电针调控VD大鼠HIF-1α/CXCR4/NF-κB和HO-1/SOD信号通路交互改善认知的机制

韩磊; 杨琳; 王建波; 张家豪; 李阳; 曲怡; 薛亚楠; 田跃龙; 王俊超; 刘继东

doi:10.12404/j.issn.1671-1815.2601102

科学技术与工程 ›› 2026, Vol. 26 ›› Issue (22) :9305-9315 DOI: 10.12404/j.issn.1671-1815.2601102

电针调控VD大鼠HIF-1α/CXCR4/NF-κB和HO-1/SOD信号通路交互改善认知的机制

韩磊 ¹ ,
杨琳 ¹ ,
王建波 ² ,
张家豪 ³ ,
李阳 ⁴ ,
曲怡 ² ,
薛亚楠 ¹ ,
田跃龙 ¹ ,
王俊超 ¹ ,
刘继东 ¹^,^*

作者信息 +

Mechanism of Electroacupuncture Regulation of HIF-1α/CXCR4/NF-κB and HO-1/SOD Signaling Pathway Interactions in VD Rats to Improve Cognitive Function

HAN Lei ¹ ,
YANG Lin ¹ ,
WANG Jian-bo ² ,
ZHANG Jia-hao ³ ,
LI Yang ⁴ ,
QU Yi ² ,
XUE Ya-nan ¹ ,
TIAN Yue-long ¹ ,
WANG Jun-chao ¹ ,
LIU Ji-dong ¹^,^*

Author information +

文章历史 +

摘要

观察电针对血管性痴呆(vascular dementia,VD)大鼠海马组织HIF-1α/CXCR4/NF-κB、HO-1/SOD通路的影响,探讨其改善认知障碍的机制。SPF级雄性SD大鼠随机分为假手术组、模型组、电针组和假电针组,以双侧颈总动脉结扎法(2-VO)制备VD模型。电针组给予“百会”“足三里”“三阴交”干预,15 min /次,1 次/d,共14 d;假电针组浅刺不通电;假手术组和模型组仅固定。Morris水迷宫和Y迷宫检测认知记忆能力;HE染色观察海马形态;实时荧光定量PCR法检测HIF-1α、CXCR4 mRNA表达;Western blot法检测HIF-1α、CXCR4蛋白及p-NF-κB/NF-κB比值;免疫组化法检测TNF-α、IL-6、IL-1β平均吸光度;免疫荧光法检测HO-1平均荧光强度;ELISA法检测丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、活性氧(reactive oxygen species,ROS)含量;荧光探针法观察ROS积聚。与假手术组相比,模型组和假电针组逃避潜伏期延长,穿越平台次数及自发交替率减少(P<0.01);海马神经元排列紊乱;HIF-1α、CXCR4 mRNA和蛋白表达及p-NF-κB/NF-κB比值升高(P<0.01);TNF-α、IL-6、IL-1β表达增高(P<0.01);HO-1荧光强度降低(P<0.01);MDA增高(P<0.05)、SOD降低(P<0.01)、ROS增高(P<0.05)。与模型组相比,电针组逃避潜伏期缩短,穿越平台次数和自发交替率增加(P<0.01);海马损伤减轻;HIF-1α、CXCR4表达及p-NF-κB/NF-κB比值降低(P<0.01);TNF-α、IL-6、IL-1β表达降低(P<0.01);HO-1荧光强度增强(P<0.01);MDA减少(P<0.05)、SOD增高(P<0.01)、ROS减少(P<0.05)。电针“百会”“足三里”“三阴交”可能通过平衡HIF-1α/CXCR4/NF-κB炎性通路与HO-1/SOD抗氧化通路的交互,减轻神经炎症,提高抗氧化应激水平,修复海马神经元损伤,从而改善VD大鼠认知功能。

