黄芪甲苷抑制Fas/FasL信号通路减轻创伤性脑损伤大鼠神经功能缺损和神经元凋亡

doi:10.11958/20231226

天津医药 ›› 2024, Vol. 52 ›› Issue (5): 469-474.doi: 10.11958/20231226

黄芪甲苷抑制Fas/FasL信号通路减轻创伤性脑损伤大鼠神经功能缺损和神经元凋亡

陈惠刚(), 池小锋, 封娣, 米娅莉^△()

张家口学院护理学院临床护理教研室（邮编075061）

收稿日期:2023-08-14 修回日期:2023-10-31 出版日期:2024-05-15 发布日期:2024-05-09
通讯作者: ^△ E-mail：876794652@qq.com
作者简介:陈惠刚（1980），男，副教授，主要从事解剖学和组织胚胎学方面研究。E-mail：chenhuigang627@163.com
基金资助:
河北省医学科学研究课题计划项目(20220572)

Astragaloside inhibits Fas/FasL signaling pathway to reduce neural dysfunction and neuronal apoptosis in traumatic brain injury of rats

CHEN Huigang(), CHI Xiaofeng, FENG Di, MI Yali^△()

Clinical Nursing Teaching and Research Office of the School of Nursing at Zhangjiakou University, Zhangjiakou 075061, China

Received:2023-08-14 Revised:2023-10-31 Published:2024-05-15 Online:2024-05-09
Contact: ^△ E-mail：876794652@qq.com

陈惠刚, 池小锋, 封娣, 米娅莉. 黄芪甲苷抑制Fas/FasL信号通路减轻创伤性脑损伤大鼠神经功能缺损和神经元凋亡[J]. 天津医药, 2024, 52(5): 469-474.

CHEN Huigang, CHI Xiaofeng, FENG Di, MI Yali. Astragaloside inhibits Fas/FasL signaling pathway to reduce neural dysfunction and neuronal apoptosis in traumatic brain injury of rats[J]. Tianjin Medical Journal, 2024, 52(5): 469-474.

摘要/Abstract

摘要：

目的探究黄芪甲苷通过Fas/FasL信号通路对创伤性脑损伤（TBI）大鼠神经功能缺损和神经元凋亡的影响。方法将大鼠随机分为5组：假手术组、模型组、黄芪甲苷组（20 mg/kg）、Fas沉默组[4 μg Fas小干扰RNA（siRNA）慢病毒载体]、黄芪甲苷+Fas沉默组（20 mg/kg黄芪甲苷+4 μg Fas siRNA慢病毒载体），每组10只。除假手术组外，其余各组大鼠制作TBI模型。各组按照对应剂量进行给药干预，每日1次，持续7 d。水迷宫实验检测大鼠神经功能缺损；荧光定量PCR法检测脑组织Fas、FasL mRNA表达；苏木精-伊红、β-微管蛋白Ⅲ（Tuj1）免疫荧光、TUNEL染色分别观察脑组织病理变化、神经元活性及神经元凋亡；Western blot法检测脑组织中Fas/FasL通路、胱天蛋白酶-3（Caspase-3）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）表达。结果与假手术组比较，模型组大鼠脑组织细胞间隙增加、变性明显，第1~5天大鼠逃避潜伏期，神经元凋亡率，Caspase-3、Bax蛋白，Fas、FasL mRNA和蛋白水平升高，穿越平台次数、Tuj1阳性细胞数目、Bcl-2蛋白水平降低（P<0.05）。与模型组比较，黄芪甲苷组、Fas沉默组大鼠脑组织细胞间隙缩小，变性减轻，第1~5天大鼠逃避潜伏期，神经元凋亡率，Caspase-3、Bax蛋白，Fas、FasL mRNA和蛋白水平降低，穿越平台次数、Tuj1阳性细胞数目、Bcl-2蛋白水平升高（P<0.05）。黄芪甲苷组和Fas沉默组大鼠脑组织病理变化和上述指标差异无统计学意义（P>0.05）。与黄芪甲苷组和Fas沉默组比较，黄芪甲苷+Fas沉默组大鼠脑组织病理损伤进一步降低，神经元凋亡率，Caspase-3、Bax蛋白，Fas、FasL mRNA和蛋白水平降低，Tuj1阳性细胞数目、Bcl-2蛋白水平升高（P<0.05）。结论黄芪甲苷通过抑制Fas/FasL信号通路介导的凋亡途径减轻TBI大鼠神经功能缺损和神经元凋亡。

