| ObjectiveThe purpose of this experimental study includes two parts.1.The effects of astragalus membranaceus and ligustrazine injection on Ca2+influx,mitochondrial membrane potential and apoptosis in Sprague Dawley(SD)rat cortical neurons on Oxygen-glucose deprivation/reperfusion(OGD/R)model and its possible molecular mechanism were studied in vitro.2.The effects of NMDA receptor regulated by astragalus membranaceus and ligustrazine injection on neurological deficit,oxidative stress,apoptosis and synaptic regulatory protein induced by cerebral ischemia-reperfusion in C57bl/6 mice were studied in vivo,and the protective mechanism of astragalus membranaceus and ligustrazine injection on cerebral ischemia-reperfusion injury was discussed.The purpose of this study is to provide a research basis for the treatment of cerebral ischemia-reperfusion injury with traditional Chinese medicine.Methods1.The effects of astragalus membranaceus and ligustrazine injection on mitochondrial function,calcium concentration and neuronal apoptosis were expounded by constructing the OGD/R model of primary cortical neurons in SD rats.(1)Primary SD rat cortical neurons were cultured and identified,and OGD3h/R24h model was established after maturation.(2)Cell viability was determined by CCK-8 method to explore the appropriate concentration of astragalus injection,ligustrazine injection and MK-801.(3)The experiment was divided into five groups:control group,OGD/R model group,OGD/R+Ast+Lig group,OGD/R+MK801 group,OGD/R+MK-801+Ast+Lig.(4)To detect the apoptosis of neurons in each group by TUNEL method.(5)To detect the effect of astragalus membranaceus and ligustrazine injection on mitochondrial function and calcium influx of neurons in each group by JC-1 and Fluo-4AM kit.(6)The expression and changes of neuronal apoptosis factors Bcl-2,Bax,Caspase-3,Caspase-9 and Caspase-12 in each group were detected by Western Blot,qRT-PCR,immunofluorescence to understand the protective mechanism of astragalus membranaceus and ligustrazine injection on neurons after oxygen-glucose deprivation injury.2.The MCAO/R model of C57b1/6 mice was randomly divided into 5 groups:sham group,MCAO/R group,Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group,Drug intervention was carried out after the model was made,and observation was carried out in 24h and 48h time points.(1)To observe the neurobehavioral changes of each group by Longa score,the changes of infarct volume by TTC staining.(2)To observe the changes of SOD activity and MDA content in cerebral ischemic area of mice in each group by SOD and MDA kits.(3)The expression of apoptosis-related proteins(Bcl-2,Bax,Caspase-3,Caspase-9,Caspase-12)and NR2A,NR2B,synaptic-related proteins(PSD-95,SYN,GAP-43)were detected by Western Blot.(4)The expression of apoptotic factors Caspase-3,Caspase-9 and Caspase-12 were detected by immunohistochemistry.(5)The mRNA expression levels of apoptosis-related factors(Bcl-2,Bax,Caspase-3,Caspase-9,Caspasel2)and NR2A,NR2B,synaptic-related factors(PSD-95,SYN,GAP-43)in each group were verified by qRT-PCR,and the effects of astragalus membranaceus and ligustrazine injection on apoptosis and synaptic regulation in mice with cerebral ischemia-reperfusion injury were observed.Results1.Culture and identification of primary SD rat cortical neuronsOn the 7th day after primary culture,it was observed under microscope that the cell body of neuron was round,fusiform,triangular or polygonal,with obvious nucleolus,full cross-linking of processes,network at the end and mature neurons.The percentage of Neun positive and DAPI positive was identified by immunofluorescence,and the purity of neurons was(94.35±2.73)%.2.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on cell viability of OGD/R model of primary rat cortical neuronsCCK-8 showed that the suitable concentrations of astragalus injection,ligustrazine injection and MK-801 were 0.2mg/mL,120μm and 5μm,respectively.After drug intervention on OGD/R model,it was found that compared with control group,the cell activity of OGD/R group decreased to less than 50%.Compared with OGD/R,the cell activity of Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group increased significantly(P<0.001),and the increase was the most obvious in Ast+Lig+MK-801 group.3.