沉默Salusin-β对糖尿病大鼠内皮功能障碍的影响及机制研究

doi:10.11958/20221272

天津医药 ›› 2023, Vol. 51 ›› Issue (5): 491-497.doi: 10.11958/20221272

沉默Salusin-β对糖尿病大鼠内皮功能障碍的影响及机制研究

左宪宏¹(), 张婷婷¹^,^△(), 李月琴², 赵佳琪²

1.张家口学院（邮编075000）
2.河北北方学院附属第一医院内分泌科

收稿日期:2022-08-15 修回日期:2022-11-01 出版日期:2023-05-15 发布日期:2023-05-05
通讯作者: △E-mail:147257761@qq.com
作者简介:左宪宏（1983），女，讲师，主要从事病理方面研究。E-mail：zuoxianhong384@163.com
基金资助:
河北省医学科学研究课题计划项目(20201432)

Effect and mechanism of silencing Salusin-β on endothelial dysfunction in diabetes rats

ZUO Xianhong¹(), ZHANG Tingting¹^,^△(), LI Yueqin², ZHAO Jiaqi²

1. Zhangjiakou University, Zhangjiakou 075000, China
2. Department of Endocrinology, the First Hospital Affiliated to Hebei North University

Received:2022-08-15 Revised:2022-11-01 Published:2023-05-15 Online:2023-05-05
Contact: △E-mail:147257761@qq.com

左宪宏, 张婷婷, 李月琴, 赵佳琪. 沉默Salusin-β对糖尿病大鼠内皮功能障碍的影响及机制研究[J]. 天津医药, 2023, 51(5): 491-497.

ZUO Xianhong, ZHANG Tingting, LI Yueqin, ZHAO Jiaqi. Effect and mechanism of silencing Salusin-β on endothelial dysfunction in diabetes rats[J]. Tianjin Medical Journal, 2023, 51(5): 491-497.

摘要/Abstract

摘要：

目的分析短发夹RNA（shRNA）沉默心血管调节肽（Salusin-β）对糖尿病（DM）大鼠内皮功能障碍的影响及其潜在的机制。方法 52只SD大鼠随机抽取12只为正常对照组（NC组），其余大鼠采用喂食高糖高脂饲料和腹腔注射链脲佐菌素的方法制备DM模型。将36只建模成功的大鼠分为DM组、Ad-Scr shRNA组和Ad-Salusin-β shRNA组，每组12只。Ad-Scr shRNA组大鼠尾静脉注射腺病毒空载体（Ad-scramble shRNA），Ad-Salusin-β shRNA组大鼠尾静脉注射编码Salusin-β shRNA的腺病毒载体（Ad-Salusin-β shRNA）。每2周注射1次，NC组和DM组注射等量生理盐水。4周后，检测大鼠空腹血糖（FBG）和血清Salusin-β水平以及胸主动脉血管舒张功能；酶联免疫吸附试验检测大鼠胸主动脉肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β、丙二醛（MDA）、超氧化物歧化酶（SOD）水平；二氢乙锭（DHE）染色检测胸主动脉中活性氧（ROS）水平；苏木精-伊红染色观察大鼠胸主动脉组织病理学变化；荧光定量PCR检测大鼠胸主动脉Salusin-β和还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（NOX2）mRNA表达水平；Western blot法检测大鼠胸主动脉NOX2、核因子κB p65（NF-κB p65）蛋白表达。结果与NC组相比，DM组大鼠FBG、血清Salusin-β水平升高，胸主动脉TNF-α、IL-6、IL-1β、MDA、ROS水平升高，胸主动脉内膜-中膜厚度增厚，Salusin-β mRNA、NOX2 mRNA和蛋白、细胞核NF-κB p65蛋白水平升高，Ach诱发的内皮依赖性血管舒张功能下降，胸主动脉SOD和细胞质NF-κB p65蛋白表达水平降低（P<0.05）。与DM组和Ad-Scr shRNA组相比，Ad-Salusin-β shRNA组大鼠FBG和血清Salusin-β表达下降，胸主动脉TNF-α、IL-6、IL-1β、MDA、ROS表达下降，胸主动脉内膜-中膜厚度变薄，Salusin-β mRNA、NOX2 mRNA和蛋白、细胞核NF-κB p65蛋白表达水平降低，Ach诱发的内皮依赖性血管舒张功能升高，胸主动脉SOD和细胞质NF-κB p65蛋白表达水平升高（P<0.05）。结论 shRNA沉默Salusin-β可减轻DM大鼠内皮功能障碍，其作用机制可能与抑制NOX2/ROS/NF-κB信号通路活化有关。

