电针调控Nrf2/HO-1通路对缺血缺氧性脑损伤大鼠小胶质细胞活化的影响

doi:10.11958/20220792

摘要/Abstract

摘要：

目的探究电针调控核因子E2相关因子2（Nrf2）/血红素加氧酶1（HO-1）通路对缺血缺氧性脑损伤（HIBD）大鼠小胶质细胞活化的影响。方法随机将40只SD大鼠均分为假手术组、模型组、电针组、电针+全反式维甲酸组。除假手术组外其余组均通过颈总动脉结扎及缺氧建立HIBD大鼠模型，假手术组大鼠仅暴露左侧颈总动脉及迷走神经，不进行结扎及缺氧处理。造模结束后，电针组于百会穴进行电针刺激；电针+全反式维甲酸组大鼠经电针刺激后腹腔注射全反式维甲酸（7 mg/kg）。通过Longa评分进行大鼠神经行为学评分；旷场及水迷宫实验检测大鼠自主活动能力和认知功能；TUNEL法检测大鼠神经细胞凋亡情况；免疫组化法检测大鼠脑组织中小胶质细胞标志蛋白离子钙结合衔接分子1（Iba1）表达；试剂盒检测大鼠脑组织中白细胞介素10（IL-10）、IL-1β、活性氧（ROS）及丙二醛（MDA）水平；Western blot检测脑组织CD68、诱导型一氧化氮合酶（iNOS）和精氨酸酶（Arginase）及Nrf2/HO-1通路相关蛋白表达。结果与假手术组比较，模型组大鼠神经行为学评分、逃避潜伏期、大脑皮质神经细胞凋亡率、Iba1、IL-1β、MDA水平、ROS荧光强度、CD68、iNOS表达增加，运动路程、站立次数、穿越平台次数、IL-10、Arginase、Nrf2及HO-1水平降低（P<0.05）；电针可降低HIBD大鼠神经行为学评分、逃避潜伏期、神经细胞凋亡率、Iba1、IL-1β、MDA水平、ROS荧光强度、CD68、iNOS表达，增加运动路程、站立次数、穿越平台次数、IL-10、Arginase、Nrf2及HO-1水平（P<0.05）；电针+全反式维甲酸组可逆转电针对上述指标的影响。结论电针可能通过激活Nrf2/HO-1通路抑制HIBD大鼠小胶质细胞过度活化，并促进其由M1型向M2型转变。

关键词: 缺氧缺血，脑, 动物，新生, 电针, 核因子NF-E2相关因子, 血红素加氧酶1, 小胶质细胞

Abstract:

Objective To explore the effect of electroacupuncture regulating nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway on the activation of microglia in rats with hypoxia-ischemia brain damage (HIBD). Methods Forty SD rats were randomly divided into the sham operation group, the model group, the electroacupuncture group and the electroacupuncture + all-trans retinoic acid group. Rat model of HIBD was established by common carotid artery ligation and hypoxia in the three groups except the sham operation group. In the sham operation group, only the left common carotid artery and vagus nerve were exposed no ligation and hypoxia treatment. After modeling, electroacupuncture stimulation at Baihui point was given to the electroacupuncture group. Rats in the electroacupuncture + all-trans retinoic acid group were given all-trans retinoic acid (7 mg/kg) intraperitoneally after electroacupuncture stimulation. Neurobehavioral scores of rats were evaluated by Longa score. Open field and water maze experiments were used to detect the behavioral changes and cognitive function of rats. TUNEL method was used to detect the apoptosis of nerve cells. Immunohistochemical method was used to detect the expression of microglia marker protein ionized calcium binding adapter molecule 1 (Iba1) in rat brain. Levels of interleukin-10 (IL-10), IL-1β, reactive oxygen species (ROS) and malondialdehyde (MDA) in rat brain were detected with kits. Western blot assay was used to detect the expression levels of CD68, inducible nitric oxide synthase (iNOS), arginase and Nrf2/HO-1 pathway related proteins in brain tissue. Results Compared with the sham operation group, the neurobehavioral score, the escape latency, cerebral cortex nerve cell apoptosis rate, the expression levels of Iba1, IL-1β and MDA, ROS fluorescence intensity, CD68 and iNOS were increased in the model group, and the exercise distance, number of standing times, the number of crossing platform times, levels of IL-10, arginase, Nrf2 and HO-1 decreased (P<0.05). Electroacupuncture can reduce neurobehavioral score, the escape latency, nerve cell apoptosis rate, expression levels of Iba1, IL-1β, MDA, ROS fluorescence intensity, CD68 and iNOS, and increase exercise distance, number of standing times, number of crossing platform times, levels of IL-10, arginase, Nrf2 and HO-1 (P<0.05). The effect of electroacupuncture on the above indicators could be reversed in the electroacupuncture + all-trans retinoic acid group. Conclusion Electroacupuncture may inhibit the hyperactivation of microglia in rats with HIBD by activating the Nrf2/HO-1 pathway, and promote the transition from M1 type to M2 type.