关键词: 脑损伤, 创伤性, 黄芪甲苷, Fas受体, 神经元

Abstract:

Objective To explore the effects of astragaloside on neural dysfunction and neuronal apoptosis of traumatic brain injury (TBI) in rats through Fas/FasL signaling pathway. Methods Fifty rats were randomly divided into 5 groups: the sham operation group, the model group, the astragaloside group (20 mg/kg), the Fas silencing group [4 μg Fas small interfering RNA (siRNA) lentiviral vector] and the astragaloside+Fas silencing group (20 mg/kg astragaloside+4 μg Fas siRNA lentiviral vector). Each group consisted of 10 rats. Except the sham operation group, the other groups of rats were established TBI rat model. Each group was received medication intervention according to the corresponding dosage, once a day, for 7 days. Water maze test was used to detect the nerve function defect in rats. Fluorescence quantitative PCR was used to detect expression levels of Fas and FasL messenger RNA (mRNA) in brain tissue. Hematoxylin-eosin, β-tubulin III (Tuj1) immunofluorescence and TUNEL staining were used to observe pathological changes, neuronal activity and apoptosis of brain tissue, respectively. Western blot assay was used to detect expression levels of Fas/FasL pathway, Caspase-3, Bcl-2 associated X protein (Bax) and B lymphoblastoma-2 (Bcl-2) protein in brain tissue. Results Compared with the sham operation group, the cell gap was increased and degeneration was obvious in brain tissue in the model group. The escape latency of rats on days 1-5, neuronal apoptosis rate, Caspase-3, Bax protein, Fas, FasL mRNA and protein levels were increased (P<0.05). The number of times crossing platforms, the number of Tuj1 positive cells and Bcl-2 protein were decreased (P<0.05). Compared with the model group, in the astragaloside group and the Fas silencing group, the cell gap in brain tissue was narrowed and degeneration was reduced, the escape latency of rats on days 1-5, neuronal apoptosis rate, Caspase-3, Bax protein, Fas, FasL mRNA and protein levels were decreased, and the number of crossing platforms, the number of Tuj1 positive cells and Bcl-2 protein level were increased (P<0.05). There were no significant differences in pathological changes of brain and the above indexes between the astragaloside group and the Fas silencing group (P>0.05). Compared with the astragaloside group and the Fas silencing group, the pathological damage of brain tissue of rats was further reduced in the astragaloside+Fas silencing group, neuronal apoptosis rate, Caspase-3, Bax protein, Fas, FasL mRNA and protein levels were decreased, and the number of Tuj1 positive cells and Bcl-2 protein were increased (P<0.05). Conclusion Astragaloside may reduce nerve function deficit and neuronal apoptosis in TBI rats by inhibiting Fas/FasL signaling pathway mediated apoptosis pathway.

Key words: brain injuries, traumatic, astragaloside Ⅳ, Fas receptor, neurons

中图分类号:

R651.15，R286.1

图/表 10

表1 QRT-PCR引物序列

Tab.1 QRT-PCR primer sequence

基因名称	引物序列（5'→3'）	产物大小/bp
Fas	上游：TGCACAGAATTGAAGGAGTA	216
	下游：ATGGTTTCACGACTGGAGGT	216
FasL	上游：GACAGCAGTGCCACTTCATC	159
	下游：TTAAGGCTTTGGTTGGTGAA	159
GAPDH	上游：GGGAATAGTAGCTGTCAAATT	91
	下游：AATGACAGCTACCAGTCCCTT	91

表2 各组大鼠Morris水迷宫实验结果比较（n=10，$\bar{x}±s$）

Tab.2 Comparison of Morris water maze test results between five groups of rats

组别	逃避潜伏期/s					穿越平台次数/次
组别	第1天	第2天	第3天	第4天	第5天	穿越平台次数/次
假手术组	49.28±9.51	43.76±8.62	38.52±7.73	31.75±6.59	28.64±5.67	7.25±1.34
模型组	83.71±16.08^a	77.68±15.26^a	71.54±14.36^a	65.29±13.54^a	51.37±10.63^a	3.58±0.73^a
黄芪甲苷组	53.66±10.64^b	48.72±9.58^b	41.65±8.31^b	35.94±7.24^b	27.86±5.29^b	5.34±1.06^b
Fas沉默组	52.63±10.59^b	46.28±9.27^b	43.16±8.51^b	35.22±6.98^b	27.06±4.32^b	5.55±1.29^b
黄芪甲苷+Fas沉默组	46.59±9.35	41.20±8.27	34.59±6.85	28.61±5.54	22.36±4.15	6.52±1.27
F	17.217^＊＊	20.025^＊＊	23.807^＊＊	30.490^＊＊	31.068^＊＊	14.318^＊＊