关键词: 糖尿病,2型, 疾病模型,动物, 心血管调节肽, 内皮功能障碍, 还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2, 活性氧, 核因子κB

Abstract:

Objective To analyze the effect of short hairpin RNA (shRNA) silencing Salusin-β on endothelial dysfunction in diabetes mellitus (DM) rats and its potential mechanism. Methods Twelve of 52 SD rats were randomly selected as the normal control group (NC group). The rest of rats were fed high sugar and high fat diet and intraperitoneally injected streptozotocin 60 mg/kg to prepare DM model. Thirty-six rats were successfully modeled and were divided into the DM group, the Ad-Scr shRNA group and the Ad-Salusin-β shRNA group, with 12 rats in each group. Adenovirus empty vector encoding scramble shRNA (Ad-scramble shRNA) was injected into the tail vein of rats in the Ad-Scr shRNA group, and adenoviral vector encoding Salusin-β shRNA (Ad-Salusin-β shRNA) was injected into the tail vein of rats in the Ad-Salusin-β shRNA group. The drug was administered once every 2 weeks. The NC group and the DM group were injected with the same volume of normal saline. After 4 weeks, fasting blood glucose (FBG) and serum Salusin-β levels and the vasodilation function of thoracic aorta were measured. Enzyme linked immunosorbent assay was used to detect levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, malondialdehyde (MDA) and superoxide dismutase (SOD) in thoracic aorta. Dihydroethidium (DHE) staining was used to detect the level of reactive oxygen species (ROS) in the thoracic aorta. Hematoxylin-eosin staining was used to observe histopathological changes of thoracic aorta. Fluorescence quantitative PCR was used to detect mRNA levels of Salusin-β and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) in thoracic aorta. Western blot assay was used to detect expression levels of NOX2 and nuclear factor-κB p65 (NF-κB p65) protein in thoracic aorta. Results Compared with the NC group, the FBG and serum Salusin-β were increased, TNF-α, IL-6, IL-1β, MDA and ROS in thoracic aorta were also increased, and intima-media thickness of the thoracic aorta was thickened in the DM group. Salusin-β mRNA, NOX2 mRNA and protein, nuclear NF-κB p65 protein levels were increased in the DM group. Ach induced endothelium dependent vasodilation, thoracic aorta SOD, cytoplasmic NF-κB p65 protein levels were decreased (P<0.05). Compared with the DM group and the Ad-Scr shRNA group, FBG and serum Salusin-β decreased, thoracic aorta TNF-α, IL-6, IL-1β, MDA and ROS decreased, intima-media thickness of thoracic aorta became thinner in the Ad-Salusin-β shRNA group. Salusin-β mRNA, NOX2 mRNA and protein, nuclear NF-κB p65 protein levels were decreased in the Ad-Salusin-β shRNA group, and Ach induced endothelium dependent vasodilation, thoracic aorta SOD, cytoplasmic NF-κB p65 protein levels were increased (P<0.05). Conclusion shRNA silencing Salusin-β can reduce endothelial dysfunction in DM rats, and its mechanism may be related to the inhibition of the activation of NOX2/ROS/NF-κB signaling pathway.

