摘要
目的 探讨钾通道Kv1.3敲除对C57BL/6小鼠创伤性脑损伤(TBI)后神经功能障碍及神经炎症的影响。方法 C57BL/6小鼠和纯合Kv1.3基因敲除(Kv1.3 KO)C57BL/6小鼠采用经典的控制性皮质撞击模型构建小鼠TBI模型,设置假手术组、C57BL/6小鼠TBI模型组(TBI组)和Kv1.3 KO C57BL/6小鼠TBI模型组(TBI+Kv1.3 KO组)。造模1、2和3周,实时荧光定量PCR测定海马组织Kv1.3、白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)和IL-10 mRNA表达水平。造模1和3周,Western印迹检测海马组织Kv1.3蛋白表达水平;造模3周,Western印迹检测海马组织IL-1β、IL-6、TNF-α和IL-10蛋白表达水平;造模3周,免疫荧光法测定海马区小胶质细胞标志物离子钙结合衔接蛋白1(IBA1)分别与Kv1.3、IL-1β和TNF-α共标的细胞数;造模3周,膜片钳记录原代小胶质细胞Kv1.3通道电流。造模1和3周,通过行为学神经功能损伤评分(NSS)、爬杆和滚轮实验考察TBI小鼠的运动功能;造模3周,开放场、Morris水迷宫和Y迷宫实验考察TBI小鼠的认知功能。结果 与假手术组比较,造模1、2和3周,TBI组海马组织Kv1.3和IL-1β mRNA表达水平显著升高,TNF-α mRNA的表达水平仅在造模2和3周后明显升高,而IL-6和IL-10 mRNA表达水平无明显变化;造模1和3周,TBI组海马组织Kv1.3蛋白表达水平显著升高;造模3周,TBI组海马组织IL-1β和TNF-α蛋白表达水平显著升高,IL-6和IL-10蛋白表达水平无明显变化;造模3周,TBI组原代小胶质细胞Kv1.3电流密度显著升高。造模3周,TBI组海马区小胶质细胞标志物IBA1分别与Kv1.3、IL-1β和TNF-α共标细胞数均显著高于假手术组。与假手术组相比,造模1和3周,TBI组小鼠NSS得分显著升高、小鼠完全掉头成功率显著降低及爬至杆底的时间显著增加;造模3周,TBI组小鼠在开放场中活动的总距离、在开放场中间区域的活动时间、穿越平台次数、在目标象限活动的时间以及自发交替率均显著减少。与TBI组相比,造模1和3周,TBI+Kv1.3 KO组小鼠NSS得分显著降低、小鼠完全掉头成功率显著升高及爬至杆底的时间明显降低;造模3周,TBI+Kv1.3 KO组小鼠在滚轴上持续跑动所坚持的时间、在开放场中活动的总距离、在中央区域停留时间、穿越平台次数、在目标象限活动时间以及自发交替率均显著增加。造模1和3周,与TBI组相比,TBI+Kv1.3 KO组小鼠海马组织中炎症因子IL-1β和TNF-α的m RNA表达水平显著降低。结论 钾通道Kv1.3敲除可缓解TBI后C57BL/6小鼠的神经功能障碍和神经炎症。
OBJECTIVE To investigate the effects of potassium channel Kv1.3 knockout(Kv1.3 KO)on neurological dysfunction and neuroinflammation in C57BL/6 mice following traumatic brain injury(TBI).METHODS C57BL/6 mice and homozygous Kv1.3 KO C57BL/6 mice were subjected to the classic controlled cortical impact model to establish a TBI model.The experimental groups included the sham surgery group,C57BL/6 TBI model group(TBI group),and a Kv1.3 KO C57BL/6 TBI model group(TBI+Kv1.3 KO group).At 1,2,and 3 weeks post-modeling,real-time quantitative PCR was used to measure the mRNA expression levels of Kv1.3,interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),and IL-10 in hippocampal tissues.At 1 and 3 weeks post-modeling,Western blotting was performed to detect Kv1.3 protein expressions in the hippocampus.At 3 weeks post-modeling,Western blotting was used to assess the protein levels of IL-1β,IL-6,TNF-α,and IL-10 in hippocampal tissues.Additionally,immunofluorescence was employed to quantify cells co-labeled with the microglial marker ionized calcium-binding adapter molecule 1(IBA1)and Kv1.3,IL-1β,or TNF-αin the hippocampus.Patch-clamp recordings were conducted to measure Kv1.3 channel currents in primary microglia at 3 weeks post-modeling.Neurological function was evaluated at 1 and 3 weeks post-modeling using the neurological severity score(NSS),pole climbing,and rotarod tests.Cognitive function was assessed at 3 weeks post-modeling via open field,Morris water maze,and Y-maze tests.RESULTS Compared with the sham group,the TBI group exhibited significantly elevated mRNA expression levels of Kv1.3 and IL-1βin the hippocampus at 1,2 and 3 weeks post-modeling,while IL-6 and IL-10 mRNA levels showed no significant changes.Notably,TNF-αmRNA expressions demonstrated a significant increase only at 2 and 3 weeks post-modeling.At 1 and 3 weeks post-modeling,Kv1.3 protein expres-sions in the hippocampus were significantly higher in the TBI group.At 3 weeks post-modeling,hippo-campal IL-1βand TNF-αprotein levels were markedly increased in the TBI group,whereas IL-6 and IL-10 protein levels did not change significantly.Moreover,Kv1.3 current density in primary microglia was signifi-cantly enhanced in the TBI group at 3 weeks post-modeling.Immunofluorescence analysis revealed that the number of IBA1-positive microglia co-labeled with Kv1.3,IL-1β,or TNF-αin the hippocampus was significantly larger in the TBI group than in the sham group at 3 weeks post-modeling.Behaviorally,the TBI group exhibited significantly higher NSS scores,lower success rates in full turn attempts,and longer times taken to descend the pole at 1 and 3 weeks post-modeling compared with the sham group.At 3 weeks post-modeling,TBI mice also demonstrated reduced total movement distance in the open field,decreased time spent in the central zone,fewer platform crossings,less time in the target quadrant,and lower spontaneous alternation rates.In contrast,the TBI+Kv1.3 KO group showed signifi-cantly improved outcomes compared with the TBI group:lower NSS scores,higher success rates in full turns,and shorter time taken to descend the pole at 1 and 3 weeks post-modeling.At 3 weeks post-modeling,the TBI+Kv1.3 KO group displayed longer rotarod endurance,increased total movement dis-tance in the open field,more time spent in the central zone,higher platform crossings,greater target quadrant exploration time,and improved spontaneous alternation rates.Furthermore,at 1 and 3 weeks post-modeling,the TBI+Kv1.3 KO group exhibited significantly reduced mRNA expression levels of the inflammatory cytokines IL-1βand TNF-αin the hippocampus compared with the TBI group.CONCLU-SION Potassium channel Kv1.3 knockout mitigates neurological dysfunction and neuroinflammation in C57BL/6 mice following TBI.
作者
陈星星
陈正薰
张蝶
江浩鹏
陶杰
汤乐乐
袁易
CHEN Xingxing;CHEN Zhengxun;ZHANG Die;JIANG Haopeng;TAO Jie;TANG Lele;YUAN Yi(Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062,China;Eye and ENT Hospital of Fudan University,Shanghai 200126,China;Ruijin Hospital Nanxiang,Shanghai 200025,China)
出处
《中国药理学与毒理学杂志》
北大核心
2025年第6期401-411,共11页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(82074162)。
