survival rate of OGD/R model PC12 cells.Also,CaSR expression was detected by Western blot.Results:1.In vivo experiment:(1)Neurological deficit:Compared with Sham group,the neurological function score of MCAO/R group was significantly increased(P<0.05).Compared with MCAO/R group,the neurological function scores of rats in AST-IV group and NPS-2143 group were significantly decreased(P<0.05).(2)Cerebral infarction volume:Compared with Sham group,rats in MCAO/R group showed extensive infarction on the left side of brain tissue(P<0.05).Compared with MCAO/R group,the cerebral infarction volume of rats in AST-IV group and NPS-2143group decreased significantly(P<0.05).(3)Pathological changes of brain tissue:(1)H&E staining results:There were no significant changes in the morphological structure of brain tissue in Sham group.The MCAO/R group showed neuronal apoptosis and necrosis in ischemic brain tissue,and a large number of neuronal cells were lost(P<0.05).Compared with MCAO/R group,AST-IV group and NPS-2143 group showed significant reduction in brain histopathological changes(P<0.05).(2)Nissl staining results:The nerve cells of Sham group rats were normal and nissite was abundant.In MCAO/R group,the nerve cells in ischemic brain tissue were severely damaged,and the number of nissite was decreased with shallow staining(P<0.05).Compared with MCAO/R group,AST-IV group and NPS-2143 group showed less morphological changes and more nissite(P<0.05).(4)Protein expression of CaSR:Western blot results showed that,compared with Sham group,the expression levels of CaSRwere up-regulated(P<0.05).Compared with MCAO/R group,the expression levels of CaSR in AST-IV group and NPS-2143 group were down-regulated(P<0.05).2.In vitro experiment:(1)Survival rate of PC12 cells:Compared with the Control group,the survival rate of PC12 cells in OGD/R group was significantly decreased(P<0.05).Compared with OGD/R group,different concentrations of Astragaloside IV could improved the survival rate of PC12 cells after OGD/R significantly(P<0.05),among which Astragaloside IV at concentration of 100?mol/L had the best protective effects on OGD/R PC12 cells.And NPS-2143 also significantly increased the survival rate of PC12 cells,compared with OGD/R group(P<0.05).And 100?mol/L concentration of Astragaloside IV was selected for subsequent experimental study.(2)PC12 cell morphology:PC12 cells in Control group showed normal adherent growth,normal cell morphology and strong refractive index.Compared with Control group,PC12cells in OGD/R group were less adheren,cell body swelling,roundness,less prominent and poor refractive index.Compared with OGD/R group,the growth state of PC12 cells in AST-IV group and NPS-2143 group improved.(3)Protein expression levels of CaSR in PC12 cells:Western blot results showed that,compared with Control group,the expression levels of CaSR up-regulated(P<0.05).Compared with MCAO/R group,the expressions of CaSRin AST-IV group and NPS-2143 group were down-regulated(P<0.05).Summary:1.Astragaloside IV and CaSR inhibitor NPS-2143 can significantly reduce the neurological function score of MCAO/R model rats,reduce the volume of cerebral infarction in rats,reduce nerve cell injury in brain tissue,down-regulate the peotein expression of CaSR,and play a neuroprotective role for CIRI.2.Astragaloside IV can significantly improve the survival rate of OGD/R model PC12 cells and improve the cell growth state,and down-regulate the peotein expression of CaSR.CaSR inhibitor NPS-2143 has similar effects to Astragaloside IV.3.Astragaloside IV may play its neuroprotective role against CIRI by inhibiting the expression of CaSR.Part two Astragaloside IV reduce cell apoptosis induced by cerebral ischemia-reperfusion injury by inhibiting the expression of calcium-sensing receptorsObjective:To observe the protective effect of Astragaloside IV and CaSR inhibitor NPS-2143 on cell apoptosis induced by CIRI,and to investigate whether Astragaloside IV can reduce CIRI induced apoptosis by inhibiting CaSR expression.Methods:MCAO/R model of SD rats and OGD/R model of PC12 cells were established to simulate the process of CIRI.Modeling and grouping methods was set as in