芦丁对慢性脑低灌注大鼠海马组织神经元损伤及RhoA/ROCK信号通路的影响

doi:10.11958/20220710

天津医药 ›› 2022, Vol. 50 ›› Issue (12): 1270-1275.doi: 10.11958/20220710

芦丁对慢性脑低灌注大鼠海马组织神经元损伤及RhoA/ROCK信号通路的影响

李艳¹(), 刘琼², 黄萱¹, 刘丽江¹^,^△()

1 江汉大学医学院病理学与病理生理学教研室（邮编430100）
2 江汉大学附属医院（武汉市第六医院）病理科

收稿日期:2022-05-09 修回日期:2022-06-02 出版日期:2022-12-15 发布日期:2022-12-30
通讯作者: 刘丽江 E-mail:liyanyan975@163.com;1359175284@qq.com
作者简介:李艳（1975），女，高级实验师，主要从事病理生理学方面研究。E-mail：liyanyan975@163.com
基金资助:
湖北省中医药中西医结合科研项目(鄂卫生计生通〔2017〕20号-11)

Effects of rutin on hippocampal neuron injury and RhoA/ROCK signaling pathway in rats with chronic cerebral hypoperfusion

LI Yan¹(), LIU Qiong², HUANG Xuan¹, LIU Lijiang¹^,^△()

1 Department of Pathology and Pathophysiology, Medical College of Jianghan University, Wuhan 430100, China
2 Department of Pathology, Affiliated Hospital of Jianghan University (Wuhan Sixth Hospital)

Received:2022-05-09 Revised:2022-06-02 Published:2022-12-15 Online:2022-12-30
Contact: LIU Lijiang E-mail:liyanyan975@163.com;1359175284@qq.com

李艳, 刘琼, 黄萱, 刘丽江. 芦丁对慢性脑低灌注大鼠海马组织神经元损伤及RhoA/ROCK信号通路的影响[J]. 天津医药, 2022, 50(12): 1270-1275.

LI Yan, LIU Qiong, HUANG Xuan, LIU Lijiang. Effects of rutin on hippocampal neuron injury and RhoA/ROCK signaling pathway in rats with chronic cerebral hypoperfusion[J]. Tianjin Medical Journal, 2022, 50(12): 1270-1275.

摘要/Abstract

摘要：

目的探讨芦丁对慢性脑低灌注大鼠海马组织神经元损伤及Ras同源基因家族成员A（RhoA）/Rho相关卷曲螺旋蛋白激酶（ROCK）信号通路的影响。方法将SD大鼠随机分为假手术组、模型组、芦丁低剂量组（20 mg/kg）、芦丁中剂量组（40 mg/kg）、芦丁高剂量组（80 mg/kg）、RhoA抑制剂组（Rhosin hydrochloride，40 mg/kg），每组9只。除假手术组外，其余各组大鼠均通过结扎颈动脉构建慢性脑低灌注大鼠模型，按照各组给药剂量进行干预处理，每天给药1次，持续4周。采用Morris水迷宫实验测定大鼠的认知功能和记忆能力，苏木素-伊红染色观察大鼠海马组织神经元状态，TUNEL染色检测大鼠海马组织神经元凋亡情况，透射电镜观察大鼠海马组织神经元超微结构，蛋白免疫印迹法检测大鼠海马组织凋亡相关蛋白[胱天蛋白酶-3（Caspase-3）、B淋巴细胞瘤-2相关X蛋白（Bax）]及RhoA/ROCK通路相关蛋白表达情况。结果与假手术组相比，模型组大鼠海马组织神经元数量减少，神经元超微结构、细胞器结构严重受损，逃避潜伏期、海马组织神经元凋亡率、凋亡相关蛋白（Caspase-3、Bax）、RhoA、ROCK1、ROCK2蛋白表达显著升高（P<0.05），穿越平台次数显著减少（P<0.05）；与模型组相比，芦丁各剂量组及RhoA抑制剂组大鼠神经元数量显著增多，神经元超微结构、细胞器受损等均得到一定的恢复，逃避潜伏期、海马组织神经元凋亡率、凋亡相关蛋白（Caspase-3、Bax）、RhoA、ROCK1、ROCK2蛋白表达显著降低（P<0.05），穿越平台次数显著增多（P<0.05），且芦丁各剂量组呈剂量依赖效应；芦丁高剂量组与RhoA抑制剂组上述指标比较差异均无统计学意义。结论芦丁可能通过抑制RhoA/ROCK信号通路缓解慢性脑低灌注引起的大鼠神经元损伤。

