Effect Of Effective Parts Of Dracocephalum Moldavica On Hbmecs After OGD/R Injury

Objective:To study the protective effect of the effective parts of Dracocephalum moldavica on cerebral microvascular endothelial cells against oxygen and glucose deprivation/re-oxygenation（OGD/R）injury in vitro,and to explore the role and mechanism of necroptosis in the OGD/R injury of the effective parts of Dracocephalum moldavica.Methods:The caspase inhibitor Z-VAD-FMK combined with OGD/R injury to establish a model of programmed necrosis of HBMECs to simulate the process of cerebral ischemia-reperfusion injury.HBMECs were randomly divided into five groups:control group（Control）,Z-VAD-FMK combined with OGD/R model group（Model）,model+EPDM low,medium and high dose groups(25μg·m L^-1,50μg·m L^-1,100μg·m L^-1).（1）Cell proliferation and cytotoxicity detection kits were used to detect cell viability;lactate dehydrogenase kits were used to detect LDH content;Hoechst 33342/PI fluorescent double staining was used to detect cell programmed necrosis rate;（2）Microscopic observation Cell morphology;NO content detected by NO assay kit;cell migration and monolayer endothelial cell permeability detected by Transwell method;VE-Cadherin expression detected by immunofluorescence,SRC,endothelial ET-1,Claudin 5,e NOS detected by western blot,and the protein expression of phosphorylated e NOS;（3）DCFH-DA probe,Calcein AM probe and JC-1 probe were used to detect intracellular ROS release,MPTP opening and MMP respectively;enzyme-linked immunosorbent assay was used to detect TNF-The release ofα,IL-1βand IL-6;the expression of programmed necrosis-related proteins was detected by immunoblotting.Results:（1）Compared with the control group,Z-VAD-FMK combined with OGD/R model group could reduce the activity of HBMECs,increase the release of LDH,and increase the rate of programmed necrosis（P<0.01）;（2）The FMK combined with OGD/R model group showed worse cell morphology,decreased cell migration,decreased NO release,increased monolayer permeability,decreased expression of VE-Cadherin,decreased expression of SRC,Claudin 5,phosphorylated e NOS,and decreased ET-1 expression（P<0.01）;（3）Compared with the control group,the Z-VAD-FMK combined with OGD/R model group increased the production of ROS,opened MPTP,decreased MMP,increased the secretion of TNF-α,IL-1βand IL-6,and expressed the expressions of RIP3 and PGAM5.increased,the p-MLKL/MLKL ratio increased.However,EPDM pretreatment could partially reverse the changes of the above factors（P<0.05）.Conclusion:EPDM has a protective effect on HBMECs under cerebral ischemia-reperfusion model.It can reduce cell necrosis rate,promote cell proliferation and migration,enhance vasomotor regulation ability,and maintain barrier function by enhancing tight junctions and adhesive junctions.Related mechanisms In order to protect mitochondrial function by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening,and thereby protect HBMECs from cerebral ischemia-reperfusion injury,it can provide a valuable scientific basis for EPDM in the treatment of cerebral ischemia-related diseases.