丹酚酸B对创伤后应激障碍模型大鼠认知功能和GSK-3β/β-Catenin信号通路的影响

doi:10.11958/20230179

摘要/Abstract

摘要：

目的探讨丹酚酸B（Sal B）是否可通过调节糖原合成酶激酶-3β/β-连环蛋白（GSK-3β/β-Catenin）信号通路改善创伤后应激障碍（PTSD）模型大鼠认知功能。方法 60只大鼠随机分为正常组、PTSD组、Sal B低剂量组（10 mg/kg）、Sal B高剂量组（20 mg/kg）和GSK-3β抑制剂组（30 mg/kg CHIR-99021），每组12只。除正常组外，其余组大鼠采用单一延长应激法构建PTSD大鼠模型。旷场实验、Morris水迷宫实验评估大鼠认知功能；Nissl染色观察海马神经元病理学变化；TUNEL染色检测海马神经元凋亡；Western blot检测海马组织中裂解的胱天蛋白酶3（cleaved caspase-3）、B细胞淋巴瘤基因-2相关X蛋白（Bax）、原癌基因（c-Myc）、细胞周期蛋白D1（Cyclin D1）、总GSK-3β（t-GSK-3β）、磷酸化GSK-3β（p-GSK-3β）、总β-Catenin（t-β-Catenin）、磷酸化β-Catenin（p-β-Catenin）蛋白表达。结果与PTSD组比较，Sal B低剂量组、Sal B高剂量组和GSK-3β抑制剂组大鼠爬行格数、站立次数、运动总距离、跨越原平台次数增多，逃避潜伏期、首次跨越原平台时间缩短，海马神经元凋亡率以及海马组织中Bax、cleaved caspase-3、t-GSK-3β、p-β-Catenin蛋白表达水平降低，Cyclin D1、c-Myc、p-GSK-3β、t-β-Catenin蛋白表达水平升高（P<0.05）。结论 Sal B能够减轻PTSD模型大鼠海马神经元凋亡、损伤并可改善其认知功能障碍，抑制GSK-3β/β-Catenin信号通路。

关键词: 应激障碍，创伤后, 丹参酸B, 认知功能障碍, 糖原合成酶激酶3, β连环素, 神经元, 细胞凋亡

Abstract:

Objective To investigate whether salvianolic acid B (Sal B) can improve the cognitive function in rats with post-traumatic stress disorder (PTSD) by regulating GSK-3β/β-Catenin signal pathway. Methods Sixty rats were randomly grouped into the normal group, the PTSD group, the Sal B low-dose group (10 mg/kg), the Sal B high-dose group (20 mg/kg) and the GSK-3β inhibitor group (30 mg/kg CHIR-99021), with 12 rats in each group. In addition to the normal group, rats in other groups were constructed PTSD rat models by using single prolonged stress (SPS) method. Open field test and Morris water maze test were applied to evaluate the cognitive function of rats. Nissl staining was applied to observe the pathological changes of hippocampal neurons. TUNEL staining was applied to detect the apoptosis of hippocampal neurons. Western blot assay was applied to detect the expression of cleared caspase-3, B-cell lymphoma gene-2-associated X protein (Bax), proto-oncogene (c-Myc), Cyclin D1, total GSK-3β (t-GSK-3β), phosphorylated GSK-3β (p-GSK-3β), total β-Catenin (t-β-Catenin) and phosphorylated β-catenin (p-β-Catenin) proteins in hippocampus. Results Compared with the PTSD group, the number of crawling spaces, standing times, total movement distance and times of crossing the original platform of rats were higher in the Sal B low-dose group, the Sal B high-dose group and the GSK-3β inhibitor group. The escape latency and the time to cross the original platform for the first time were shorter, the apoptosis rate of hippocampal neurons and the expression levels of Bax, cleaved caspase-3, t-GSK-3β and p-β-Catenin proteins in hippocampus were lower, and the expression levels of Cyclin D1, c-Myc, p-GSK-3β, t-β-Catenin proteins were higher (P<0.05). Conclusion Sal B can reduce the apoptosis and damage of hippocampal neurons in rats with PTSD and improve cognitive dysfunction in rats, and inhibit the GSK-3β/β-Catenin signal pathway.