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on apoptosis of primary rat cortical neurons in OGD/R modelCompared with control group,the proportion of apoptosis of Tunel positive cells in OGD/R group increased significantly,reaching 41.582 ±2.801%.After drug treatment,compared with OGD/R,the proportion of apoptotic cells in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group decreased significantly,and the most significant decrease was(34.430 ±2.163)%in Ast+Lig+MK-801 group.4.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on mitochondrial membrane potential of primary rat cortical neurons in OGD/R modelJC-1 fluorescence probe was used to detect the changes of mitochondrial membrane potential in each group.In control group,the membrane potential was basically normal,and the green fluorescent monomer increased significantly in OGD/R group,indicating that there were more early apoptotic cells(P<0.001).Compared with OGD/R group,the mitochondrial membrane potential increased in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group and the early apoptotic cells decreased relatively(P<0.001),especially in Ast+Lig+MK-801 group.5.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on intracellular Ca2+ concentration in OGD/R model of primary rat cortical neuronsCompared with control group,the fluorescence intensity of Ca2+in OGD/R group increased significantly(P<0.001),reached(43.413±2.825)%,and decreased significantly in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group compared with OGD/R group,and the decrease was the most significant in Ast+Lig+MK-801 group(34.513 ± 1.875)%.6.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on apoptosis factors in OGD/R model of primary rat cortical neuronsThe results of immunofluorescence and WB showed as follows.Compared with control group,the expression of pro-apoptotic protein Caspase-3,Caspase-9,Caspase-12 and Bax was significantly up-regulated,while the expression of anti-apoptotic protein Bcl2 was significantly down-regulated in OGD/R group.After treatment,compared with OGD/R,Caspase-9,Caspase-12 and Bax protein decreased significantly in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group,but the expression of Caspase-3 was down-regulated only in Ast+Lig+MK-801 group(P<0.05).The expression of Bcl2 was significantly up-regulated in Ast+Lig group(P<0.05).The results of qRT-PCR were basically consistent with those proteins.7.Effect of astragalus membranaceus and ligustrazine injection and MK-801 on neurological function score of MCAO/R model in C57bl/6 mice at 24h and 48hAt 24h and 48h after reperfusion,the results of Longa score showed that there were different degrees of neurological impairment in all groups except sham group.Compared with the MCAO/R group,the behavioral score decreased after administration,but there was no significant change in each group(P>0.05).However,at the time point of 48h of reperfusion,the neurological injury in the Ast+Lig+MK-801 group was significantly decreased compared with the MCAO/R group(P<0.05).8.Effect of astragalus membranaceus and ligustrazine injection and MK-801 on infarct area in MCAO/R model of C57bl/6 mice at 24h and 48hTTC staining showed that there was no infarction in sham group,but the infarct area in MCAO/R24h group and MCAO/R48h group was significantly increased(P<0.05).Compared with MCAO/R group,Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group,the infarct area decreased significantly at both time points(P<0.05).It was also found that the effect of infarct size reduction in the Ast+Lig+MK-801 group was better than that in the single use of one of them.9.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on SOD and MDA in 24h and 48h MCAO/R model of C57b1/6 miceCompared with sham group,SOD activity decreased significantly in MCAO/R 24h group and MCAO/R 48h group(P<0.05),and compared with MCAO/R group,SOD activity in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group increased significantly at both time points(P<0.05),and the effect was most obvious in Ast+Lig+MK-801 group.10.