藏红花素对自发性高血压大鼠血管内皮功能障碍及动脉粥样硬化的作用及其ROCK/JNK信号通路机制

doi:10.13481/j.1671-587X.20220614

摘要/Abstract

摘要：

目的探讨藏红花素（CR）对自发性高血压（SH）大鼠血管内皮功能障碍（ED）和动脉粥样硬化（As）的影响，阐明其可能的作用机制。方法选取10只Wistar Kyoto大鼠作为正常组，40只SH大鼠随机分为模型组和低（25 mg·kg^－1·d^－1）、中（50 mg·kg^－1·d^－1）及高剂量（100 mg·kg^－1·d^－1）CR组，每组10只，正常组和模型组大鼠腹腔注射等量生理盐水。检测各组大鼠血压和血脂水平、动脉粥样硬化指数（AI）及24 h尿样8-异构前列腺素F2α（8-iso-PGF2α）肾脏排泄量，HE染色观察各组大鼠主动脉组织形态表现，ELISA法检测各组大鼠血清谷胱甘肽过氧化物酶（GSH-Px）活性和丙二醛（MDA）、一氧化氮（NO）、内皮素1（ET-1）及血栓素B2（TXB2）水平，实时荧光定量PCR（RT-qPCR）法检测各组大鼠主动脉组织中RhoA/Rho相关激酶1（ROCK1）、ROCK2和C-Jun氨基酸末端激酶（JNK）mRNA表达水平，Western blotting法检测各组大鼠主动脉组织中ROCK1、ROCK2、JNK和磷酸化JNK（p-JNK）蛋白表达水平。结果与正常组比较，模型组大鼠收缩压与舒张压，24 h尿8-iso-PGF2α肾脏排泄量，血清总胆固醇（TC）、甘油三酯（TG）和低密度脂蛋白胆固醇（LDL-c）水平，AI，MDA、ET-1及TXB2水平、主动脉组织中ROCK1、ROCK2和JNK mRNA表达水平及ROCK1、ROCK2、JNK和p-JNK蛋白表达水平升高（P<0.05），血清高密度脂蛋白胆固醇（HDL-c）、GSH-Px和NO水平降低（P<0.05）。模型组大鼠主动脉血管壁粗糙、增厚及大量泡沫细胞和坏死物质分布。与模型组比较，低、中和高剂量CR组大鼠收缩压与舒张压，24 h尿8-iso-PGF2α肾脏排泄量，血清TC、TG和LDL-c水平，AI，MDA、ET-1和TXB2水平，主动脉组织中ROCK1、ROCK2和JNK mRNA表达水平以及ROCK1、ROCK2、JNK和p-JNK蛋白表达水平降低（P<0.05），血清GSH-Px 活性和HDL-c及NO水平升高（P<0.05）。低剂量CR组大鼠主动脉血管壁粗糙、增厚及泡沫细胞与动脉粥样坏死物质分布可见不同程度减少。与低剂量CR组比较，中剂量CR组大鼠收缩压和舒张压，24 h尿8-iso-PGF2α肾脏排泄量，血清TC、TG和LDL-c水平，AI，MDA、ET-1及TXB2水平，主动脉组织中ROCK1、ROCK2及JNK mRNA表达水平及ROCK1、ROCK2、JNK和p-JNK蛋白表达水平降低（P<0.05），血清GSH-Px活性和HDL-c及NO水平升高（P<0.05）。中剂量CR组大鼠主动脉血管壁粗糙、增厚及泡沫细胞与动脉粥样坏死物质分布有所减少；与中剂量CR组比较，高剂量CR组大鼠收缩压和舒张压，24 h尿8-iso-PGF2α肾脏排泄量，血清TC、TG和LDL-c水平，AI，MDA、ET-1及TXB2水平，主动脉组织中ROCK1、ROCK2及JNK mRNA表达水平及ROCK1、ROCK2、JNK和p-JNK蛋白表达水平降低（P<0.05），血清GSH-Px活性和HDL-c及NO水平升高（P<0.05）。高剂量CR组大鼠主动脉血管壁动脉粥样病理形态明显改善。结论 CR可改善SH大鼠ED及As，其作用机制可能与抑制ROCK/JNK信号通路激活有关。

关键词: 藏红花素, 自发性高血压, 血管内皮功能障碍, 动脉粥样硬化, Rho蛋白激酶, C-Jun氨基酸末端激酶

Abstract:

