黄连解毒汤通过抑制NF-κB信号通路调控神经元改善缺血性脑卒中后认知功能障碍OA

Objective:To investigate the effects of Huanglian Jiedutang(HLJDT)on cognitive function in mice with ischemic stroke(IS)and to elucidate whether its neuroprotective effects are mediated by inhibition of the nuclear factor-κB(NF-κB)signaling pathway and subsequent suppression of NF-κB-regulated neuronal apoptosis.Methods:An IS model was established using middle cerebral artery occlusion(MCAO).Sixty C57BL/6J mice were randomly assigned to five groups(n=12 per group),i.e.,sham operation,model,HLJDT low-dose(3.9 g·kg-1·d-1),HLJDT high-dose(7.8 g·kg-1·d-1),and Ginkgo biloba extract(GBE,31.2 mg·kg-1·d-1).Post-operatively,neurological deficit scores(Longa score),cerebral infarct volume assessed by 2,3,5-triphenyltetrazolium chloride(TTC)staining,and brain water content were evaluated.Learning and memory were assessed using new object recognition(NOR)and fear conditioning(FC)tests.Hippocampal pathology was examined via hematoxylin and eosin(HE)staining.Immunofluorescence detected expression of glial fibrillary acidic protein(GFAP,astrocyte marker),cellular oncogene Fos(c-Fos,neuronal activation marker),and glutamate decarboxylase 65(GAD65).Western blot measured nuclear factor-κB inhibitor protein α(IκBα),phosphorylated IκBα(p-IκBα),NF-κB p65,phosphorylated NF-κB p65(p-NF-κB p65),ionic calcium binding adapter molecule 1(Iba-1),tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and apoptosis-related proteins,such as cleaved cysteinyl aspartate-specific protease 3(Caspase-3),B-cell lymphoma 2(Bcl-2),and Bcl-2-associated X protein(Bax).Real-time quantitative PCR(Real-time PCR)was used to assess mRNA levels of Iba-1,TNF-α,IL-1β,NF-κB p65,cleaved Caspase-3,Bax,and Bcl-2.Results:Compared with the sham group,the model group exhibited significantly increased neurological deficit scores,brain water content,and cerebral infarct volume(P＜0.01).Hippocampal CA1 neurons were disorganized,showing nuclear pyknosis and karyolysis.NOR exploration time and FC freezing time were significantly reduced(P＜0.01).GFAP and c-Fos expression were increased,while GAD65 expression was decreased(P＜0.01).Cleaved Caspase-3 and Bax were upregulated,Bcl-2 was downregulated,and the Bax/Bcl-2 ratio was elevated(P＜0.01).Expression levels of p-IκBα,p-NF-κB p65,IL-1β,TNF-α,and Iba-1 were significantly increased(P＜0.01).Compared with the model group,HLJDT high-dose,low-dose,and GBE groups showed significant improvements in all parameters(P＜0.01).Among them,the HLJDT high-dose group showed the most pronounced neuronal structural recovery and superior performance in NOR and FC tests(P＜0.01).In this group,GFAP and c-Fos decreased,GAD65 increased(P＜0.01),apoptosis-related protein expression was reversed,and NF-κB signaling and related inflammatory factor expression were suppressed(P＜0.01).Conclusion:HLJDT ameliorates cognitive dysfunction in mice after IS,potentially by inhibiting the NF-κB signaling pathway,thereby reducing neuroinflammation and hippocampal neuronal apoptosis.