关键词: 芦丁, 神经元, rho相关激酶类, 细胞凋亡, 认知功能障碍, 记忆, 慢性脑低灌注

Abstract:

Objective To investigate the effects of rutin on hippocampal neuron injury and Ras homolog gene family member A/Rho-associated coiled-coil protein kinase (RhoA/ROCK) signaling pathway in chronic cerebral hypoperfusion rats. Methods SD rats were randomly divided into the sham operation group, the model group, the rutin low dose group (20 mg/kg), the rutin medium dose group (40 mg/kg), the rutin high dose group (80 mg/kg) and RhoA inhibitor group (Rhosin hydrochloride, 40 mg/kg), with 9 rats in each group. Except the sham operation group, rats in the other groups were subjected to chronic cerebral hypoperfusion by ligating carotid artery. Intervention treatment was carried out according to the dosage of each group. The drug was administered once a day for 4 weeks. Morris water maze test was used to determine the cognitive function and memory ability of rats. Hematoxylin eosin staining was used to observe the status of hippocampal neurons. TUNEL staining was used to detect the apoptosis of hippocampal neurons. Transmission electron microscope was used to observe the ultrastructure of hippocampal neurons. Western blot assay was used to detect the expression levels of apoptosis related proteins [Caspase-3, B cell lymphoma-2 associated X protein (Bax)] and RhoA/ROCK pathway related proteins in hippocampus of rats in each group. Results Compared with the sham operation group, the number of neurons in hippocampus decreased in the model group, the ultrastructure and organelle structure of neurons were seriously damaged, the escape latency, apoptosis rate of hippocampal neurons, expression levels of apoptosis related proteins (Caspase-3, Bax), RhoA, ROCK1 and ROCK2 were significantly increased (P<0.05), and the number of platform crossings was significantly decreased (P<0.05). Compared with the model group, the number of hippocampal neurons significantly increased in the rutin treatment groups and RhoA inhibitor group, the ultrastructure of neurons and the damage of organelles recovered to a certain extent, the escape latency, apoptosis rate of hippocampal neurons, expression levels of apoptosis related proteins (Caspase-3, Bax), RhoA, ROCK1 and ROCK2 were significantly decreased (P<0.05), and the number of platform crossings was significantly increased (P<0.05). There was a dose-dependent effect in the rutin treatment groups. There were no significant differences in above indexes between the rutin high dose group and the RhoA inhibitor group (P>0.05). Conclusion Rutin can alleviate the neuronal injury induced by chronic cerebral hypoperfusion in rats, which may be achieved by inhibiting RhoA/ROCK signaling pathway.

Key words: rutin, neurons, rho-associated kinases, apoptosis, cognitive dysfunction, memory, chronic cerebral hypoperfusion

中图分类号:

R743

图/表 8

表1 各组大鼠逃避潜伏期和穿越平台次数比较（n=9，$\bar{x}\pm s$）

Tab.1 Comparison of incubation period and number of swimming across the platform of rats between the six groups

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	穿越平台次数（次）
假手术组	37.10±7.38	36.12±6.59	35.28±7.03	34.15±7.11	17.62±3.67
模型组	85.06±10.75^a	83.94±10.52^a	82.31±10.58^a	81.85±11.94^a	4.96±1.03^a
芦丁低剂量组	72.55±9.02^ab	71.83±9.87^ab	70.50±9.68^ab	69.33±10.52^ab	6.53±1.35^ab
芦丁中剂量组	62.83±8.74^abc	61.06±9.33^abc	59.22±8.36^abc	57.60±10.58^abc	9.18±2.15^abc
芦丁高剂量组	45.44±9.17^abcd	44.26±8.94^abcd	43.81±8.07^abcd	42.40±9.00^abcd	13.12±2.65^abcd
RhoA抑制剂组	47.08±8.99^abcd	46.52±7.99^abcd	45.60±9.70^abcd	44.28±8.94^abcd	14.61±3.10^abcd
F	36.835^＊＊	37.389^＊＊	35.664^＊＊	30.731^＊＊	34.785^＊＊