Methods Ten Wistar Kyoto rats were selected as normal group， and 40 SH rats were randomly divided into model group and low （25 mg·kg^－1·d^－1）， medium （50 mg·kg^－1·d^－1）， and high doses of CR （100 mg·kg^－1·d^－1） groups， and there were 10 rats in each group. The rats in normal group and model group were intraperitoneally injected with the same amount of normal saline. After administration， the blood pressure， blood lipid levels，atherosclerosis index （AI）， 24 h urine 8-iso-prostaglandin F2α （8-iso-PGF2α） renal excretion of the rats in various groups were detected； HE staining was used to observe the morphology of aorta tissue of the rats in various groups；the serum activities of glutathione peroxidase （GSH-Px） and the serum levels of malondialdehyde （MDA）， nitric oxide （NO）， endothelin-1 （ET-1），and thromboxane B2 （TXB2） of the rats in various groups were detected by ELISA method；the expression levels of RhoA/ RhoA/Rho related kinase 1 （ROCK1）， ROCK2 and C-Jun amino acid terminal kinase （JNK） mRNA in aorta tissue of the rats in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR） method；Western blotting mehod was used to detect the expression levels of ROCK1， ROCK2， JNK，and phophorylated JNK（p-JNK） proteins in aorta tissue of the rats in various groups. Results Compared with normal group， the systolic blood pressure and diastolic blood pressure， 24 h urine 8-iso-PGF2α renal excretion， levels of serum total cholesterol（TC），triglyceride（TG） and low density lipoprotein cholesterol（LDL-c）， AI，levels of MDA， ET-1 and TXB2， expression levels of ROCK1， ROCK2， and JNK mRNA， expression levels of ROCK1， ROCK2， JNK， and p-JNK proteins in aorta tissue of the rats in model group were increased （P<0.05），the serum acitivity of GSH-Px and serum levels of high density lipoprotein cholesterol（HDL-c） and NO were decreased （P<0.05）. In model group， the aortic wall was rough and thickened， and a large number of foam cells and atherosclerotic necrosis substances were distributed. Compared with model group， the systolic blood pressure and diastolic blood pressure，24 h urine 8-iso-PGF2α renal excretion， serum levels of TC， TG，and LDL-c， AI， levels of MDA， ET-1，and TXB2， expression levels of ROCK1， ROCK2 and JNK mRNA，expression levels of ROCK1， ROCK2， JNK， and p-JNK proteins in aorta tissue of the rats in low， medium， and high doses of CR groups were decreased （P<0.05）， and the serum activity of GSH-Px and serum levels of HDL-c and NO were increased （P<0.05）. In low dose of CR group， the roughness and thickening of the aortic wall and the distribution of the foam cells and atherosclerotic necrosis substances in the aorta tissue were decreased to varying degrees.Compared with low dose of CR group， the systolic blood pressure and diastolic blood pressure，24 h urine 8-iso-PGF2α renal excretion， serum levels of TC， TG， and LDL-c， AI， levels of MDA， ET-1， and TXB2，expression levels of ROCK1， ROCK2 and JNK mRNA in aora tissue， expression levels of ROCK1， ROCK2， JNK， and p-JNK proteins in aorta tissue of the rats in medium dose of CR group were decreased （P<0.05）， while the serum activity of GSH-Px and serum levels of HDL-c and NO were increased （P<0.05）. In medium dose of CR group， the roughness and thickening of the aortic wall and the distribution of the foam cells and atherosclerotic necrosis substances in the aorta tissue were reduced. Compared with medium dose of CR group，the systolic blood pressure and diastolic blood pressure， 24 h urine 8-iso-PGF2α renal excretion， serum levels of TC， TG， and LDL-c， AI， levels of MDA， ET-1， and TXB2， expression levels of ROCK1， ROCK2，JNK mRNA in aorta tissue， expression levels of ROCK1， ROCK2， JNK， and p-JNK proteins in aorta tissue of the rats in high dose of CR group were decreased （P<0.05）， while the serum activity of GSH-Px and serum levels of HDL-c and NO were increased （P<0.05）. The atherosclerotic pathomorphology of aorta wall of the rats in high dose of CR group was significantly improved. Conclusion CR can improve ED and As in the SH rats， and its mechanism may be related to inhibiting the activation of ROCK/JNK signaling pathway. Objective To investigate the effect of crocin （CR） on vascular endothelial dysfunction （ED） and atherosclerosis （As） in the rats with spontaneously hypertension （SH），and to clarify its possible mechanism.

