痫得安丸联合丙戊酸钠通过TNF-α/NF-κB信号通路抑制卒中后癫痫神经炎症的体内外研究OA

Objective To investigate the therapeutic effect and mechanism of Xiande'an Pills(XAP)combined with sodium valproate(VPA)on post-stroke epilepsy(PSE)through in vivo and in vitro studeies.Methods(1)Animal experiment:A post-ischemic stroke epilepsy model was established in rats using unilateral middle cerebral artery occlusion combined with common carotid artery ligation(MCA/CCAo).Forty successfully modeled epileptic rats were randomly divided into the model group,VPA group,XAP group,and XAP+VPA combination group.Ten additional rats underwent sham surgery(vessel exposure only followed by wound closure)as the sham group.After 4 weeks of administration,seizure frequency and duration were recorded.Hippocampal histopathology was observed by hematoxylin-eosin(HE)staining,Nissl bodies were assessed by Nissl staining,and levels of glutamate(Glu),γ-aminobutyric acid(GABA),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)in the brain were measured.mRNA and protein expression of TNF-α,TNF receptor 1(TNFR1),nuclear factor-κB(NF-κB)p65,phosphorylated p65(p-p65),cyclooxygenase-2(Cox2),matrix metalloproteinase-9(MMP9),and c-Fos in hippocampal tissue were detected by RT-qPCR and Western Blot.(2)Cell experiment:RAW264.7 cells were seeded in 96-well plates and divided into blank control,lipopolysaccharide(LPS),VPA,XAP,and XAP+VPA combination groups.After 6 hours of incubation,LPS was added for 24 hours.mRNA and protein expression of IL-1β,TNF-α,TNFR1,NF-κB p65,p-p65,Cox2,and MMP9 in RAW264.7 cells were detected by RT-qPCR and Western Blot.Results(1)Animal experiment:Compared with the sham group,the model group showed significantly increased seizure frequency and duration(P＜0.01),disorganized pyramidal neuron arrangement,cell atrophy,and deepened staining in the hippocampal CA1 region,significantly reduced Nissl body count,decreased GABA content in serum and brain,increased Glu content,Glu/GABA ratio,and IL-1β and TNF-α levels(P＜0.01),upregulated hippocampal mRNA expression of TNF-α,TNFR1,NF-κB p65,Cox2,MMP9 and c-Fos(P＜0.01),and increased protein expression of TNF-α,TNFR1,p-p65,Cox2 and MMP9(P＜0.05 or P＜0.01).The XAP+VPA groups exhibited significantly reduced seizure frequency and duration,demonstrated superior improvements in neuronal arrangement regularity,cell body contour clarity,uniform nuclear staining,and Nissl body distribution,increased GABA content brain,decreased Glu content,Glu/GABA ratio,and IL-1β and TNF-α levels,downregulated hippocampal mRNA expression levels of TNF-α,TNFR1,NF-κB p65,Cox2,MMP9 and c-Fos,and decreased protein expression levelsof TNF-α,TNFR1,p-p65,Cox2 and MMP9,the differences being statistically significant compared with the model group(P＜0.05 or P＜0.01),and the curative effects were superior to the XAP group or VPA group(P＜0.05 or P＜0.01).(2)Cell experiment:After LPS stimulation,all treatment groups showed decreased mRNA expression of IL-1β,Cox2,and MMP9,and decreased protein expression of TNF-α,p-p65,Cox2,and MMP9 compared to the LPS group(P＜0.05 or P＜0.01).The VPA and XAP+VPA groups exhibited decreased TNF-α mRNA expression(P＜0.05)and TNFR1 protein expression(P＜0.01)compared to the LPS group.The XAP+VPA group showed lower MMP9 mRNA and p-p65 protein expression than the VPA group(P＜0.01),and lower TNF-α and TNFR1 protein expression than the XAP group(P＜0.05).Conclusion The combination of XAP and VPA alleviates seizures,neuroinflammatory responses in rats with post-stroke epilepsy,potentially through inhibition of the TNF-α/NF-κB signaling pathway.