图1 各组大鼠海马组织神经元结构变化（HE染色，×400）

Fig.1 Changes of hippocampal neuron structure of rats in each group (HE staining, ×400)

表2 各组大鼠海马组织神经元凋亡率和凋亡相关蛋白Caspase-3、Bax表达比较（n=3，$\bar{x}\pm s$）

Tab.2 Comparison of neuronal apoptosis rate and expression levels of apoptosis-related proteins Caspase-3 and Bax in hippocampal neurons of rats between the six groups

组别	凋亡率（%）	凋亡相关蛋白
组别	凋亡率（%）	Caspase-3	Bax
假手术组	2.84±0.59	0.21±0.04	0.23±0.05
模型组	30.25±5.31^a	1.26±0.18^a	1.30±0.15^a
芦丁低剂量组	19.13±3.96^ab	0.90±0.13^ab	0.97±0.14^ab
芦丁中剂量组	11.26±2.73^abc	0.61±0.10^abc	0.66±0.12^abc
芦丁高剂量组	6.29±1.33^abcd	0.41±0.06^abcd	0.40±0.10^abcd
RhoA抑制剂组	5.87±1.24^abcd	0.39±0.07^abcd	0.43±0.10^abcd
F	35.114^＊＊	39.040^＊＊	36.955^＊＊

图2 各组大鼠海马组织神经元凋亡情况（TUNEL染色，×400）

Fig.2 Apoptosis of hippocampal neurons of rats in each group (TUNEL staining, ×400)

图3 各组大鼠海马组织神经元凋亡相关蛋白（Caspase-3、Bax）表达情况 A：假手术组；B：模型组；C：芦丁低剂量组；D：芦丁中剂量组；E：芦丁高剂量组；F：RhoA抑制剂组。

Fig.3 Expression levels of apoptosis-related proteins (Caspase-3 and Bax) in hippocampal neurons of rats in each group

图4 各组大鼠海马组织神经元超微结构观察（醋酸铀和柠檬酸铅染色，×30 000）

Fig.4 Ultrastructural observation of hippocampal neurons of rats in each group (stained with Uranium acetate and lead citrate, ×30 000)

表3 各组大鼠海马组织RhoA、ROCK1、ROCK2蛋白表达比较（n=3, $\bar{x}\pm s$）

Tab.3 Comparison of RhoA, ROCK1 and ROCK2 protein expressions in hippocampus of rats between the six groups

组别	RhoA	ROCK1	ROCK2
假手术组	0.11±0.03	0.21±0.05	0.17±0.03
模型组	1.08±0.12^a	1.28±0.14^a	1.34±0.20^a
芦丁低剂量组	0.79±0.11^ab	0.99±0.12^ab	0.95±0.15^ab
芦丁中剂量组	0.53±0.09^abc	0.71±0.09^abc	0.63±0.11^abc
芦丁高剂量组	0.32±0.06^abcd	0.41±0.07^abcd	0.33±0.06^abcd
RhoA抑制剂组	0.30±0.05^abcd	0.43±0.07^abcd	0.36±0.07^abcd
F	55.651^＊＊	53.794^＊＊	42.043^＊＊

图5 各组大鼠海马组织RhoA、ROCK1、ROCK2蛋白表达情况 A：假手术组；B：模型组；C：芦丁低剂量组；D：芦丁中剂量组；E：芦丁高剂量组；F：RhoA抑制剂组。

Fig.5 Expression levels of RhoA, ROCK1 and ROCK2 proteins in hippocampus of rats in each group

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芦丁对慢性脑低灌注大鼠海马组织神经元损伤及RhoA/ROCK信号通路的影响

Effects of rutin on hippocampal neuron injury and RhoA/ROCK signaling pathway in rats with chronic cerebral hypoperfusion

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