Rho/ROCK信号通路在阻塞性睡眠呼吸暂停低通气综合征大鼠急性缺血性卒中发生中的作用

doi:10.13481/j.1671-587X.20210514

吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (5): 1178-1186.doi: 10.13481/j.1671-587X.20210514

Rho/ROCK信号通路在阻塞性睡眠呼吸暂停低通气综合征大鼠急性缺血性卒中发生中的作用

嵇朋¹,郭向东²,孙根¹,寇启星¹,屈雪萍¹,孙雅静¹,江利敏³()

^1.河南省郑州市第三人民医院河南大学肿瘤医院神经内科，河南郑州 450000
^2.河南中医药大学第一附属医院耳鼻喉科，河南郑州 450000
^3.河南中医药大学第一附属医院体检中心，河南郑州 450000

收稿日期:2020-12-22 出版日期:2021-09-28 发布日期:2021-10-26
通讯作者: 江利敏 E-mail:zhandechao5563@163.com
作者简介:嵇朋（1978－），男，河南省三门峡市人，副主任医师，医学硕士，在读博士研究生，主要从事脑血管病基础和临床方面的研究。
基金资助:
河南省科技厅科技攻关项目(182102310172);河南省医学科技攻关计划联合共建项目(2018020745)

Role of Rho/ROCK signaling pathway in occurrence of acute ischemic stroke in rats with obstructive sleep apnea hypopnea syndrome

Peng JI¹,Xiangdong GUO²,Gen SUN¹,Qixing KOU¹,Xueping QU¹,Yajing SUN¹,Limin JIANG³()

^1.Department of Neurology，Third People’s Hospital of Zhengzhou City，Henan Provine，Cancer Hospital，Henan University，Zhengzhou 450000，Henan，China
^2.Department of Otolaryngology，First Affiliated Hospital，Henan University of Traditional Chinese Medicine，Zhengzhou 450000，China
^3.Physical Examination Center，First Affiliated Hospital，Henan University of Traditional Chinese Medicine 450000，Zhengzhou 450000，China

Received:2020-12-22 Online:2021-09-28 Published:2021-10-26
Contact: Limin JIANG E-mail:zhandechao5563@163.com

摘要/Abstract

摘要： 目的

研究小GTP结合蛋白（Rho）/Rho相关卷曲螺旋形成蛋白激酶（ROCK）信号通路在阻塞性睡眠呼吸暂停低通气综合征（OSAHS）大鼠急性缺血性卒中发生中的作用，为OSAHS后急性缺血性卒中病变的防治提供参考。

方法

50只SD大鼠随机分为假手术组、OSAHS组、卒中组、OSAHS后卒中组和Rho抑制剂组（C3转移酶5 μg·kg^－1·d^－1腹腔注射），每组10只。采用慢性间歇性低氧建立OSAHS模型，线栓法阻断大脑中动脉建立急性脑卒中模型。观察各组大鼠建模后的一般情况和脑皮质区组织病理形态表现，计算脑梗死体积。比较各组大鼠呼吸频率和神经功能损伤评分，采用实时荧光定量PCR（RT-qPCR）法和Western blotting法检测各组大鼠脑组织中肌球蛋白轻链（MLC）、Rho和ROCK mRNA及蛋白表达水平。

结果

与假手术组比较，其他各组大鼠均有精神状态不佳和脑皮层结构损伤等改变，其中OSAHS后卒中组大鼠变化最严重，Rho抑制剂组变化较轻。与假手术组比较，OSAHS组大鼠呼吸频率增加（P<0.05）；与OSAHS组比较，卒中组大鼠呼吸频率降低（P<0.05）；与卒中组比较，OSAHS后卒中组大鼠呼吸频率和Longa评分增加（P<0.05），Rho抑制剂组大鼠Longa评分降低（P<0.05）；与OSAHS后卒中组比较，Rho抑制剂组大鼠呼吸频率与和Longa评分均降低（P<0.05）。与假手术组比较，OSAHS组大鼠逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05）；与OSAHS组比较，卒中组大鼠逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05）；与卒中组比较，OSAHS后卒中组大鼠逃避潜伏期延长（P<0.05），穿越平台次数减少（P<0.05）；与OSAHS后卒中组比较，Rho抑制剂组大鼠逃避潜伏期缩短（P<0.05），穿越平台次数增加（P<0.05）。与卒中组比较，OSAHS后卒中组大鼠脑梗死体积增大（P<0.05）；与OSAHS后卒中组比较，Rho抑制剂组大鼠脑梗死体积缩小（P<0.05）。与假手术组比较，OSAHS组大鼠脑组织中Rho和ROCK mRNA和蛋白表达水平及p-MLC/MLC比值升高（P<0.05）；与OSAHS组比较，卒中组大鼠脑组织中Rho和ROCK mRNA及蛋白表达水平及其p-MLC/MLC比值升高（P<0.05）；与卒中组比较，OSAHS后卒中组大鼠脑组织中Rho和ROCK mRNA及蛋白相表达水平及其p-MLC/MLC比值升高（P<0.05）；与OSAHS后卒中组比较，Rho抑制剂组大鼠脑组织中Rho和ROCK mRNA及蛋白表达水平及其p-MLC/MLC比值降低（P<0.05）。

