| In recent years,domestic and foreign scholars have found a new type of cell death,necroptosis,which is mediated by the death receptor and regulated by a series of signal transduction pathway.It is a caspase-independent cell death,and both have the characteristics of necrosis and apoptosis.This type of cell death was first identified in the model of middle cerebral artery ischemia-reperfusion injury(IRI)in mice,and subsequently several experiments have confirmed that necroptosis was involved in brain damage caused by ischemia and hypoxia.Panax notoginseng is China’s famous traditional Chinese medicine,which is widely used in the treatment of ischemic stroke in clinical.Panax notoginseng saponin(PNS)is the main pharmacodynamic component of traditional Chinese medicine Panax notoginseng.Preliminary study found that Panax notoginseng saponin treatment can significantly reduce the score of neurological function of experimental cerebral ischemia in rats,reduce the volume of cerebral infarction and brain edema.The role of main protection is closely related to the inhibition of vascular inflammation after cerebral ischemia,the protection of vascular endothelial function and the promotion of vascular repair.Therefore,on the basis of previous studies,firstly making necroptosis model which is reduced by ischemia-reperfusion injury in brain microvascular endothelial cells(BMECs)by utilizing oxygen-glucose deprivation(OGD)and reintroduction conditions and the intervention of the casepase inhibitor z-VAD-FMK;secondly observing the effects of panax notoginseng saponins on necroptosis which is reduced by ischemia-reperfusion injury on BMECs and the expression of some related molecules on RIP 1-RIP3-MLKL signaling pathway;resultly to reveal the molecular mechanism of panax notoginseng saponin protecting endothelial cells in ischemia-reperfusion injury.Objective:To investigate the effect of panax notoginseng saponin on necroptosis of cerebral microvascular endothelial cells induced by ischemia-reperfusion injury and the regulation of RIP 1-RIP3-MLKL signaling pathway and mitochondrial injury,reveal the molecular mechanism of panax notoginseng saponin protecting endothelial cells in ischemia-reperfusion injury,provide a new scientific interpretation for the pharmacological mechanism of panax notoginseng saponin in the treatment of ischemic stroke.Methods:1.Establishment of necroptosis model which is reduced by ischemia-reperfusion injury in brain microvascular endothelial cells:First,primary rat BMECs were cultured in oxygen-glucose deprivation and reintroduction conditions for various time points to screen a optimal condition of in vitro ischemia-reperfusion injury.Then,z-VAD-FMK,the casepase inhibitor,was administrated at 20μmol/L concentration on the ischemia-reperfusion injured BMECs.The cell activity was detected by CCK-8.The ultra-stucture was observed using the transmission electron microscope.The death mode of BMECs was detected using Annexin V-FITC/PI(propidium iodide)double staining.2.To observe the effect of panax notoginseng saponin on necroptosis of BMECs in ischemia-reperfusion injury:the brain microvascular endothelial cells transmitting to the third generation were randomly divided into 4 groups:normal group,IRI+z-VAD-FMK group,IRI+Z-VAD-FMK+PNS group and IRI+z-VAD-FMK+Nec-1 group.In addition to the normal group,the remaining three groups are made models according to the above method of modeling.In IRI+z-VAD-FMK+PNS group,the cells were administered 3h before modeling and the modeling process by PNS.The final concentration of PNS was 22μg/ml.In IRI+z-VAD-FMK+Nec-1 group,cells were added to Nec-1 at a concentration of 10μmol/L 30 min before modeling and during modeling.At the end of the model,the cells were treated with CCK-8,the cell ultrastructure was observed by transmission electron microscopy.The cell death was detected by flow cytometry using Annexin V-FITC/PI double staining.3.Effects of PNS on RIP1-RIP3-MLKL signaling pathway in cerebral microvascular endothelial cells injured by ischemia-reperfusion injury:The third