一叶萩碱对大鼠脑缺血再灌注损伤后神经功能的影响

doi:10.11958/20221801

摘要/Abstract

摘要：

目的基于Toll样受体4（TLR4）/核转录因子-κB（NF-κB）通路，探讨一叶萩碱（SE）对大鼠脑缺血再灌注损伤（CIRI）后神经功能的影响及相关机制。方法 100只成年SD大鼠随机分为假手术组（Sham组），模型组（CIRI组），SE低、中、高剂量组，每组20只。采用线栓法建立大鼠CIRI模型，建模成功后立即腹腔注射低、中、高剂量（20 mg/kg、40 mg/kg、80 mg/kg）的SE，连续3 d，Sham组与CIRI组给予等量生理盐水。术后24、48、72 h，参照Longa法对各组大鼠进行神经功能缺损评分；术后72 h，干湿重法测定大鼠脑组织含水量，2，3，5-氯化三苯基四氮唑（TTC）染色评估大鼠脑梗死体积百分比，Western blot检测大鼠脑组织离子钙结合衔接分子1（Iba-1）、TLR4、NF-κB p65和p-NF-κB p65蛋白表达，酶联免疫吸附试验（ELISA）检测大鼠脑组织白细胞介素（IL）-1β、肿瘤坏死因子-α（TNF-α）和IL-6水平，免疫荧光染色检测小胶质细胞活化和神经元存活情况。结果与Sham组相比，CIRI组大鼠神经功能缺损评分、脑组织含水量升高，脑梗死体积百分比增大，脑组织中TLR4、p-NF-κB p65、Iba-1、IL-1β、TNF-α和IL-6表达水平升高（P<0.05）。中、高剂量的SE可降低大鼠CIRI后神经功能缺损评分和损伤后脑组织含水量，降低脑梗死体积百分比及损伤区域TLR4、p-NF-κB p65、Iba-1蛋白表达水平及IL-1β、TNF-α和IL-6水平（P<0.05）。结论 SE可通过抑制TLR4/NF-κB通路激活降低大鼠CIRI后神经炎症反应，发挥脑保护作用。

关键词: 脑缺血, 再灌注损伤, TLR4/NF-κB通路, 一叶萩碱, 炎性因子

Abstract:

Objective To explore the effect and mechanism of securinine (SE) on cerebral ischemia reperfusion injury (CIRI) in rats based on toll-like receptor 4 (TLR4)/nuclear transcription factor-kappa B (NF-κB) pathway. Methods A total of 100 male or female adult SD rats were randomly divided into the sham group, the CIRI group and the SE low-, medium- and high-dose (20 mg/kg, 40 mg/kg, 80 mg/kg) groups, with 20 rats in each group. The rat model of CIRI was established by thread occlusion. Rats were treated with SE (20 mg/kg, 40 mg/kg and 80 mg/kg) immediately after successful modeling for 3 consecutive days. Rats in the sham group and the CIRI group were given the same amount of normal saline. At 24 h, 48 h and 72 h after operation, the neurological deficits of rats were measured according to Longa score. At 72 h after operation, brain tissue water content was evaluated by wet-dry weight method, and the percentage of cerebral infarction volume was assessed by triphenyltetrazolium chloride (TTC) staining. Protein expression levels of Iba-1，TLR4, NF-κB p65 and p-NF-κB p65 in brain tissue were determined by Western blot assay. Expression levels of inflammatory factors such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). Level of microglia activation and the number of surviving neurons were examined by immunofluorescence staining. Results Compared with the sham group, neurological function scores, brain tissue water content and the percentage of cerebral infarction volume were significantly increased in the CIRI group (all P<0.05). Expression levels of TLR4, p-NF-κB p65, Iba-1, IL-1β, TNF-α and IL-6 in brain tissue were significantly increased (all P<0.05). However, medium- and high-doses of SE could significantly alleviate the neurological deficits, reduce the water content of brain tissue and the size of cerebral infarction volume, decrease expression levels of TLR4, p-NF-κB p65, Iba-1, IL-1β, TNF-α and IL-6 in the injured area of brain tissue in rats. Conclusion SE can alleviate CIRI-induced neuroinflammatory response via inhibiting TLR4/NF-κB pathway activation, thereby conferring a protective role in brain of rats.