Abstract

The effects of electroacupuncture on the HIF-1α/CXCR4/NF-κB and HO-1/SOD pathways in the hippocampal tissue of rats with VD (vascular dementia) were investigated to explore the mechanisms underlying its improvement of cognitive impairment. SPF-grade male SD rats were randomly divided into a sham surgery group, a model group, an electroacupuncture group, and a sham electroacupuncture group. A VD model was established by bilateral common carotid artery ligation (2-VO). Electroacupuncture was administered at the “Baihui” “Zusanli” and “Sanyinjiao” acupoints in the electroacupuncture group for once every 15 minutes, once daily, for 14 days. Shallow needling without electrical stimulation was performed in the sham electroacupuncture group. Only fixation was applied in the sham surgery and model groups. Cognitive memory abilities were assessed by the Morris water maze and Y-maze tests. Hippocampal morphology was observed by HE staining. HIF-1α and CXCR4 mRNA expression were detected by real-time quantitative PCR. HIF-1α and CXCR4 protein levels and the p-NF-κB/NF-κB ratio were detected by Western blot. The mean optical density of TNF-α, IL-6, and IL-1β was measured by immunohistochemistry. The mean fluorescence intensity of HO-1 was measured by immunofluorescence. Levels of MDA(malondialdehyde), SOD(superoxide dismutase), and ROS(reactive oxygen species) were measured by ELISA. ROS accumulation was assessed by a fluorescent probe assay. Compared with the sham surgery group, in the model and sham electroacupuncture groups, escape latency was prolonged, platform crossing frequency was reduced, and spontaneous alternation rate is decreased (P<0.01). Hippocampal neuronal organization is disordered. HIF-1α and CXCR4 mRNA and protein expression and the p-NF-κB/NF-κB ratio are increased (P<0.01). TNF-α, IL-6, and IL-1β expression is increased (P<0.01). HO-1 fluorescence intensity is decreased (P <0.01). MDA levels are elevated (P<0.05), SOD activity is decreased (P<0.01), and ROS levels are increased (P<0.05). Compared with the model group, in the electroacupuncture group, escape latency is shortened, the number of platform crossings is increased, and spontaneous alternation rate is increased (P <0.01). Hippocampal damage is reduced. HIF-1α and CXCR4 expression and the p-NF-κB/NF-κB ratio are decreased (P <0.01). TNF-α, IL-6, and IL-1β expression is decreased (P<0.01). HO-1 fluorescence intensity is increased (P<0.01). MDA is decreased (P<0.05), SOD is increased (P <0.01), and ROS is decreased (P<0.05). Neuroinflammation may be alleviated, antioxidant stress resistance may be enhanced, and hippocampal neuronal damage may be repaired by electroacupuncture at the “Baihui” “Zusanli” and “Sanyinjiao” acupoints through balancing the interaction between the HIF-1α/CXCR4/NF-κB inflammatory pathway and the HO-1/SOD antioxidant pathway, thereby improving cognitive function in VD rats.

Graphical abstract

关键词

血管性痴呆 / 电针 / 通路交互 / 认知能力 / 神经炎症 / 抗氧化应激

Key words

vascular dementia / electroacupuncture / pathway interaction / cognitive function / neuroinflammation / antioxidant stress

引用本文

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韩磊, 杨琳, 王建波, 张家豪, 李阳, 曲怡, 薛亚楠, 田跃龙, 王俊超, 刘继东. 电针调控VD大鼠HIF-1α/CXCR4/NF-κB和HO-1/SOD信号通路交互改善认知的机制[J]. 科学技术与工程, 2026, 26(22): 9305-9315 DOI:10.12404/j.issn.1671-1815.2601102

HAN Lei, YANG Lin, WANG Jian-bo, ZHANG Jia-hao, LI Yang, QU Yi, XUE Ya-nan, TIAN Yue-long, WANG Jun-chao, LIU Ji-dong. Mechanism of Electroacupuncture Regulation of HIF-1α/CXCR4/NF-κB and HO-1/SOD Signaling Pathway Interactions in VD Rats to Improve Cognitive Function[J]. Science Technology and Engineering, 2026, 26(22): 9305-9315 DOI:10.12404/j.issn.1671-1815.2601102

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血管性痴呆(vascular dementia,VD)是由脑血管疾病引起的,以记忆力下降、执行功能减退、情绪或行为异常为主要表现的认知障碍综合征^[1]。其发病率在痴呆类型中位列第二,仅次于阿尔茨海默病。随着人口老龄化进程加快,在降低自身生活质量的同时,也给家庭及社会带来沉重压力与经济负担^[2-3]。目前,临床治疗以使用胆碱酯酶抑制剂、NMDA(N-methyl-D-aspartate receptor antagonist)受体拮抗剂等对症药物为主,缺乏针对病因的根治性手段,所以研究中医特色的干预手段及潜在的治疗靶点具有重要临床价值与意义^[4]。

