Huoxue Rongluo Prescription Combined With Edaravone And Bromocriptine Inhibits Pyroptosis Of Human Brain Microvascular Endothelial Cells After Oxygen-glucose Deprivation/reoxygenatio

Objective: Investigation of the anti-pyroptosis effect of the combination of Huoxue Rongluo decoction with edaravone dextanol injection in cerebral ischemia-reperfusion injury based on NLRP3/Caspase1 signaling pathway.Methods: Through in vitro experiments,human brain microvascular endothelial(HCMEC/D3)cells were divided into blank group,model group,edaravone dextanol group,Huoxue Rongluo decoction group and combined drug group.An in vitro model of cerebral ischemia/reperfusion injury was constructed by oxygen glucose deprivation/reoxygenation(OGD/R)induction.The in vitro model of cerebral ischemia/reperfusion injury was assessed by scanning electron microscopy;the effect of edaravone dextran injection on cell viability and the optimal dose of drug intervention were detected by CCK8 method;the expression level of NLRP3/Caspase1 pathway protein was detected by Western blot method;the expression of GSDMD,a key protein of focal death,was detected by immunofluorescence method;and the level of IL-1β and TNF-αin cell supernatant was detected by ELISA method.Results: 1.In vitro model of cerebral ischemia/reperfusion injury: human brain microvascular endothelial cells(HCMEC/D3)were induced by oxygen glucose deprivation/reoxygenation(OGD/R),and the presence of focal vesicles on the plasma membrane could be seen under scanning electron microscopy,suggesting that the in vitro model of cerebral ischemia/reperfusion injury was successfully established.2.Cell viability assay: compared with edaravone dextanol blank stock solution,edaravone dextanol stock solution(5,10,20,30 m L/L)inhibited cell viability(P<0.05 or P<0.01),compared with edaravone dextanol blank dilution solution,edaravone dexcamphorol dilution solution(5,10,20 m L/L)had no significant inhibitory effect on cells,oxygen glucose deprivation/ After reoxygenation(OGD/R)induction,edaravone dextanol dilution intervention group(5,10 and 20 ml/L)could promote cell viability recovery to different degrees(P<0.05 or P<0.01),with the best effect in the 20 ml/L intervention group.3.Protein expression: compared with the blank group,the protein expression levels of the NLRP3/caspase1 inflammatory pathway in the model group were increased(P < 0.01);Compared with the model group,the protein expression levels of the NLRP3 / caspase1 inflammatory pathway were significantly downregulated(P < 0.01)in the edaravone dextanol group,the Huoxue Rongluo decoction group,and the combination group,in which the combination group exhibited better effects than the monotherapy group(P < 0.05 or P < 0.01).4.Fluorescence expression situation: compared with the blank group,the fluorescence intensity of GSMDM,a key protein of pyroptosis,was enhanced in the model group(P < 0.01);Compared with the model group,the fluorescence intensity of GSMDM protein was significantly attenuated(P < 0.01)in the edaravone dextanol group,the Huoxue Rongluo decoction group,and the combination group,in which the combination was more effective than the monotherapy group(P < 0.05 or P < 0.01).5.Levels of inflammatory factors:Compared with the blank group,the concentrations of inflammatory factors IL-1β and TNF-α in the cell supernatant of the model group increased(P < 0.01);compared with the model group,the concentrations of inflammatory factors IL-1β and TNF-α in the cell supernatant of the edaravone dextanol group,the Huoxue Rongluo decoction group and the combination group were significantly reduced(P< 0.01),with the best effect in the combination group(P< 0.01).Conclusion: The synergistic effect of Huoxue Rongluo decoction and edaravone dextanol injection can reduce human cerebral microvascular endothelial cell scorching injury after cerebral ischemia-reperfusion by inhibiting the expression of NLRP3/Caspase1 inflammatory pathway.