摘要
目的探讨补肾开窍方对自发性高血压大鼠[SHR,一种注意缺陷多动障碍(ADHD)模型]注意力缺陷和工作记忆障碍的改善作用,并揭示其通过调控氧糖剥夺/再复氧(OGD/R)条件下小胶质细胞的活化状态,以及抑制NF-κB/NLRP3炎症小体信号通路发挥神经保护作用的机制。方法将50只SHR大鼠随机分为模型组(SHR)、补肾开窍方低、中、高剂量组(LOW、MID、HIGH)及哌醋甲酯(MPH)组,每组10只,Wistar Kyoto(WKY)大鼠作为对照,灌胃4周后采用Y迷宫行为测试及免疫荧光检测。同时体外采用OGD/R模型,将原代小胶质细胞分成对照组、OGD/R模型组、补肾开窍方含药血清干预组(LOW、MID、HIGH)及MPH组,通过CCK8法、Western blot技术、TMRE荧光探针、免疫荧光观察原代小胶质细胞及神经元相关指标。结果与SHR组相比,LOW、MID、HIGH组自发轮替率升高(0.4840 vs.0.6280、0.4840 vs.0.6600、0.4840 vs.0.6620,均P<0.001)。前额叶i-NOS水平(1.801 vs.1.358、1.303、1.175,均P<0.001)及炎症因子IL-1β(pg/mL)(44.72±3.190 vs.31.65±1.348、20.85±0.934、16.21±0.723,均P<0.001)和IL-6水平(pg/mL)降低(13.180±0.655 vs.7.527±0.095、5.457±0.170、3.757±0.076,P<0.001)。含药血清及MPH干预后小胶质细胞中p-NF-κB-p65、p-IκBα、NLRP3表达量可减少(NLRP3:1.360±0.148 vs.0.798±0.068、0.654±0.052、1.097±0.077,均P<0.05;p-NF-κB-p65:1.301±0.087 vs.0.557±0.064、0.775±0.051、0.943±0.093,P<0.001;p-IκBα:1.097±0.117 vs.0.722±0.051、0.725±0.049、0.760±0.031,均P<0.001),线粒体膜电位升高(36.48±2.50 vs.74.26±2.14、79.46±2.01、101.5±0.61,均P<0.001)。药物干预组条件培养基干预后的神经元TUNEL+/DAPI+细胞比例降低(56.75±1.15 vs.47.52±0.08、45.73±1.97、43.63±0.45,均P<0.001),且突触生长良好。结论补肾开窍方可以通过调控小胶质细胞NF-κB/NLRP3通路发挥其抗炎与神经保护作用,从而改善ADHD样行为缺陷。
Objective To investigate the therapeutic effects of Bushen Kaiqiao Formula(BSKQF)on attention deficit and spatial memory impairment in spontaneously hypertensive rats(SHRs),a widely used animal model for attention-deficit hyperactivity disorder(ADHD).The potential neuroprotective mechanism was explored with a focus on microglial activation under oxygen-glucose deprivation/reoxygenation(OGD/R)conditions and regulation of neuroinflammation via the NF-κB/NLRP3 inflammasome signaling pathway.Methods Fifty SHRs were randomly divided into a model group(SHR),low-dose(LOW),medium-dose(MID),high-dose(HIGH)BSKQF treatment groups,and a methylphenidate(MPH)group(n=10 each).Wistar-Kyoto(WKY)rats served as the control group.After 4 weeks of gavage,behavioral changes were assessed using the Y-maze test.Immunofluorescence was used to assess molecular indicators in the prefrontal cortex.Primary microglia were divided into control,OGD/R model,BSKQF serum-treated(LOW,MID,HIGH),and MPH groups.CCK-8,Western blot,TMRE mitochondrial potential probes,and immunofluorescence assays were used to detect key molecular and cellular markers in microglia and neurons.Results Compared to SHRs,BSKQF treatment significantly increased the spontaneous alternation rate in Y-mate tests(0.4840 vs.0.6280、0.4840 vs.0.6600、0.4840 vs.0.6620,P<0.001).In the prefrontal cortex of SHRs,iNOS,IL-1β,and IL-6 levels were significantly elevated,while BSKQF treatment significantly reduced these markers[i-NOS:1.801 vs.1.358、1.303、1.175,P<0.001;IL-1β(pg/mL):44.72±3.190 vs.31.65±1.348、20.85±0.934、16.21±0.723,P<0.001;IL-6(pg/mL):13.180±0.655 vs.7.527±0.095、5.457±0.170、3.757±0.076,P<0.001].After treatment with BSKQF and MPH,the expression levels of p-NF-κB-p65,p-IκBαand NLRP3 in microglia were reduced accompanied by an increase in mitochondrial membrane potential(NLRP3:1.360±0.148 vs.0.798±0.068、0.654±0.052、1.097±0.077,P<0.05;p-NF-κB-p65:1.301±0.087 vs.0.557±0.064、0.775±0.051、0.943±0.093,P<0.001;p-IκBα:1.097±0.117 vs.0.722±0.051、0.725±0.049、0.760±0.031,P<0.001);mitochondrial membrane potential(36.48±2.50 vs.74.26±2.14、79.46±2.01、101.5±0.91,P<0.001).Medium from BSKQF-treated groups showed significantly reduced apoptosis and preserved synaptic integrity(56.75±1.15 vs.47.52±0.08、45.73±1.97、43.63±0.45,P<0.001).Conclusion BSKQF exerts anti-inflammatory and neuroprotective effects in ADHD-like rats by modulating microglial activation and inhibiting the NF-κB/NLRP3 inflammasome pathway,thereby improving behavioral deficits associated with ADHD.
作者
章嘉琦
孙茹欣
杨宇婷
朱康琳
王静
倪新强
黄敏
ZHANG Jia-qi;SUN Ru-xin;YANG Yu-ting;ZHU Kang-lin;WANG Jing;NI Xin-qiang;HUANG Min(Department of Neurosurgery,the Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen 518107,Guangdong,China;不详)
出处
《广东医学》
2025年第6期801-809,共9页
Guangdong Medical Journal
基金
国家自然科学基金资助项目(82074492,82374517)
深圳市科技创新委员会项目(JCYJ20230807094808017)。
作者简介
章嘉琦,山大学2022级硕士研究生,2022年始参与广州中医药大学第四临床医学院倪新强课题组科学研究,由黄敏、倪新强共同指导;通信作者:黄敏,E-mail:huangm78@mail.sysu.edu.cn;通信作者:倪新强,E-mail:nxq2bio@hotmail.com。