generation rat brain microvascular endothelial cells and the 7 generations later human brain microvascular endothelial cells were randomly divided into 4 groups:normal group,IRI+z-VAD-FMK group,IRI+z-VAD-FMK+PNS group and IRI+z-VAD-FMK+Nec-1 group.The method of administration is the same as above.The mRNA and protein phosphorylation levels of RIP1,RIP3 and MLKL in RIP1-RIP3-MLKL signaling pathway were detected by real-time PCR(RT-PCR)and Western Blotting.4.Effects of PNS on mitochondrial damage in cerebral microvascular endothelial cells after ischemia-reperfusion:the cells are grouped the same as above.The mRNA and protein levels of PGAM5 and Drpl were detected by RT-PCR and Western Blotting.By JC-1 staining,the effects of panax notoginseng saponins on mitochondrial membrane potential of cerebral microvascular endothelial cells injured by ischemia-reperfusion injury were observed by flow cytometry and fluorescence inverted phase contrast microscope respectively.Results:1.OD value decreased significantly(P<0.01)when OGD 2h and reintroduction 8h.The cell injury was significant and the injury occurred mainly at 4-8h after reperfusion,which was consistent with the characteristics of ischemia-reperfusion injury.So this time was determined as the best time point to simulate ischemia-reperfusion injury.The addition of z-VAD-FMK increased the necrosis of the cells,decreased the ratio of cells in quadrant Q2,but the necroptosis-specific inhibitor Nec-1 significantly reduced the ratio of Q2 quadrant cells(P<0.05),suggesting that necroptosis occurred in cells.2.Panax notoginseng saponin can reduce the necroptosis of cerebral microvascular endothelial cells in ischemia-reperfusion injury:compared with the IRI+z-VAD-FMK group,the OD value of IRI+z-VAD-FMK+PNS group and IRI+z-VAD-FMK+Nec-1 group increased significantly(P<0.01,P<0.01).The cell damage was improved;the nucleus and cell membrane were more complete and the mitochondrial injury was improved.The ratio of Q2 quadrant cells was significantly decreased(P<0.05),Q3 quadrant ratio increasing significantly(P<0.01),which was statistically significant.3.Panax notoginseng saponin has a down-regulation of RIP1-RIP3-MLKL signaling pathway in cerebral microvascular endothelial cells injured by ischemia-reperfusion injury:after modeling,the RIP1-RIP3-MLKL signal pathway was activated.The mRNA and protein phosphorylation expression of RIP1,RIP3 and MLKL in brain microvascular endothelial cells were significantly increased.Compared with IRI+z-VAD-FMK group,panax notoginseng saponin can reduce the phosphorylation levels and mRNA expression of three signal factors,which has the similar effect with Nec-1.4.Panax notoginseng saponin can reduce mitochondrial damage in brain microvascular endothelial cells after ischemia-reperfusion:after modeling,the mRNA and protein expression of PGAM5 and Drpl in rat brain microvascular endothelial cells were significantly increased and the mitochondrial membrane potential decreased.Compared with IRI+z-VAD-FMK group,the mRNA and protein expression of PGAM5 and Drpl in IRI+z-VAD-FMK+PNS group and IRI+z-VAD-FMK+Nec-1 group were significantly increased and the mitochondrial membrane potential decreased.Conclusion:1.After the intervention of OGD and reintroduction combined with z-VAD-FMK in BMECs,the death characteristics of cells were consistent with necroptosis,suggesting that the method can prepare necroptosis model.2.The total saponins of panax notoginseng can effectively reduce the occurrence of necroptosis in brain microvascular endothelial cells induced by the above method,and its intrinsic mechanism is related to the inhibition of RIP1-RIP3-MLKL signaling pathway,decreasing the expression of downstream PGAM5 and Drpl and mitigating mitochondrial damage.This may be one of the molecular mechanisms of panax notoginseng saponin in endothelial protection in ischemic brain injury.This conclusion provides a modern biological basis for the role of "Tongluo" in traditional Chinese medicine of panax notoginseng. |
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