现代研究证实:HIF-1α/CXCR4/NF-κB通路与HO-1/SOD通路存在交互,组成了细胞应对缺氧、炎症与氧化应激等复杂微环境的经典调控网络。在VD的病理进程中,炎性通路的过度激活与抗氧化通路抑制形成恶性循环,即二者交互失衡是脑内组织损伤、认知记忆能力衰退的驱动因素^[5-6]。具体而言:VD中脑内的缺氧微环境可激活HIF-1α/CXCR4/NF-κB炎性通路的过表达,促进TNF-α、IL-6、IL-1β等因子大量释放,抑制抗氧化HO-1/SOD 通路,削弱机体抗氧化能力,导致活性氧(reactive oxygen species,ROS)清除能力下降,加重海马体等脑区结构损伤^[7]。陈永衡等^[8]发现VD患者血浆丙二醛(malondialdehyde,MDA)及血清同型半胱氨酸(Hcy)含量升高,血清超氧化物歧化酶(superoxide dismutase,SOD)含量降低,亦佐证了氧化应激与炎性反应在VD发病机制中的协同作用。此外,临床研究表明:针刺“百会”“足三里”“三阴交”等穴位能够提升阿尔茨海默病(Alzheimer's disease,AD)患者的认知能力^[9]。针刺作为最具有中医特色的干预手段,具有能同时干预多靶点、多通路的优势,但电针是否通过上述机制发挥疗效尚未完全明确。

因此,现以假电针组为对照,观察电针“百会”“足三里”“三阴交”对VD大鼠海马组织HIF-1α/CXCR4/NF-κB与HO-1/SOD通路的影响,阐释电针通过调控通路信号交互、抑制炎症反应、修复氧化应激损伤,改善认知功能的机制,为临床提供科学数据。

1 材料与方法

1.1 实验动物与分组

SPF(specific pathogen-free)级雄性SD(Sprague-Dawley)大鼠40只,体重200 g±20 g,辽宁长生生物科技公司生产,实验动物质量许可证号:[SCXK(辽)2025-0001],室温22 ℃±4 ℃、湿度50%~60%、12 h/12 h光照和黑暗条件下进行实验,自由进食饮水。经7 d适应性喂养后,采用随机数字表法将所有大鼠分为假手术组10只,造模组30只。

1.2 主要试剂与仪器

HIF-1α(赛维尔,GB114936)、CXCR4(华安生物,ER1802-28)、NF-κB(赛维尔,GB11997)、p-NF-κB(赛维尔,GB113882)、TNF-α(赛维尔,GB11188)、IL-6(赛维尔,GB11117)、IL-1β(赛维尔,GB11113)、HO-1(赛维尔,GB11104);动物组织直扩PCR试剂盒(赛维尔,GB11117)、组织切片活性氧检测试剂盒(BB-460513,上海贝博生物科技有限公司);戊巴比妥钠(南京生兴生物技术有限公司);华佗牌SDZ-Ⅲ型电针仪(苏州医疗用品厂有限公司);一次性无菌针灸针(0.30 mm×13 mm,北京中研太和);包埋机(武汉俊杰电子有限公司,JB-P5);石蜡切片机(上海徕卡仪器有限公司,RM2016);蛋白电泳仪及转膜仪(上海天能);酶标仪(美国赛默飞);实时荧光定量 PCR 仪(美国赛默飞);M8数字切片扫描成像系统(Precipoint,FM34F056),光谱型微光扫描共聚焦显微镜系统(FV10i)。

1.3 造模方法

采用双侧颈总动脉结扎法实施血管性痴呆大鼠模型构建^[10]。术前12 h禁食禁水,腹腔注射3%戊巴比妥钠(45 mg/kg)麻醉后,仰卧位,颈部剃毛备皮,常规消毒,于颈部正中线作一约1.5 cm的皮肤切口,钝性分离皮下组织,用玻璃分针分离双侧颈总动脉、神经及膜组织,4号线结扎双侧颈总动脉,结扎后缝合伤口,碘伏消毒^[11]。假手术组大鼠仅剥离双侧颈总动脉,不结扎。术后7 d采用Morris水迷宫检测大鼠逃避潜伏期,以假手术组的逃避潜伏期作为参考,造模大鼠的逃避潜伏期延长率>20%,判定造模成功^[12]。本次实验将30只大鼠进行VD造模,手术中死亡2只,剔除不符合标准的大鼠4只,最终剩余24只大鼠造模成功,假手术组未出现死亡情况。将24只大鼠再次使用随机数字表法分为模型组、电针组、假电针组,8只/组。