图1 各组大鼠脑组织病理变化（HE染色，×200）箭头示变性的脑组织细胞。

Fig.1 Pathological changes of brain tissue of rats in each group (HE staining, ×200)

图2 各组大鼠脑组织Tuj1免疫荧光染色结果（×200）

Fig.2 Results of Tuj1 immunofluorescence staining in brain tissue of rats in each group (×200)

图3 各组大鼠脑组织Tuj1阳性细胞数目比较 A—E分别为假手术组、模型组、黄芪甲苷组、Fas沉默组及黄芪甲苷+Fas沉默组；a与假手术组比较，b与模型组比较，c与黄芪甲苷组比较，d与Fas沉默组比较，P<0.05。

Fig.3 Comparison of the number of Tuj1 positive cells in brain tissue between five groups of rats

图4 各组大鼠脑组织神经元凋亡情况（TUNEL染色，×200）

Fig.4 Apoptosis of neurons in brain tissue of rats in each group (TUNEL staining, ×200)

图5 各组大鼠脑组织Caspase-3、Bax、Bcl-2蛋白表达情况 A：假手术组；B：模型组；C：黄芪甲苷组；D：Fas沉默组；E：黄芪甲苷+Fas沉默组。

Fig.5 Expression levels of Caspase-3, Bax and Bcl-2 protein in brain tissue of rats in each group

表3 各组大鼠脑组织神经元凋亡率和凋亡相关蛋白表达比较（n=10，$\bar{x}±s$）

Tab.3 Comparison of neuronal apoptosis rate and apoptosis-related protein expression in brain tissue of rats between five groups

组别	凋亡率/%	凋亡相关蛋白表达
组别	凋亡率/%	Caspase-3	Bax	Bcl-2
假手术组	1.91±0.37	1.24±0.26	0.67±0.13	2.05±0.40
模型组	16.52±3.06^a	3.06±0.61^a	2.38±0.46^a	0.53±0.11^a
黄芪甲苷组	10.57±2.43^b	1.80±0.37^b	1.46±0.27^b	0.94±0.22^b
Fas沉默组	10.63±2.55^b	1.83±0.32^b	1.39±0.22^b	0.96±0.20^b
黄芪甲苷+ Fas沉默组	6.72±0.83^cd	1.05±0.19^cd	0.88±0.16^cd	1.45±0.34^cd
F	64.597^＊＊	43.080^＊＊	58.528^＊＊	45.138^＊＊

图6 各组大鼠脑组织Fas、FasL蛋白表达情况 A：假手术组；B：模型组；C：黄芪甲苷组；D：Fas沉默组；E：黄芪甲苷+Fas沉默组。

Fig.6 The expression of Fas and FasL protein in brain tissue of rats in each group

表4 各组大鼠脑组织Fas、FasL mRNA和蛋白表达比较（n=10，$\bar{x}±s$）

Tab.4 Comparison of Fas and FasL mRNA and protein expression in brain tissue of rats between five groups

组别	Fas		FasL
组别	mRNA	蛋白	mRNA	蛋白
假手术组	1.00±0.04	0.25±0.04	1.00±0.03	0.94±0.17
模型组	3.58±0.72^a	1.56±0.30^a	5.21±1.02^a	2.03±0.43^a
黄芪甲苷组	2.23±0.46^b	0.79±0.16^b	3.39±0.66^b	1.57±0.32^b
Fas沉默组	2.17±0.46^b	0.77±0.13^b	3.22±0.60^b	1.55±0.31^b
黄芪甲苷+ Fas沉默组	1.32±0.25^cd	0.43±0.08^cd	2.24±0.45^cd	1.21±0.26^cd
F	49.998^＊＊	89.858^＊＊	59.218^＊＊	21.098^＊＊

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黄芪甲苷抑制Fas/FasL信号通路减轻创伤性脑损伤大鼠神经功能缺损和神经元凋亡

Astragaloside inhibits Fas/FasL signaling pathway to reduce neural dysfunction and neuronal apoptosis in traumatic brain injury of rats

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