Exploring the Mechanism of Electroacupuncture Serum on the Blood-Brain Barrier After Ischemic Stroke Based on m6A Methylation of LncRNA H19 Regulating the S1PR2/TLR4/NLRP3 Pathway
Objective To investigate the protective mechanism of electroacupuncture(EA)serum against blood-brain barrier(BBB)damage after ischemic stroke from regulating the S1PR2 pathway via m6A methylation of lncRNA H19.Methods Preparation of EA serum:the focal cerebral ischemia/reperfusion model rats were established,and three EA interventions were performed at 5 min after ischemia,24 h and 48 h after reperfusion,with acupuncture at Renzhong and Baihui acupoints for 30 min.EA serum was taken from the rats after the three EA interventions.Cellular experiments:The BBB model in which brain microvascular endothelial cells and pericytes were co-cultured was divided into five groups,including the control,model,EA,EA combined with METTL3 overexpression and EA combined with lncRNA H19 overexpression groups.Except for the control group,the remaining groups were deprived of oxygen and sugar for 2 h and then reoxygenated and reoxygenated for 24 h.EA serum,METTL3 and lncRNA H19 overexpression lentivirus were given at the same time of reoxygenation and reoxygenation.In this experiment,transendothelial cell resistance(TEER)and Na-F were used to detect the function of BBB;the colorimetry was perform to detect the quantification of total RNA m6A modification;Western blot was used to detect the METTL3,ZO-1,S1PR2,TLR4 and NLRP3 protein levels;and qPCR was used to detect the METTL3,ZO-1,lncRNA H19,S1PR2,TLR4,NLRP3 and NLRP3 mRNA levels.Results Compared with the model group,the TEER was increased(P<0.01);Na-F value was decreased(P<0.01);total RNA m6A modification level was decreased(P<0.01);METTL3,S1PR2,TLR4,NLRP3,and lncRNA H19 expression were downregulated(P<0.01),and ZO-1 protein and mRNA expression was upregulated(P<0.01)in the EA group.Compared with the EA group,the TEER was decreased(P<0.05);the Na-F value was increased(P<0.01);the level of total RNA m6A modification was increased(P<0.01),and the expression of METTL3,S1PR2,TLR4,NLRP3,and lncRNA H19 was were up-regulated(P<0.05),and ZO-1 protein and mRNA expression were down-regulated(P<0.01)in the EA combined METTL3 and lncRNA H19 overexpression groups.Conclusion EA serum has a protective role on the co-culture BBB model after oxygen-glucose deprivation,and this effect may be that EA regulates the methylation process of target gene lncRNA H19 through METTL3,thereby inhibiting the S1PR2/TLR4/NLRP3 signaling pathway,and attenuating the BBB injury after cerebral ischemia.
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