1.4 干预方法

电针组大鼠在清醒固定状态下,选取“百会”“足三里”和“三阴交”穴进行电针干预,参照《实验针灸学》大鼠腧穴定位,“百会”位于大鼠顶骨正中;“足三里”位于后肢胫骨粗隆外侧膝至踝间上1/5处,“三阴交”位于后肢内踝上方5 mm处。选取0.25 mm×25 mm一次性无菌针灸针,“百会”针体与头皮呈15°平刺,深度2 mm,“足三里”垂直进针,深度4 mm,“三阴交”向后45°斜刺,深度4 mm。设置为疏密波,在频率50 Hz的频率下,电流强度以针身轻微颤动和大鼠的承受为宜(1~2 mA),15 min/次,1次/日,连续14 d。假电针组将电针浅刺皮下,不提插捻转,不通电。假手术组及模型组大鼠仅做固定,不施加其他处理。

1.5 取材

各组大鼠腹腔注射3%戊巴比妥钠(50 mg/kg)麻醉,随机选取4只大鼠,4%多聚甲醛心脏灌流,取全脑用于HE、免疫组化、免疫荧光染色;剩余4只大鼠取全脑,分离海马,-80 ℃超低温冰箱保存,进行Western blot、聚合酶链式反应(polymerase chain reaction,PCR)、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)、活性氧(reactive oxygen species,ROS)检测。

2 观察指标及检测方法

2.1 行为学检测

2.1.1 Morris水迷宫

采用Morris水迷宫进行定位航行实验、空间探索能力检测。实验总时长为6 d,分为2个阶段。第一阶段定位航行测试中,将圆形水池分为4个象限,在第Ⅱ象限设置1个顶部低于水面2 cm的透明平台,水温控制在23 ℃±2 ℃。检查时,将大鼠从4个不同象限面向池壁置入水中,以其到达平台所需时间作为逃避潜伏期。若在120 s内未到达,则由操作人员带至平台停留10 s,此时记录逃避潜伏期为最大值120 s。第二阶段空间探索,于第6 天移除平台,各组大鼠自第Ⅳ象限固定点入水,记录60 s内穿越原平台区域次数。

2.1.2 Y迷宫

所有大鼠以同一方向放置于Y迷宫的中央起始区,记录大鼠进入迷宫各臂的先后顺序。当大鼠连续进入3个不同的迷宫臂时,即视为1次自发交替行为,统计每只大鼠在5 min内的自发交替行为次数,按照以下公式计算:自发交替率=[自发交替次数/(总进臂次数-2)]×100%。

2.2 HE染色法观察海马组织形态

全脑经4%多聚甲醛固定后,乙醇梯度及二甲苯脱水,石蜡包埋,设置切片厚度为5 μm^[13],再经二甲苯及梯度乙醇脱蜡后,苏木素染色5 min,盐酸乙醇分化30 s、伊红染色2 min,透明化处理,中性树胶封片,光学显微镜下观察脑内海马区神经元形态。

2.3 实时荧光定量PCR检测海马组织HIF-1α、CXCR4的mRNA表达

取50 mg海马组织,经TRIZOL裂解、氯仿萃取、异丙醇沉淀、75%乙醇洗涤提取总RNA,检测RNA浓度及纯度^[14],以总RNA为模板反转录为cDNA。以cDNA为模板,扩增目的基因HIF-1α、CXCR4。扩增条件为:95 ℃预变性 10 min,95 ℃变性 15 s,60 ℃退火 60 s,延伸60 s,40个循环。以GAPDH为内参,2^-ΔΔC_t法计算HIF-1α、CXCR4 mRNA的相对表达量。引物序列如表1所示。

