Studies On Protective Effects And Mechanism Of Harpagide From Scrophularia Radix On Cerebral Ischemia-Reperfusion Injury In Neurons Base On Inhibition Of Endoplasmic Reticulum Stress

Objective Cerebrovascular disease has been harming human seriously,and pathophysiolog--ical processes of cerebral ischemia reperfusion injury plays an increasingly vital role in the treatments.In our present study,the injury model was established by oxygen-glucose deprivation and reoxygenation（OGD/R）model simulating cerebral ischemia-reperfusion injury in vitro,to observe the apoptosis and endoplasmic reticulum stress related changes of rat cerebral cortical neurons;to investigate whether and how the neuroprotective effects of harpagide from scrophularia radix are mediated by the inhibition of endoplasmic reticulum stress（ERS）,after oxygen-glucose deprivation and reoxygenation injury;to explore molecular neuroprotective mechanism of harpagide base on the interaction of calcium homeostasis and ERS in PC12 cells,after giving thapsigragin（TG）and inhibiting SERCA 2a expression by sh SERCA.Methods（1）Research of calcium homeostasis and ERS on rat cerebral cortical neurons injuryed by OGD/RThe primary cortical neurons were randomly divided into control group,OGD2h/R0h group,OGD2h/R6h group,OGD 2h/R12h group,OGD 2h/R24h group,OGD 2h/R48h group.The cell morphology was observated,the cell apoptosis was detected by FCM.The vitality of SERCA was detected,and[Ca²⁺]_i was detected by LSCM.The expression of SERCA and ERS related factors were evaluated by RT-PCR and Western-Blot.It was aimed to explore the interaction of calcium homeostasis and ERS during different reperfusion time intervals.（2）Neuroprotective effects of harpagide from scrophularia radix on OGD/R injury in rat cerebral cortical neuronsIn apoptosis detection,the primary cortical neurons were randomly divided into control group,OGD/R group,harpagide（10μM,50μM,100μM,150μM）+OGD/R groups;in MTT test,the primary cortical neurons were randomly divided into control group,OGD/R group,harpagide（50μM,100μM）groups,4-PBA group,OGD/R+4-PBA group,OGD/R+harpagide（50μM,100μM）groups.In other tests,the primary cortical neurons were randomly divided into control group,OGD/R group,harpagide（50μM）group,4-PBA group,OGD/R+4-PBA group,OGD/R+harpagide（50μM）group.The cell morphology was observated,the cell survival rate was detected by MTT method,the cell apoptosis was detected by FCM.[Ca²⁺]_i was detected by LSCM.The expression of ERS related factors were evaluated by RT-PCR and Western-Blot.It was aimed to explore neuroprotective effects of harpagide on OGD/R injury.（3）Thapsigragin was given to explore neuroprotective mechanism of harpagide on OGD/R injury in PC12 cells.In MTT test,PC12 cells were divided into control group,OGD/R group,harpagide（10μM,50μM,100μM,150μM）+OGD/R groups.In other tests,PC12 cells were divided into control group,harpagide group,OGD/R group,TG group,OGD/R+harpagide group,TG+harpagide group,a total of 6 groups.The cell morphology was observated,the cell survival rate was detected by MTT method,the cell apoptosis was detected by FCM.[Ca²⁺]_i was detected by LSCM.The expression of SERCA and ERS related factors were evaluated by RT-PCR and Western-Blot.（4）To explore neuroprotective mechanism of harpagide on OGD/R injury in PC12 cells,after inhibiting SERCA 2a expression by sh SERCA.PC12 cells were divided into control group,OGD/R group,harpagide（50μM）group,harpagide（50μM）+OGD/R group;PC12/SERCA^-cells were divided into control group,OGD/R group,harpagide（50μM）group,harpagide（50μM）+OGD/R group;PC12/NC cells were divided into control group,OGD/R group,harpagide（50μM）group,harpagide（50μM）+OGD/R group.The cell morphology was observated,the cell survival rate was detected by MTT method,the expression of ERS related fators were evaluated by RT-PCR and Western-Blot.Results（1）Research of calcium homeostasis and ERS on rat cerebral cortical neurons injuryed by OGD/RNSE staining and MAP-2 staining could reflect the growth of neurons,the dyeing results showed that the cells were with high purity,and the purity was 90%-94%in five visions.Apoptosis experiment results showed that:compared with the control group,the apoptotic rates were significantly increased（P<0.01）at 12h reperfusion time.The vitality of SERCA showed that:the vitality of SERCA decreased with reperfusion time gradually.A sudden drop occured at 12 h to 48 h reperfusion time.[Ca²⁺]_i measurement results showed that:reperfusion rapidly enhanced intracellular[Ca²⁺]_i in individual cells with nearly a time-dependent manner.Compared with control group,[Ca²⁺]_i in rat cerebral cortical cells displayed significant difference at 6h,12 h,24 h and 48 h（P<0.05,P<0.01）.PCR results showed that:compared with the control group,the expression of SERCA m RNA was significantly down regulated in 6h group.At 12h,24 h and 48 h,SERCA levels were further decreased（P<0.01）.GRP78 m RNA kept rising from 0 h to 24 h post reperfusion（P<0.05 at 6 h and 12 h,P<0.01 at 24 h）.The GRP78 m RNA has decrease at 48h（P<0.01）.Caspase-12 m RNA and CHOP m RNA were increased throughout the course of reperfusion（P<0.01）.And the Western-Blot results are consistent with PCR results.