Key words: Crocin, Spontaneous hypertension, Vascular endothelial dysfunction, Atherosclerosis, Rho protein kinase, C-jun amino acid terminal kinase

中图分类号:

R544.1

余淑华,刘朏,吴倩倩,杨东伟. 藏红花素对自发性高血压大鼠血管内皮功能障碍及动脉粥样硬化的作用及其ROCK/JNK信号通路机制[J]. 吉林大学学报(医学版), 2022, 48(6): 1481-1489.

Shuhua YU,Wei LIU,Qianqian WU,Dongwei YANG. Effect of crocetin on vascular endothelial dysfunction and atherosclerosis in spontaneously hypertensive rats and its ROCK/JNK signaling pathway mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(6): 1481-1489.

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Group	Systolic blood pressure	Diastolic blood pressure
Normal	113.35±4.26	72.27±4.25
Model	187.68±12.03^*	116.91±5.27^*
CR Low dose	168.76±10.03^△	108.17±4.63^△
Medium dose	137.93±8.11^△＃	97.25±5.91^△＃
High dose	124.22±6.74^△＃○	82.78±4.02^△＃○

Group	TC[c_B/（mmol·L^－1)]	TG[c_B/（mmol·L^－1)]	LDL-c[c_B/（mmol·L^－1)]	HDL-c[c_B/（mmol·L^－1)]	AI
Normal	1.08±0.11	0.62±0.06	0.76±0.05	0.94±0.08	0.13±0.03
Model	2.25±0.16^*	1.48±0.09^*	2.33±0.15^*	0.44±0.05^*	4.08±0.57^*
CR Low dose	2.07±0.12^△	1.36±0.06^△	2.18±0.08^△	0.52±0.03^△	2.87±0.39^△
Medium dose	1.78±0.10^△＃	1.14±0.06^△＃	1.77±0.09^△＃	0.68±0.04^△＃	1.59±0.18^△＃
High dose	1.42±0.09^△＃○	0.85±0.06^△＃○	1.32±0.10^△＃○	0.87±0.04^△＃○	0.62±0.09^△＃○

Group	MDA[c_B/(nmol·L^－1）]	GSH-Px[c_B/(μmol·L^－1）]	NO[c_B/(μmol·L^－1）]	ET-1 [ρ_B/(ng·L^－1）]	TXB2[ρ_B/(ng·L^－1）]
Normal	8.12±0.21	109.52±9.68	22.10±2.18	65.85±4.31	32.61±3.10
Model	15.27±1.08^*	58.24±3.68^*	13.58±0.72^*	114.78±5.95^*	58.99±4.60^*
CR Low dose	14.12±0.62^△	65.09±4.03^△	14.54±1.10^△	103.70±5.64^△	53.33±3.77^△
Medium dose	11.86±0.84^△＃	78.39±6.20^△＃	16.66±1.14^△＃	87.86±6.96^△＃	46.06±3.69^△＃
High dose	9.52±0.81^△＃○	98.67±7.51^△＃○	19.96±0.94^△＃○	72.46±4.69^△＃○	35.86±3.24^△＃○

Group	ROCK1	ROCK2	JNK
Normal	1.00±0.01	1.01±0.01	1.02±0.01
Model	5.87±0.54^*	4.88±0.59^*	4.72±0.54^*
CR Low dose	5.26±0.32^＃	3.95±0.24^＃	4.11±0.26^＃
Medium dose	3.94±0.47^△＃	2.89±0.21^△＃	2.77±0.15^△＃
High dose	1.96±0.18^△＃○	1.71±0.34^△＃○	1.62±0.26^△＃○

Group	ROCK1	ROCK2	JNK	p-JNK
Normal	0.09±0.02	0.18±0.03	0.38±0.03	0.16±0.02
Model	0.64±0.05^*	0.65±0.05^*	0.92±0.04^*	0.53±0.05^*
CR Low dose	0.54±0.05^＃	0.57±0.03^＃	0.86±0.04^＃	0.42±0.05^＃
Medium dose	0.38±0.05^△＃	0.41±0.02^△＃	0.79±0.04^△＃	0.37±0.03^△＃
High dose	0.19±0.03^△＃○	0.24±0.04^△＃○	0.58±0.04^△＃○	0.31±0.03^△＃○