part 1.In vivo experiment:Transmission electron microscopy was adopted to observe the ultrastructure of nerve cells in cortical brain tissue of ischemic area,and the expression of CaSR protein was detected by immune-fluorescence,and the expression of apoptosis-related proteins Caspase 3,Bcl-2 and Bax were detected by Western blot.In vitro experiment:apoptosis rate was detected by flow cytometry,and protein expression levels of Caspase 3,Bcl-2 and Bax were detected by Western blot to detect the effect of Astragaloside IV and CaSR inhibitor NPS-2143 on CIRI-induced apoptosis.Results:1.In vivo experiment:(1)Ultrastructural changes of nerve cells in brain tissue:The results of transmission electron microscopy showed that the structure of nerve cells in Sham group was normal.In MCAO/R group,the nerve nuclei were irregular,with increased heterochromatin in the nucleus,edge aggregation to the inner side of the nuclear membrane,and even nuclear membrane fracture and dissolution,increased intracellular vacuolar structure.Compared with MCAO/R group,AST-IV group and NPS-2143 significantly reduced the ultrastructural changes of nerve cells.(2)Expression of CaSR in neurons:Immunofluorescence double-labeled staining results showed that,in Sham group,the number of neurons in the brain tissue was more and the expression of CaSR was less.CaSR in MCAO/R group was up-regulated compared with Sham group.Compared with MCAO/R group,the expression level of CaSR in AST-IV group and NPS-2143 group was down-regulated.(3)Protein expression of Caspase 3,Bcl-2 and Bax:Western blot results showed that,compared with Sham group,the expression levels of Caspase 3 were up-regulated(P<0.05),and Bcl-2/Bax decreased(P<0.05).Compared with MCAO/R group,the expression levels of Caspase 3 in AST-IV group and NPS-2143 group were down-regulated(P<0.05),and Bcl-2/Bax increased(P<0.05).2.In vitro experiment:(1)Apoptosis rate:Compared with the Control group,the apoptosis rate of PC12 cells in OGD/R group was significantly increased(P<0.05).Compared with OGD/R group,the apoptosis rate of PC12 cells in AST-IV group and NPS-2143 group was significantly decreased(P<0.05).(2)Protein expression levels of Caspase 3,Bcl-2 and Bax:Western blot results showed that,compared with Control group,the expression levels of Caspase 3 up-regulated(P<0.05),and Bcl-2/Bax decreased(P<0.05).Compared with MCAO/R group,the expressions of Caspase 3 in AST-IV group and NPS-2143 group were down-regulated(P<0.05),and t Bcl-2/Bax increased(P<0.05).Summary:1.Astragaloside IV and CaSR inhibitor NPS-2143 can significantly inhibit MCAO/R-induced increase of CaSR on nerve cells,reduce expression of Caspase 3,increase Bcl-2/Bax,and alleviate apoptosis of rat brain cells induced by MCAO/R.2.Astragaloside IV and CaSR inhibitor NPS-2143 can significantly reduce OGD/R-induced apoptosis rate of PC12 cells,reduce expression of Caspase 3,increase Bcl-2/Bax,alleviate the apoptosis rate of PC12 cells induced by OGD/R.3.Astragaloside IV may alleviate CIRI-induced apoptosis by inhibiting CaSR expression.Part three Mechanism of Astragaloside IV inhibiting calcium-sensing receptor to reduce apoptosis after cerebral ischemia-reperfusion injuryObjective:To investigate the mechanism of Astragaloside IV inhibiting calcium-sensing receptor to reduce apoptosis after cerebral ischemia-reperfusion injury.Methods:The SD rat MCAO/R model was established to simulate the CIRI process.Rats were randomly divided into Sham group,model group(MCAO/R),astragaloside IV group(AST-IV),CaSR activator group(GdCl3),Astragaloside IV+CaSR activator group(AST-IV+GdCl3)and CaSR inhibitor group(NPS-2143).Astragaloside IV group,GdCl3 group,AST-IV+GdCl3group and NPS-2143 group were given 20 mg·kg-1,GdCl3 30 mol·kg-1,Astragaloside 20 mg·kg-1+GdCl3 30 mol·kg-1,and NPS-2143 10 mol·kg-1intraperitoneally at the same time of reperfusion.Cell apoptosis was observed by TUNEL staining,and calcium ion concentration in serum and brain tissue was detected by microplate ssay.Mitochondrial structure was observed by transmission electron microscopy.The expression of apoptosis inducing factor(AIF)protein was detected by Western blot.The OGD/R model of PC12 