（2）Neuroprotective effects of harpagide from scrophularia radix on OGD/R injury in rat cerebral neuronsDetermined by MTT experiment results showed that:compared with the control group,survival rate of primary cortical neurons were significantly decreased in OGD/R group（P<0.01）;harpagide in 50μM,100μM dose groups could increase the cell survival rate（P<0.01）.Apoptosis experiment results showed that:compared with the control group,OGD/R could increase the apoptosis rate in primary cortical neurons（P<0.01）.Compared with the OGD/R group,the apoptosis rate in 50μM harpagide+ODG/R and 4-PBA+OGD/R groups were significantly decreased（P<0.01）,the apoptosis rate in 100μM harpagide+ODG/R groups was also decreased（P<0.05）.compared with the control group,[Ca²⁺]_i in OGD/R group was significantly increased（P<0.01）,GRP78,Caspase-12 and CHOP in OGD/R group was significantly increased（P<0.01）;compared with OGD/R group,[Ca²⁺]_i in 50μM harpagide+OGD/R group was further decreased（P<0.01）,[Ca²⁺]_i in 4-PBA+OGD/R group was decreased,but had no significant difference;GRP78、Caspase-12 in harpagide+OGD/R group and 4-PBA+OGD/R group were significantly decreased（P<0.05）.CHOP expression in 50μM harpagide+OGD/R group was significantly decreased（P<0.05）,CHOP expression in 4-PBA group was decreased,but had no significant difference.（3）Thapsigragin（TG）was given to explore neuroprotective mechanism of harpagide on OGD/R injury in PC12 cellsDetermined by MTT experiment results showed that:compared with the control group,survival rate of PC12 cells were significantly decreased in OGD/R group（P<0.01）;harpagide in 50μM,100μM dose groups could increase the cell survival rate（P<0.01）.Apoptosis experiment results showed that:compared with the control group,OGD/R and TG could increase the apoptosis rate in PC12 cells（P<0.01）.Compared with the OGD/R group,the apoptosis rate in harpagide+ODG/R group was significantly decreased（P<0.01）.Compared with TG group,the apoptosis rate in harpagide+TG group was significantly decreased（P<0.01）.Compared with the control group,[Ca²⁺]_i in OGD/R and TG groups were significantly increased（P<0.01）;SERCA in OGD/R and TG group were significantly decreased（P<0.01）,ERS related factors in OGD/R and TG groups were significantly increased（P<0.01）.Compared with OGD/R group,[Ca²⁺]_i in harpagide+OGD/R group was further decreased（P<0.01）,SERCA in harpagide+OGD/R group was significantly increased（P<0.05）,ERS related factors in harpagide+OGD/R group were significantly decreased（P<0.05,P<0.01）;compared with TG group,[Ca²⁺]_i in harpagide+TG group was further decreased（P<0.01）,SERCA in harpagide+TG group was significantly increased（P<0.01）,expression of ERS related factors in harpagide+TG group were significantly decreased（P<0.05,P<0.01）.（4）To explore neuroprotective mechanism of harpagide on OGD/R injury in PC12 cells,after inhibiting SERCA 2a expression by sh SERCA.The lentivirus expression vector targeted at SERCA was successfully constructed.Stable transfected PC12 cells line was established.The effective interference verification revealed that sh SERCA could significantly reduce the m RNA and protein level of SERCA（P<0.01）.Determined by MTT experiment results showed that:compared with the control group,survival rate of PC12 and PC12/NC cells were significantly decreased in OGD/R group（P<0.01）;harpagide in 50μM dose group could increase the cell survival rate（P<0.01）;compared with same group in PC12 cells,survival of PC12/SERCA^-cells in harpagide+OGD/R group had significantly difference（P<0.05）.PCR results showed that:compared with the control groups,GRP78 m RNA,Caspase-12m RNA,CHOP m RNA in OGD/R groups of PC12 and PC12/NC were significantly increased（P<0.01）;compared with OGD/R group,GRP78 m RNA,Caspase-12 m RNA,CHOP m RNA in harpagide+OGD/R group were significantly decreased（P<0.05）.GRP78 m RNA,Caspase-12 m RNA,CHOP m RNA of PC12/SERCA^-cells in 50μM harpagide+OGD/R group was significantly increased（P<0.05）,compared with the same group of PC12.Western-Blot results showed that:compared with the control groups,GRP78,Caspase-12 and CHOP in OGD/R groups of PC12 and PC12/NC were significantly increased（P<0.01）;compared with OGD/R group,GRP78,Caspase-12 and CHOP in harpagide+OGD/R group were significantly decreased（P<0.05,P<0.01）.The levels of GRP78,Caspase-12 and CHOP in PC12/SERCA^-cells in 50μM harpagide+OGD/R group was significantly increased（P<0.05）,compared with the same group of PC12.Conclusion（1）Simulated ischemia and reperfusion models were successfully built through OGD/R in vitro.Neuronal apoptosis can be induced by the OGD/R model.Endoplasmic reticulum stress was involved in the apoptosis.（2）Harpagide could improve cell viability and reduce apoptosis rate vitro,and could significantly decrease[Ca²⁺]_i and the expression of ERS-related factors in rat cerebral cortical neurons injured by OGD/R,suggesting that harpagide had protective effects on rat cerebral cortical neurons by inhibiting ERS.（3）Harpagide could reduce apoptosis rate and decrease the expression of ERS-related factors in PC12 cells injured by TG,suggesting harpagide had direct inhibitory effect on ERS.Harpagide could improve expression of SERCA m RNA and protein induced by TG,indicated the protective effects against cerebral ischemia reperfusion injury of harpagide may be related to direct intervention of SERCA,inhibition of ERS,reducing cell apoptosis.（4）Harpagide had no obviously improve in cell viability in PC12/SERCA^-injured by oxygen-glucose deprivation,indicated that harpagide protected cerebral ischemia reperfusion injury from cell apoptosis through decreasing ERS via SERCA.