Key words: stress disorders, post-traumatic, Danshensuan B, cognitive dysfunction, glycogen synthase kinase 3, beta Catenin, neurons, apoptosis

中图分类号:

R285.5

图/表 12

表1 各组大鼠旷场实验中爬行格数、站立次数和运动总距离比较

Tab.1 Comparison of the number of crawling cells, standing times and total distance of exercise in open field experiment between the five groups of rats （n=12，$\bar{x}±s$）

组别	爬行格数/个	站立次数/次	运动总距离/cm
正常组	76.33±4.68	14.25±1.46	2 436.18±90.29
PTSD组	48.21±3.95^a	5.17±0.85^a	1 059.67±52.43^a
Sal B低剂量组	61.83±4.40^b	9.08±1.02^b	1 836.29±71.85^b
Sal B高剂量组	72.50±4.06^bc	12.47±1.25^bc	2 345.35±83.86^bc
GSK-3β抑制剂组	69.58±4.12^bc	11.67±1.18^bc	2 308.42±89.27^bc
F	82.233^＊＊	108.915^＊＊	635.255^＊＊

表2 各组大鼠Morris水迷宫实验中逃避潜伏期比较

Tab.2 Comparison of escape latency in Morris water maze experiment between the five groups of rats （n=12，s，$\bar{x}±s$）

组别	1 d	2 d
正常组	31.59±4.42	23.47±4.03
PTSD组	75.64±6.83^a	70.95±6.92^a
Sal B低剂量组	56.78±5.97^b	49.26±5.84^b
Sal B高剂量组	43.15±5.62^bc	35.24±5.16^bc
GSK-3β抑制剂组	46.06±4.98^bc	37.58±5.34^bc
F	104.545^＊＊	126.256^＊＊
组别	3 d	4 d
正常组	15.26±3.15	8.95±2.64
PTSD组	67.08±6.54^a	61.24±6.71^a
Sal B低剂量组	41.65±5.23^b	33.17±4.38^b
Sal B高剂量组	30.57±4.31^bc	24.97±3.97^bc
GSK-3β抑制剂组	33.19±4.58^bc	27.12±3.65^bc
F	182.284^＊＊	217.942^＊＊

表3 各组大鼠Morris水迷宫实验中跨越原平台次数和首次跨越原平台时间比较

Tab.3 Comparison of the times of crossing the original platform and the time of crossing the original platform for the first time in Morris water maze experiment between the five groups of rats （n=12，$\bar{x}±s$）

组别	跨越原平台次数/次	首次跨越原平台时间/s
正常组	9.25±1.38	10.68±1.29
PTSD组	3.42±0.85^a	39.45±3.18^a
Sal B低剂量组	4.83±0.96^b	27.84±2.86^b
Sal B高剂量组	7.67±1.25^bc	19.07±1.95^bc
GSK-3β抑制剂组	7.00±1.04^bc	21.31±2.42^bc
F	51.921^＊＊	233.936^＊＊

图1 各组大鼠海马神经元病理学变化（Nissl染色，×200）

Fig.1 Pathological changes of hippocampal neurons in each group (Nissl staining, ×200)

图2 各组大鼠海马神经元凋亡情况（TUNEL染色，×200）

Fig.2 Apoptosis of hippocampal neurons in each group (TUNEL staining, ×200)

图3 Western blot检测各组大鼠海马组织中Bax、cleaved caspase-3蛋白表达 A：正常组；B：PTSD组；C：Sal B低剂量组；D：Sal B高剂量组；E：GSK-3β抑制剂组。

Fig.3 The expression of Bax and cleaved caspase-3 protein in hippocampus of each group detected by Western blot assay

表4 各组大鼠海马神经元凋亡率以及海马组织中Bax、cleaved caspase-3蛋白表达水平比较

Tab.4 Comparison of apoptosis rate of hippocampal neurons and expression levels of Bax and cleaved caspase-3 in hippocampal tissue between the five groups of rats （n=6，$\bar{x}±s$）