cells was established to simulate the neuronal injury induced by CIRI.PC12 cells were randomly divided into normal Control group(Control),model group(OGD/R),Astragaloside IV group(AST-IV),CaSR activator group(GdCl3),Astragaloside IV+CaSR activator group(AST-IV+GdCl3)and CaSR inhibitor group(NPS-2143).AST-IV group,GdCl3 group,AST-IV+GdCl3group and NPS-2143 group were treated with100?mol/L Astragaloside IV,150?mol/L GdCl3,100?mol/L Astragaloside IV+150?mol/L GdCl3,and 25?mol/L NPS-2143 at the time of reoxygenation.The cell apoptosis rate was measured by Flow cytometry assay,intracellular and extracellular calcium concentration was measured by microplate assay,mitochondrial membrane potential(MMP)was measured by JC-1 staining,and expressions of AIF protein were measured by Western blot.Results:1.In vivo experiment:(1)Apoptosis index of nerve cells in brain tissue:TUNEL staining results:compared with Sham group,the apoptosis index in MCAO/R group increased significantly(P<0.05).Compared with MCAO/R group,the apoptosis index of AST-IV group and NPS-2143 group decreased significantly(P<0.05),Apoptosis index of GdCl3 group increased significantly(P<0.05).Compared with AST-IV group,the apoptosis index of GdCl3 group and AST-IV+GdCl3 group increased significantly(P<0.05).Compared with GdCl3 group,the apoptotic index of AST-IV+GdCl3 group and NPS-2143 group decreased significantly(P<0.05).(2)Serum calcium level:There was no significant difference in serum calcium content between the groups(P>0.05).(3)Calcium level in brain tissue:Compared with Sham group,calcium level in ischemic brain tissues of MCAO/R group was significantly increased(P<0.05).Compared with MCAO/R group,calcium level in AST-IV group and NPS-2143 group decreased significantly(P<0.05),while calcium level in GdCl3 group increased significantly(P<0.05).Compared with AST-IV group,the calcium content in GdCl3 group and the AST-IV+GdCl3 group increased significantly(P<0.05).Compared with GdCl3 group,calcium level in AST-IV+GdCl3group and NPS-2143 group was significantly decreased(P<0.05).(4)Mitochondrial structure of nerve cells in brain tissue:Transmission electron microscopy showed that the mitochondrial structure of nerve cells in the left cortex brain tissues of rats in Sham group was normal,round or rod-shaped,with intact mitochondrial membrane and developed cristae.Mitochondria in ischemic brain tissue of MCAO/R group were swollen and spherical,with incomplete mitochondrial membranes,broken cristae and even disappeared.Compared with MCAO/R group,the mitochondrial structure of AST-IV group and NPS-2143 group was improved,while the mitochondrial damage of GdCl3 group was more serious.Compared with AST-IV group,the mitochondrial damage in the GdCl3 group and AST-IV+GdCl3 group was more serious.Compared with GdCl3 group,AST-IV+GdCl3 group and NPS-2143 group had less damage of mitochondrial structure.(5)Expression of AIF protein:Western blot results showed that compared with Sham group,AIF protein expression in ischemic brain tissue of MCAO/R group was up-regulated(P<0.05).Compared with MCAO/R group,AIF protein expression in AST-IV group and NPS-2143 group was down-regulated(P<0.05),while AIF protein expression in GdCl3 group was up-regulated(P<0.05).Compared with AST-IV group,the expression of AIF protein in GdCl3 group and AST-IV+GdCl3 group was up-regulated(P<0.05).Compared with GdCl3 group,AIF protein expression in AST-IV+GdCl3 group and NPS-2143 group was down-regulated(P<0.05).2.In vitro experiment:(1)Apoptosis rate:Compared with Control group,the apoptosis rate of PC12 cells in OGD/R group was significantly increased(P<0.05).Compared with OGD/R group,apoptosis rate of AST-IV group and NPS-2143 group decreased significantly(P<0.05)and apoptosis rate of GdCl3 group increased significantly(P<0.05).Compared with the AST-IV group,the apoptosis rate of GdCl3 group and AST-IV+GdCl3 group was significantly increased(P<0.05).Compared with GdCl3 group,the apoptosis rate of AST-IV+GdCl3group and NPS-2143 group decreased obviously(P<0.05).