2.4 Western blot法检测海马组织HIF-1α、CXCR4、NF-κB、p-NF-κB的蛋白表达

取50 mg海马组织,加入500 μL RIPA裂解液,于冰浴条件下进行研磨,裂解30 min,4 ℃、3 000 r/min,离心15 min,取上清液为总蛋白。BCA法检测蛋白浓度,5×上样缓冲液,95 ℃变性8 min。冷却后,总蛋白经SDS-PAGE电泳分离后,在恒流200 mA条件下,通过湿转法转移至PVDF膜,5%脱脂奶粉室温封闭2 h,加入一抗HIF-1α(1∶1 000)、CXCR4(1∶1 000)、NF-κB p65(1∶1 000)、p-NF-κB(1∶1 000)、GAPDH(1∶1 000)4 ℃孵育过夜,TBST(tris-buffered saline with Tween 20)洗涤3次,10 min/次,加入稀释度为1∶5 000的二抗室温孵育1 h,TBST洗涤3次,10 min/次。使用ECL发光液进行显影,采集条带,Image J进行分析,目的蛋白的相对表达量为其灰度值与内参蛋白GAPDH灰度值的比值,p-NF-κB/NF-κB的比值作为NF-κB的磷酸化程度。

**2.5 免疫组织化学法检测海马组织TNF-α、IL-6、IL-1β的表达**

取HE染色过程中制作的石蜡切片,经脱蜡、水化、PBS漂洗后,柠檬酸钠缓冲液进行抗原修复,室温冷却后,3% H₂O₂室温孵育去除过氧化物酶,山羊血清封闭,清洗,分别滴TNF-α(1∶200)、IL-6(1∶200)、IL-1β(1∶200)一抗,湿盒4 ℃孵育过夜,加入二抗(1∶200),37 ℃孵1h,DAB(3,3'-diaminobenzidine)显色,苏木素复染,二甲苯透明,中性树胶封片。光学显微镜拍照,Image J分析TNF-α、IL-6、IL-1β的表达。

2.6 免疫荧光法检测海马组织HO-1的荧光强度

取全脑组织石蜡切片,二甲苯及乙醇脱蜡后,柠檬酸钠缓冲液抗原修复,H₂O₂室温孵育,滴入兔抗大鼠HO-1(1∶200),湿盒4 ℃孵过夜,磷酸盐缓冲液(phosphate-buffered saline,PBS) 清洗3次,滴入 Alexa Fluor 488标记羊抗兔二抗,室温避光孵1 h,PBS 洗3次,滴入DAPI(4',6-diamidino-2-phenylindole)染液,室温避光孵育10 min,PBS清洗3次,封片。荧光显微镜观察,Image J分析HO-1平均荧光强度。

2.7 ELISA法检测海马组织MDA、SOD、 ROS含量

取50 mg大鼠海马组织,加入450 μL预冷的PBS,组织破碎机研磨后,4 ℃、3 000 r/min离心15 min,取上清^[15-16]。按照大鼠SOD、MDA、ROS ELISA试剂盒说明书操作,酶标仪450 nm测OD值。以标准品浓度-OD值绘制标准曲线并拟合方程,结合各样品OD值计算SOD、MDA、ROS含量。

2.8 荧光探针法观察ROS的积累情况

取全脑石蜡切片,脱蜡水化后滴加200 μL染色工作液,37 ℃避光孵育1 h,清洗洗涤3次后,盖玻片封片。使用激光共聚焦显微镜捕捉图像,Image J计算荧光强度以观察脑内海马区ROS的积累情况^[17-18]。

2.9 统计学分析

实验结果以均数±标准差(

$\stackrel{-}{x}$

±s)表示,运用SPSS 26.0软件进行统计分析。对数据进行正态性检验与方差齐性检验,多组间比较选取单因素方差分析,若方差齐,LSD-t检验被采用于组间两两比较,方差不齐则采取Dunnett's T3检验,差异有统计学意义的标准为P<0.05。

3 结果

3.1 各组大鼠Morris水迷宫实验结果比较

与假手术组比较,模型组与假电针组大鼠逃避潜伏期明显延长,穿越平台次数显著减少(P<0.01);与模型组比较,电针组大鼠逃避潜伏期明显缩短,穿越平台次数增加(P<0.01),假电针组无显著差异(P>0.05),结果如图1所示。

3.2 各组大鼠Y迷宫实验结果比较

与假手术组比较,模型组与假电针组大鼠自发交替率降低(P<0.01);与模型组比较,电针组大鼠自发交替率显著升高(P<0.01),假电针组无显著差异(P>0.05),结果如图2所示。

3.3 各组大鼠大脑海马区形态比较

假手术组大鼠海马区神经元排列整齐、结构致密,细胞形态完整,细胞核清晰。模型组大鼠海马神经元排列失序、结构松散,细胞间隙增宽,部分出现核深染。与模型组比较,电针组海马神经元排列整齐,深染现象减少。假电针组海马神经元病理形态与模型组相似,无明显改善,结果如图3所示。