Key words: brain ischemia, reperfusion injury, TLR4/NF-κB pathway, securinine, inflammatory factors

中图分类号:

R743.3

图/表 8

表1 各组大鼠CIRI后神经功能缺损评分比较 ($\bar{x}\pm s$)

Tab.1 Comparison of neurological function scores after CIRI between the five groups of rats

组别	n	神经功能缺损评分/分
组别	n	24 h	48 h	72 h
Sham组	8	0.00±0.00	0.00±0.00	0.00±0.00
CIRI组	8	2.75±0.39^a	2.70±0.38^a	2.46±0.40^a
SE低剂量组	8	2.71±0.38	2.46±0.43	2.33±0.44
SE中剂量组	8	2.42±0.35	2.25±0.43^b	1.95±0.38^bc
SE高剂量组	8	2.21±0.40^b	1.83±0.44^b	1.33±0.40^bcd
F		3.687^＊	6.249^＊	12.625^＊

图1 各组大鼠CIRI后脑梗死体积百分比（TTC染色）

Fig.1 Percentage of cerebral infarction volume after CIRI in each group of rats (TTC staining)

表2 各组大鼠CIRI后脑梗死体积百分比及脑组织含水量比较（%，$\bar{x}\pm s$）

Tab.2 Comparison of the percentage of cerebral infarct volume and brain tissue water content after CIRI between the five groups of rats

组别	n	脑梗死体积百分比	n	脑组织含水量
Sham组	5	0.00±0.00	6	66.54±2.37
CIRI组	5	42.53±3.00^a	6	82.49±7.30^a
SE低剂量组	5	39.18±3.27	6	79.18±7.76
SE中剂量组	5	27.02±2.45^bc	6	73.17±8.63^b
SE高剂量组	5	16.91±2.17^bcd	6	68.78±8.33^bc
F		90.629^＊		5.204^＊

表3 各组大鼠CIRI后脑组织TLR4、NF-κB和Iba-1蛋白表达的比较（n=6，$\bar{x}\pm s$）

Tab.3 Comparison of TLR4, NF-κB p65 and Iba-1 protein expression levels of brain tissue after CIRI between the five groups of rats

组别	TLR4/ β-actin	p-NF-κB p65/ t-NF-κB p65	p-NF-κB/ β-actin	Iba-1/ β-actin
Sham组	0.36±0.03	0.66±0.05	0.45±0.05	0.29±0.05
CIRI组	0.57±0.04^a	1.19±0.06^a	0.86±0.07^a	0.76±0.06^a
SE低剂量组	0.55±0.04	1.08±0.07	0.76±0.07	0.68±0.06
SE中剂量组	0.40±0.04^bc	0.73±0.05^bc	0.49±0.05^bc	0.39±0.06^bc
SE高剂量组	0.39±0.04^bc	0.67±0.06^bc	0.47±0.05^bc	0.29±0.05^bcd
F	45.248^＊	101.488^＊	61.862^＊	92.760^＊

图2 Western blot检测各组大鼠CIRI后脑组织TLR4、NF-κB p65和Iba-1蛋白表达 A：Sham组；B：CIRI组；C：SE低剂量组；D：SE中剂量组；E：SE高剂量组。

Fig.2 Expression levels of TLR4, NF-κB p65 and Iba-1 after CIRI in each group detected by Western blot assay

图3 各组大鼠CIRI后Iba-1蛋白的表达（免疫荧光染色，×400）

Fig.3 Expression levels of Iba-1 after CIRI in each group (immunofluorescent staining，×400)

表4 各组大鼠CIRI后脑组织IL-1β、TNF-α及IL-6水平比较（ng/g，$\bar{x}\pm s$）

Tab.4 Comparison of IL-1β, TNF-α and IL-6 levels of brain tissue after CIRI between the five groups of rats

组别	n	IL-1β	TNF-α	IL-6
Sham组	5	30.20±4.67	9.19±0.87	0.43±0.11
CIRI组	5	85.67±7.39^a	18.61±1.15^a	2.64±0.32^a
SE低剂量组	5	81.64±6.87	17.27±1.31	2.42±0.24
SE中剂量组	5	55.32±6.32^bc	13.86±1.25^bc	1.64±0.21^bc
SE高剂量组	5	45.64±5.82^bcd	12.05±1.14^bcd	0.91±1.14^bcd
F		85.261^＊	66.366^＊	116.747^＊

图4 各组大鼠CIRI后脑组织神经元存活情况（免疫荧光染色，×400）

Fig.4 The number of neuronal survival after CIRI in each group (immunofluorescent staining，×400)

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