结论

OSAHS的间歇性缺氧状态可损伤神经功能，Rho/ROCK信号通路可能在OSAHS大鼠急性缺血性卒中发生中起一定作用。

关键词: 阻塞性睡眠呼吸暂停低通气综合征, 急性缺血性卒中, 小GTP结合蛋白, Rho相关卷曲螺旋形成蛋白激酶, 肌球蛋白轻链

Abstract: Objective

To study the role of small GTP binding protein （Rho）/Rho-related coiled-coil forming protein kinase （ROCK） signaling pathway in the occurrence of acute ischemic stroke in the rats with obstructive sleep apnea hypopnea syndrome （OSAHS），and to provide reference for the prevention and treatment of acute ischemic stroke after OSAHS.

Methods

Fifty SD rats were randomly divided into sham operation group， OSAHS group， stroke group， post-OSAHS stroke group， and Rho inhibitor group （C3 transferase 5 μg·kg^－1·d^－1 intraperitoneal injection）， with 10 rats in each group. The OSAHS model was established by chronic intermittent hypoxia， and the middle cerebral artery was blocked by the suture method to establish an acute ischemic stroke model. The general situation and pathomorphology of cerebral cortex tissue of the rats after modeling were observed and the volumes of cerebral infarction were calculated. The respiratory frequencies and the neurological impairment scores of the rats in various groups were compared. The expression levels of myosin light chain （MLC）， Rho and ROCK in brain tissue of the rats in various groups were detected by Real-time fluorescence quantitative PCR （RT-qPCR）and Western blotting methods.

Results

Compared with sham operation group， the rats in other groups had changes such as poor mental state and cerebral cortex structure damage， among them， the rats in post-OSAHS stroke group changed the most and the rats in Rho inhibitor group changed slightly. Compared with sham operation group，the respiratory frequency of rats in OSAHS group was increased （P<0.05）；compared with OSAHS group， the respiratory frequency of rats in stroke group was decreased （P<0.05）； compared with stroke group， the respiratory rate and Longa score of rats in post-OSAHS stroke group were increased（P<0.05）， and the Longa score of rats in Rho inhibitor group were decreased （P<0.05）； compared with post-OSAHS stroke group， the respiratory frequency and Longa score of rats in Rho inhibitor group were decreased （P<0.05）. Compared with sham operation group， the escape latency of rats in OSAHS group was prolonged（P<0.05）， and the number of crossing platform was decreased （P<0.05）； compared with OSAHS group， the escape latency of rats in stroke group was prolonged（P<0.05） and the number of crossing platform was decreased （P<0.05）； compared with stroke group， the escape latency of rats in post-OSAHS stroke group was prolonged（P<0.05）， and the number of crossing platform was decreased（P<0.05）；compared with post-OSAHS stroke group， the escape latency of rats in Rho inhibitor group was shortened（P<0.05），and the times of crossing platform was decreased（P<0.05）. Compared with stroke group， the cerebral infarction volume of rats in post-OSAHS stroke group was increased（P<0.05）； compared with post-OSAHS stroke group，the cerebral infarction volume of rats in Rho inhibitor group was decreased（P<0.05）. Compared with sham operation group， the expression levels of Rho， rock mRNA and proteins and the P-MLC / MLC ratio in brain tissue of the rats in OSAHS group were increased （P<0.05）；compared with OSAHS group， the expression levels of Rho and ROCK mRNA and proteins and the p-MLC/MLC ratio in brain tissue of the rats in stroke group were increased （P<0.05）； compared with stroke group， the expression levels of Rho and ROCK mRNA and proteins in brain tissue of the rats in post-OSAHS stroke group and the p-MLC/MLC ratio were increased （P<0.05）；compared with post-OSAHS stroke group， the expression levels of Rho and ROCK mRNA and proteins in brain tissue of the rats in Rho inhibitor group and the p-MLC/MLC ratio were decreased （P<0.05）.