Key words: hypoxia-ischemia, brain, animals, newborn, electroacupuncture, nuclear factor NF-E2-related factor, heme oxygenase 1, microglia

中图分类号:

R285.5

图/表 7

参考文献 18

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组别		旷场实验
组别		运动路程（cm）		站立次数（次）
假手术组		2 253.30±230.41		70.16±6.03
模型组		852.17±118.80^a		42.57±3.22^a
电针组		1 695.16±161.30^ab		61.07±4.04^ab
电针+全反式维甲酸组		1 233.46±127.14^abc		48.20±3.26^abc
F		133.590^＊＊		84.406^＊＊
组别	水迷宫实验
组别	穿越平台次数（次）		逃避潜伏期（s）
假手术组	8.93±1.02		14.10±1.12
模型组	2.04±0.50^a		27.96±2.17^a
电针组	6.25±0.71^ab		18.40±1.22^ab
电针+全反式维甲酸组	4.37±0.60^abc		22.70±1.46^abc
F	158.019^＊＊		146.818^＊＊

组别		旷场实验
组别		运动路程（cm）		站立次数（次）
假手术组		2 253.30±230.41		70.16±6.03
模型组		852.17±118.80^a		42.57±3.22^a
电针组		1 695.16±161.30^ab		61.07±4.04^ab
电针+全反式维甲酸组		1 233.46±127.14^abc		48.20±3.26^abc
F		133.590^＊＊		84.406^＊＊
组别	水迷宫实验
组别	穿越平台次数（次）		逃避潜伏期（s）
假手术组	8.93±1.02		14.10±1.12
模型组	2.04±0.50^a		27.96±2.17^a
电针组	6.25±0.71^ab		18.40±1.22^ab
电针+全反式维甲酸组	4.37±0.60^abc		22.70±1.46^abc
F	158.019^＊＊		146.818^＊＊

组别	细胞凋亡率（%）	Iba1阳性细胞数（个/视野）
假手术组	7.21±1.18	4.12±1.02
模型组	39.16±2.22^a	89.87±9.08^a
电针组	18.69±1.10^ab	40.64±5.36^ab
电针+全反式维甲酸组	23.64±2.25^abc	68.18±6.37^abc
F	278.843^＊＊	179.359^＊＊

组别	细胞凋亡率（%）	Iba1阳性细胞数（个/视野）
假手术组	7.21±1.18	4.12±1.02
模型组	39.16±2.22^a	89.87±9.08^a
电针组	18.69±1.10^ab	40.64±5.36^ab
电针+全反式维甲酸组	23.64±2.25^abc	68.18±6.37^abc
F	278.843^＊＊	179.359^＊＊

组别	IL-1β（mg/g）	IL-10（mg/g）	ROS荧光强度	MDA（mmol/mg）
假手术组	0.015±0.004	0.053±0.004	125.59±18.36	3.98±0.28
模型组	0.050±0.007^a	0.018±0.003^a	284.74±25.87^a	6.67±0.32^a
电针组	0.023±0.005^b	0.042±0.005^ab	180.15±15.36^ab	5.01±0.29^ab
电针+全反式维甲酸组	0.038±0.005^abc	0.030±0.004^abc	227.15±16.50^abc	5.80±0.30^abc
F	42.261^＊＊	69.167^＊＊	60.618^＊＊	73.946^＊＊