组别	神经元凋亡率/ %	Bax	cleaved caspase-3
正常组	5.89±0.96	0.18±0.04	0.27±0.05
PTSD组	57.26±7.85^a	0.79±0.06^a	0.92±0.08^a
Sal B低剂量组	32.17±5.43^b	0.51±0.06^b	0.63±0.06^b
Sal B高剂量组	20.42±2.56^bc	0.29±0.05^bc	0.41±0.05^bc
GSK-3β抑制剂组	22.05±3.18^bc	0.32±0.06^bc	0.45±0.07^bc
F	100.345^＊＊	115.510^＊＊	94.342^＊＊

图4 Western blot检测各组大鼠海马组织中Cyclin D1、c-Myc蛋白表达 A：正常组；B：PTSD组；C：Sal B低剂量组；D：Sal B高剂量组；E：GSK-3β抑制剂组。

Fig.4 The expression of Cyclin D1 and c-Myc protein in hippocampal tissue of rats in each group detected by Western blot assay

表5 各组大鼠海马组织中c-Myc、Cyclin D1蛋白表达水平比较

Tab.5 Comparison of protein expression levels of c-Myc and Cyclin D1 in hippocampal tissue between the five groups of rats （n=6，$\bar{x}±s$）

组别	Cyclin D1	c-Myc
正常组	0.69±0.06	0.57±0.05
PTSD组	0.23±0.04^a	0.18±0.03^a
Sal B低剂量组	0.40±0.05^b	0.35±0.03^b
Sal B高剂量组	0.54±0.05^bc	0.46±0.04^bc
GSK-3β抑制剂组	0.51±0.06^bc	0.42±0.05^bc
F	63.761^＊＊	74.750^＊＊

图5 Western blot检测各组大鼠海马组织中t-GSK-3β、p-GSK-3β、t-β-Catenin、p-β-Catenin蛋白表达情况 A：正常组；B：PTSD组；C：Sal B低剂量组；D：Sal B高剂量组；E：GSK-3β抑制剂组。

Fig.5 The expression of t-GSK-3β, p-GSK-3β, t-β-Catenin and p-β-Catenin in hippocampal tissue of rats in each group detected by Western blot assay

表6 各组大鼠海马组织中t-GSK-3β、p-GSK-3β、t-β-Catenin、p-β-Catenin蛋白表达水平比较

Tab.6 Comparison of protein expression levels of t-GSK-3β, p-GSK-3β, t-β-Catenin and p-β-Catenin in hippocampal tissue between the five groups of rats （n=6，$\bar{x}±s$）

组别	t-GSK- 3β	p-GSK- 3β	t-β- Catenin	p-β- Catenin
正常组	0.15±0.03	0.61±0.05	0.79±0.05	0.18±0.03
PTSD组	0.78±0.06^a	0.18±0.03^a	0.23±0.04^a	0.67±0.06^a
Sal B低剂量组	0.54±0.05^b	0.35±0.04^b	0.51±0.05^b	0.49±0.05^b
Sal B高剂量组	0.37±0.04^bc	0.45±0.04^bc	0.65±0.05^bc	0.35±0.04^bc
GSK-3β抑制剂组	0.34±0.04^bc	0.47±0.05^bc	0.66±0.06^bc	0.33±0.05^bc
F	165.088^＊＊	83.802^＊＊	107.528^＊＊	92.378^＊＊

图6 Sal B的信号通路图 Tcf/Lef：T细胞因子/淋巴细胞增强因子；FH535：Tcf抑制剂；Dkk-1：Wnt抑制剂；sFRP：分泌性卷曲相关蛋白；Wif-1：Wnt抑制因子1；APC：Wnt信号通路调节因子；Axin：轴蛋白；Dsh/Dvl：蓬乱蛋白/蓬乱蛋白Dsh同源物2；Frizzled：卷曲蛋白；LRP-5/6：低密度脂蛋白受体相关蛋白5/6。

Fig.6 Signal path diagram of Sal B

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