**3.4 各组大鼠海马组织HIF-1α、CXCR4 mRNA的相对表达量比较**

qPCR结果显示,与假手术组比较,模型组和假电针组HIF-1α、CXCR4的mRNA表达量升高(P<0.01);与模型组比较,电针组HIF-1α、CXCR4的mRNA表达量降低(P<0.01)^[14,19],假电针组无显著差异(P>0.05),结果如图4所示。

3.5 各组大鼠海马组织HIF-1α、CXCR4蛋白相对表达量及p-NF-κB/NF-κB比值的比较

与假手术组比较,模型组与假电针组HIF-1α、CXCR4的蛋白表达及p-NF-κB/NF-κB比值升高(P<0.01),与模型组比较,电针组大鼠HIF-1α、CXCR4的蛋白表达及p-NF-κB/NF-κB比值降低(P<0.01),假电针组无显著差异^[20-21](P>0.05),结果如图5所示。

3.6 各组大鼠海马组织IL-1β、IL-6、TNF-α的表达比较

与假手术组比较,模型组与假电针组大鼠海马组IL-1β、IL-6、TNF-α表达增多(P<0.01);与模型组比较,电针组IL-1β、IL-6、TNF-α表达减少(P<0.01),假电针组无显著差异(P>0.05)^[22-23],结果如图6所示。

3.7 各组大鼠海马组织HO-1平均荧光强度的比较

与假手术组大鼠比较,模型组与假电针组大鼠海马组织中HO-1平均荧光强度下降(P<0.01);与模型组比较,电针组平均荧光强度升高(P<0.01),假电针组无显著差异(P>0.05),结果如图7所示。

3.8 各组大鼠海马组织MDA、SOD、ROS的含量比较

与假手术组大鼠比较,模型组与假电针组大鼠海马组织MDA、ROS含量升高(P<0.01),SOD含量降低(P<0.01);与模型组大鼠比较,电针组大鼠MDA、ROS含量降低(P<0.01),SOD含量升高(P<0.01),假电针组无显著差异(P>0.05),结果如图8所示。

3.9 各组大鼠海马组织ROS积累情况的比较

与假手术组比较,模型组和假电针组大鼠海马组织ROS的荧光强度升高(P<0.01);与模型组大鼠比较,电针组大鼠ROS 的荧光强度减弱(P<0.01),与假电针组比较无显著差异(P>0.05),结果如图9所示。

4 讨论

中医学将VD归于“呆病”“健忘”“中风”范畴,其病位在脑,与心、脾、肾相关,以气血不足、脑络失养为本,瘀血阻滞为标,属本虚标实之证。《灵枢·大惑论》言“上气不足……故善忘”明确了气血亏虚、脑失濡养是VD发生的中医病机;《医林改错》提出“瘀血令人善忘”“脑髓渐空”,强调瘀血内阻在VD病程中的关键作用;《金匮要略》载中风“邪入于腑,即不识人”,揭示了中风是继发认知障碍的病理关联,进一步补充了VD的中医病机。基于上述病机,本次实验以“百会”“足三里”“三阴交”配伍治疗VD并阐释其作用机制。其中,百会属督脉,居巅顶而通脑髓,可充养脑髓,直击“脑失濡养”;足三里为足阳明合穴,能健脾益气、化痰祛浊,在补后天气血之时,改善瘀血之标;三阴交系肝脾肾之交会穴,可滋补肝肾,补气血不足之本,三穴协同,共奏补虚泻实、调畅脑络之效。关于VD的研究常采用双侧颈总动脉结扎法建立VD模型,以Morris水迷宫、Y迷宫检测结果以及海马组织HE染色为标准^[24-25],验证模型是否构建成功。此外,MDA是ROS攻击海马神经元细胞膜发生脂质过氧化的产物,间接反映了组织的氧化应激损伤程度^[26-27]。结果显示:模型组大鼠逃避潜伏期延长,穿越平台次数显著减少,自发交替率显著降低;同时海马神经元排列紊乱、松散,细胞间隙增大,出现核深染等结构改变,且MDA含量升高,提示VD状态下存在海马神经元损伤,影响了认知功能。经电针干预后,上述指标出现了逆转,表明刺激“百会”“足三里”“三阴交”能够有效恢复海马神经元的氧化应激损伤,改善大鼠的认知功能^[28]。