(2)Calcium level in cell culture solution supernatant:Compared with Control group,the supernatant concentration of calcium in OGD/R group decreased significantly(P<0.05).Compared with OGD/R group,the concentration of calcium in AST-IV group and NPS-2143 group increased significantly(P<0.05),and the calcium level in GdCl3 group decreased significantly(P<0.05).Compared with AST-IV group,the calcium level in GdCl3 group and AST+GdCl3 group was significantly decreased(P<0.05).Compared with GdCl3 group,the calcium level in AST-IV+GdCl3 group and NPS-2143 group increased obviously(P<0.05).(3)Intracellular calcium concentration:Compared with Control group,the concentration of calcium ions in PC12cells in OGD/R group was significantly increased(P<0.05).Compared with OGD/R group,intracellular calcium level in AST-IV group and NPS-2143group decreased significantly(P<0.05)and intracellular calcium level in GdCl3 group increased significantly(P<0.05).Compared with AST-IV group,the intracellular calcium level in GdCl3 group and AST-IV+GdCl3 group was significantly increased(P<0.05).Compared with GdCl3 group,the intracellular calcium level in AST-IV+GdCl3 group and NPS-2143 group decreased obviously(P<0.05).(4)Mitochondrial membrane potential level:Compared with Control group,the MMP level of PC12 cells in OGD/R group was significantly decreased(P<0.05).Compared with OGD/R group,MMP level of PC1 AST-IV group and NPS-2143 group was significantly increased(P<0.05)and MMP level in GdCl3 group significantly decreased(P<0.05).Compared with AST-IV group,the MMP level in GdCl3 group and AST-IV+GdCl3 group group was significantly decreased(P<0.05).Compared with GdCl3 group,the MMP level in AST-IV+GdCl3 group and NPS-2143 group increased obviously(P<0.05).(5)Expression of AIF protein:Compared with Control group,AIF expression in PC12 cells of OGD/R group was up-regulated(P<0.05).Compared with OGD/R group,AIF expression was down-regulated in AST-IV group and NPS-2143 group(P<0.05),while AIF protein expression in GdCl3 group was up-regulated(P<0.05).Compared with AST-IV group,GdCl3 group and AST-IV+GdCl3 group showed up-regulated expression of AIF(P<0.05).Compared with GdCl3group,AIF expression was down-regulated in AST-IV+GdCl3 group and NPS-2143 group(P<0.05).Summary:1.Astragaloside IV can inhibit MCAO/R-induced apoptosis by inhibiting the increase of calcium concentration in ischemic brain tissue,reducing mitochondrial structural damage,and down-regulating the expression level of apoptosis-inducing factor AIF.2.Astragaloside IV can inhibit extracellular calcium ion flow,inhibit intracellular calcium overload,inhibit the decline of mitochondrial membrane potential,down-regulate the expression level of apoptosis-inducing factor AIF,and thus inhibit OGD/R-induced PC12 cell apoptosis.Conclusions:1.Astragaloside IV can reduce the neurological function score of MCAO/R rats,reduce the volume of cerebral infarction in rats,increase the number of nerve cells and nidosomes,and reduce brain tissue damage.It can also improve the survival rate of OGD/R model PC12 cells and improve the cell growth status.Calcium-sensing receptor is involved.2.Astragaloside IV can reduce the expression level of CaSR in MCAO/R-induced rat brain tissue,inhibit the expression of Caspase 3,increase Bcl-2/Bax,and alleviate the apoptosis induced by MCAO/R-induced rat brain tissue.The apoptosis rate of PC12 cells induced by OGD/R was significantly reduced,the expression of CaSR and Caspase 3 proteins was down-regulated,and the Bcl-2/Bax was increased to alleviate the OGD/R-induced apoptosis of PC12cells.Calcium sensitive receptors are involved in the inhibition of astragaloside iv on apoptosis.3.Astragaloside IV can inhibit MCAO/R-induced CaSR protein expression in rat brain tissues,inhibit the increase of calcium ion concentration in ischemic brain tissues,reduce the damage of mitochondrial structure,down-regulate the expression level of AIF,and thus inhibit MCAO/R-induced apoptosis.In addition,astragaloside iv can inhibit OGD/R-induced PC12 cell CaSR protein expression,inhibit extracellular calcium ion flow,inhibit intracellular calcium overload,inhibit the decline of MMP,down-regulate AIF expression level,and thus inhibit OGD/R-induced PC12 cell apoptosis. |
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