Conclusion

The intermittent hypoxia of OSAHS can damage the nerve function， and Rho/ROCK signaling pathway may play a role in the occurrence of acute ischemic stroke in the OSAHS rats.

Key words: obstructive sleep apnea hypopnea syndrome, acute ischemic stroke, small GTP binding protein, Rho associated coiled-coil forming protein kinase, myosin light chain

中图分类号:

R543.5

嵇朋,郭向东,孙根,寇启星,屈雪萍,孙雅静,江利敏. Rho/ROCK信号通路在阻塞性睡眠呼吸暂停低通气综合征大鼠急性缺血性卒中发生中的作用[J]. 吉林大学学报(医学版), 2021, 47(5): 1178-1186.

Peng JI,Xiangdong GUO,Gen SUN,Qixing KOU,Xueping QU,Yajing SUN,Limin JIANG. Role of Rho/ROCK signaling pathway in occurrence of acute ischemic stroke in rats with obstructive sleep apnea hypopnea syndrome[J]. Journal of Jilin University(Medicine Edition), 2021, 47(5): 1178-1186.

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Gene	Primer sequences
Rho	F:5′-AGTAGCCACGCGTGATTACGTTGC-3′
Rho	R:5′-TTAGCCTGAAGCTGATGTACGTGA-3′
ROCK	F:5′-TAGTCCTGTAGTATGAGTGTC-3′
ROCK	R:5′-CTGTAGAGGAGATTCTCGTGA-3′
MLC	F:5′-ATGACGATGTATGGCTCTG-3′
MLC	R:5′-TGTGCTATGGCATGTGACT-3′
β-actin	F:5′-TGTACTGAGCGTGTTATGTTGTA-3′
β-actin	R:5′-GAGCTGATGTGACTCGTGATCGG-3′

Group	n	Respiratory frequency （times·min^－1）	Longa score（points）
Sham operation	9	89.25±9.22	—
OSAHS	10	133.89±11.08^*	—
Stroke	9	92.32±8.50^△	1.03±0.25
Post-OSAHS stroke	8	134.50±10.74^*#	1.33±0.30^#
Rho inhibitor	9	105.32±9.25^*△#○	0.85±0.23^#○

Group	n	Escape latency（t/s）	Number of crossing platforms（times）
Sham operation	9	25.33±3.02	6.83±1.50
OSAHS	10	31.36±3.35^*	5.21±1.03^*
Stroke	9	38.89±4.50^*△	3.05±0.70^*△
Post-OSAHS stroke	8	48.48±5.12^*△#	2.08±0.52^*△#
Rho inhibitor	9	32.41±3.50^*#○	4.89±1.05^*#○

Group	Rho	ROCK	MLC
Sham operation	0.44±0.05	0.65±0.06	0.91±0.08
OSAHS	0.55±0.08^*	0.80±0.08^*	0.88±0.10
Stroke	0.82±0.10^*△	0.95±0.10^*△	0.92±0.10
Post-OSAHS stroke	1.08±0.12^*△#	1.22±0.14^*△#	0.95±0.12
Rho inhibitor	0.25±0.05^*△#○	0.32±0.05^*△#○	0.90±0.08

Group	Rho	ROCK	p-MLC/MLC
Sham operation	0.15±0.03	0.05±0.01	0.12±0.03
OSAHS	0.22±0.04^*	0.12±0.03^*	0.30±0.05^*
Stroke	0.35±0.05^*△	0.23±0.05^*△	0.45±0.05^*△
Post-OSAHS stroke	0.45±0.05^*△#	0.50±0.06^*△#	0.70±0.07^*△#
Rho inhibitor	0.09±0.03^*△#○	0.03±0.01^*△#○	0.06±0.02^*△#○

Rho/ROCK信号通路在阻塞性睡眠呼吸暂停低通气综合征大鼠急性缺血性卒中发生中的作用

Role of Rho/ROCK signaling pathway in occurrence of acute ischemic stroke in rats with obstructive sleep apnea hypopnea syndrome

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