海马体是脑内负责记忆与学习的核心中枢,在认知维持中发挥关键作用^[29]。研究表明:在VD等缺血缺氧性脑病中,HIF-1α/CXCR4/NF-κB通路是介导炎症-氧化应激循环的起始通路^[30],该通路以脑内微缺氧为触发信号,加剧炎症-氧化的交互,破坏海马体结构与功能。其中,HIF-1α作为缺氧应答的关键分子,因脯氨酰羟化酶(prolyl hydroxylase,PHD)活性受抑制而积累,上调CXCR4启动缺氧适应性反应,CXCR4表达上升后进一步磷酸化NF-κB,增强细胞核内促炎因子转录活性,驱动TNF-α、IL-6、IL-1β等因子大量释放^[31]。本次实验结果表明:模型组大鼠海马组织中HIF-1α和CXCR4的mRNA与蛋白表达水平均升高,NF-κB磷酸化水平上升,TNF-α、IL-6、IL-1β含量增多,提示VD状态下的脑区微缺氧环境会过度激活HIF-1α/CXCR4/NF-κB通路与其相关炎性因子的释放,再次验证了上述结论。经电针“百会”“足三里”“三阴交”干预后,海马组织中上述指标降低且假针刺组指标与模型组比较没有显著差异,表明电针能够有效抑制大鼠海马组织HIF-1α/CXCR4/NF-κB信号通路的激活,降低TNF-α、IL-6、IL-1β等炎性因子释放,从而修复海马体神经细胞损伤。

氧化应激是加剧VD病理损伤的关键机制^[32],其中HO-1/SOD通路是机体对抗氧化应激的核心通路,与HIF-1α/CXCR4/NF-κB信号通路和下游TNF-α、IL-6、IL-1β等因子的异常活化存在密切交互关系。生理状态下,HIF-1α/CXCR4/NF-κB通路的适度激活,使HO-1维持低水平的基础表达,通过降解血红素生成胆红素与SOD协同清除少量ROS,二者的精细交互,维持炎性-氧化应激的动态平衡,保证组织微环境氧化应激稳态^[33-34]。但在VD病理状态下,HIF-1α/CXCR4/NF-κB通路过度释放的TNF-α、IL-6、IL-1β等炎性因子会下调HO-1与SOD表达,抑制HO-1/SOD通路的抗氧化功能,造成ROS蓄积,打破了这一平衡,加重脂质过氧化损伤^[35]。本次研究结果显示:与假手术比较,模型组大鼠海马组织HO-1的荧光强度与SOD含量降低,ROS含量升高,同时结合HIF-1α/CXCR4/NF-κB通路与炎性因子的检测结果,表明HO-1/SOD通路受到抑制,削弱了机体抗氧化能力,无法清除过量的ROS;经电针干预后,随着HO-1、SOD表达量的显著回升与ROS含量下降,提示机体抗氧化能力与清除ROS能力得到有效恢复。

5 结论

综上所述,电针“百会”“足三里”“三阴交”能够修复VD大鼠海马组织HIF-1α/CXCR4/NF-κB通路与HO-1/SOD通路的交互,通过抑制HIF-1α/CXCR4/NF-κB通路的过度激活与TNF-α、IL-6、IL-1β炎症因子释放,恢复HO-1/SOD的抗氧化,清除ROS的能力,减轻MDA引起的氧化应激损伤及神经元凋亡,改善VD认知功能^[36]。这为电针治疗血管性痴呆提供了坚实的实验依据,也为深入理解电针治疗VD的作用机制开辟了新的思路。

但本次研究仍存在一定局限性,有待后续完善:一是干预周期仅 14 d,观察时间较短,后续可延长干预与随访周期,系统评估远期疗效及安全性;二是未设置西药阳性对照组,难以客观对比疗效差异,后续将增设药物对照,明确本干预方案的临床优势;三是目前仅完成动物实验,缺乏临床证据支撑,后续可逐步开展临床试验,为研究成果的临床转化与应用提供可靠依据。

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基金资助

国家自然科学基金面上项目(82474634)

辽宁省科技计划联合项目(2023-MSLH-178)

辽宁省科技计划联合计划(2024JH2/102600199)

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Received	Published	Revised
2026-03-04	2026-08-08	2026-05-15
Online Date
2026-09-03

摘要