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on the expression of apoptotic factors in MCAO/R model of C57bl/6 mice for 24h and 48hThe results of WB showed as follows.Compared with sham group,the relative expression of Caspase-3,Caspase-9,Caspase-12 and Bax protein in MCAO/R24h group and MCAO/R48h group was significantly up-regulated,while the relative expression of Bcl-2 protein was significantly down-regulated.After drug intervention,compared with MCAO/R group,the relative expression of Caspase-12 and Bax in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group decreased significantly,while the expression of Caspase-3 did not change significantly.The expression of Caspase-9 only decreased significantly in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group(P<0.05).The expression of Bcl-2 protein was up-regulated only in Ast+Lig+MK-801 group.Compared with MCAO/R group,the expression of Caspase-9 in Ast+Lig group,MK-801 group,Ast+Lig+MK-801 group decreased significantly at 48h,while Caspase-3 and Bax in Ast+Lig group and Ast+Lig+MK-801 group decreased significantly,but there was no significant change in MK801 group.The expression of Caspase-12 only decreased significantly in Ast+Lig group.The expression of Bcl-2 protein was up-regulated only in Ast+Lig+MK-801 group.qRT-PCR was basically consistent with the above results,but the down-regulation trend of pro-apoptosis factor mRNA was more obvious in the drug group,and the up-regulation trend of anti-apoptosis factor Bcl-2 mRNA was more obvious.11.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on the expression of NR2 subunit NR2A and NR2B in MCAO/R model of C57b1/6 mice for 24h and 48hThe results of WB showed as follows.Compared with sham group,the relative expression of NR2B protein was significantly up-regulated(P<0.001)and the relative expression of NR2A protein was significantly down-regulated(P<0.001)in MCAO/R24h group.After drug intervention,compared with MCAO/R group,the expression of NR2A in each group had no significant difference,but the expression of NR2B protein in Ast+Lig,MK-801 group and Ast+Lig+MK-801 group was significantly down-regulated(P<0.001).At the 48h time point,compared with sham group,the relative expression of NR2A,NR2B protein was significantly up-regulated in 24h sham group,but the expression of NR2B protein was significantly down-regulated in Ast+Lig,MK-801 group and Ast+Lig+MK-801 group(P<0.01),but there was no significant difference in NR2A expression between in Ast+Lig,MK-801 group and Ast+Lig+MK-801 group after drug treatment.qRT-PCR is basically consistent with the above results.12.Effects of astragalus membranaceus and ligustrazine injection and MK-801 on the expression of synaptic regulatory proteins PSD95,GAP-43 and S YN in MC AO/R model of C57bl/6 mice at 24h and 48hThe results of WB showed that the relative expression of PSD-95 protein in MCAO/R24h group and MCAO/R48h group was significantly higher than that in sham group(P<0.001),while the relative expression of GAP-43 and SYN protein had no significant change(P>0.05).At 24h time point,compared with MCAO/R group,the expression of PSD-95 in Ast+Lig,MK-801 group and Ast+Lig+MK-801 group was significantly down-regulated,while the expression of GAP-43 protein was significantly up-regulated,but there was no significant difference in SYN expression among groups.At 48h time point,compared with MC AO/R group,the expression of PSD-95 in Ast+Lig,MK-801 group and Ast+Lig+MK-801 group was significantly down-regulated(P<0.001),while GAP-43 and SYN protein were significantly up-regulated(P<0.05).qRT-PCR is basically consistent with the above results.Conclusion1.In the OGD/R injury model of primary cortical neurons of SD rats,astragalus membranaceus and ligustrazine injection could increase cell viability,reduce the rate of apoptosis,inhibit the concentration of intracellular Ca2+,increase the mitochondrial membrane potential,lower the production of early apoptotic cells,up-regulate the expression of Bcl-2 and down-regulate the expression of Caspase-3,Caspase-9,Caspase-12 and Bax,and finally play the role of anti-neuronal apoptosis.2.In the injury model of MCAO/R mice,astragalus membranaceus and ligustrazine injection can reduce the area of cerebral infarction,lower the content of MDA,increase the activity of SOD,weaken the injury of oxidative stress and protect the neurological function of mice with cerebral ischemia-reperfusion injury by regulating the expression of NMDA receptor NR2 subunits(NR2A,NR2B).3.In the injury model of MCAO/R mice,astragalus membranaceus and ligustrazine injection can play an anti-apoptotic effect,which is related to regulating the protein expression levels of Bcl-2,Caspase-3,Caspase-9,Caspase-12 and Bax.At the same time,astragalus membranaceus and ligustrazine injection can regulate synapses,which is related to the regulation of PSD-95,GAP-43 and SYN protein expression. |
PDF Full Text Request