Key words: diabetes, type 2, disease model, animal, cardiovascular regulatory peptide, endothelial dysfunction, reduced nicotinamide adenine dinucleotide phosphate oxidase 2, active oxygen, nuclear factor κB

中图分类号:

R587.1

图/表 11

参考文献 21

[1]	SUN D, WANG J, TOAN S, et al. Molecular mechanisms of coronary microvascular endothelial dysfunction in diabetes mellitus: focus on mitochondrial quality surveillance[J]. Angiogenesis, 2022, 25(3):307-329. doi:10.1007/s10456-022-09835-8.
[2]	MARUHASHI T, HIGASHI Y. Pathophysiological association between diabetes mellitus and endothelial dysfunction[J]. Antioxidants (Basel), 2021, 10(8):1306-1318. doi:10.3390/antiox10081306.
[3]	LUO E F, LI H X, QIN Y H, et al. Role of ferroptosis in the process of diabetes-induced endothelial dysfunction[J]. World J Diabetes, 2021, 12(2):124-137. doi:10.4239/wjd.v12.i2.124.
[4]	YASSIEN M, FAWZY O, MAHMOUD E, et al. Serum salusin-β in relation to atherosclerosis and ventricular dysfunction in patients with type 2 diabetes mellitus[J]. Diabetes Metab Syndr, 2020, 14(6):2057-2062. doi:10.1016/j.dsx.2020.10.025.
[5]	ZHU X, ZHOU Y, CAI W, et al. Salusin-β mediates high glucose-induced endothelial injury via disruption of AMPK signaling pathway[J]. Biochem Biophys Res Commun, 2017, 491(2):515-521. doi:10.1016/j.bbrc.2017.06.126.
[6]	ZHAO M X, ZHOU B, LING L, et al. Salusin-β contributes to oxidative stress and inflammation in diabetic cardiomyopathy[J]. Cell Death Dis, 2017, 8(3):2690-2699. doi:10.1038/cddis.2017.106.
[7]	SUN H J, CHEN D, WANG P Y, et al. Salusin-β is involved in diabetes mellitus-induced endothelial dysfunction via degradation of peroxisome proliferator-activated receptor gamma[J]. Oxid Med Cell Longev, 2017, 2017:6905217. doi:10.1155/2017/6905217.
[8]	张书娅, 邵钟铭, 伍彩霞, 等. Rnd3表达改变对糖尿病大鼠内皮祖细胞生物学特性的影响[J]. 中国病理生理杂志, 2021, 37(5):834-840.
	ZHANG S Y, SHAO Z M, WU C X, et al. Effects of dysregulation of Rnd3 on general biological characteristics of endothelial progenitor cells in diabetic rats[J]. Chinese Journal of Pathophysiology, 2021, 37(5):834-840. doi:10.3969/j.issn.1000-4718.2021.05.008.
[9]	CAI Z, YUAN S, ZHONG Y, et al. Amelioration of endothelial dysfunction in diabetes:role of takeda G protein-coupled receptor 5[J]. Front Pharmacol, 2021, 12(1):637051-637060. doi:10.3389/fphar.2021.637051.
[10]	武兵兵, 马礼科, 刘秀珠, 等. 中药通过AMPK途径对糖尿病致内皮功能障碍的保护作用及机制[J]. 中药药理与临床, 2022, 38(3):225-230.
	WU B B, MA L K, LIU X Z, et al. Protective effect and mechanism of chinese medicine on endothelial cell dysfunction induced by diabetes mellitus through AMPK pathway[J]. Pharmacology and Clinics of Chinese Materia Medica, 2022, 38(3):225-230. doi:10.13412/j.cnki.zyyl.2022.03.005.
[11]	SUN H, ZHANG F, XU Y, et al. Salusin-β promotes vascular calcification via nicotinamide adenine dinucleotide phosphate/reactive oxygen species-mediated Klotho downregulation[J]. Antioxid Redox Signal, 2019, 31(18):1352-1370. doi:10.1089/ars.2019.7723.
[12]	LU Q B, DU Q, WANG H P, et al. Salusin-β mediates tubular cell apoptosis in acute kidney injury:Involvement of the PKC/ROS signaling pathway[J]. Redox Biol, 2020, 30(1):101411-101427. doi:10.1016/j.redox.2019.101411.
[13]	SUN S, ZHANG F, PAN Y, et al. A TOR2A gene product:Salusin-β contributes to attenuated vasodilatation of spontaneously hypertensive rats[J]. Cardiovasc Drugs Ther, 2021, 35(1):125-139. doi:10.1007/s10557-020-06983-1.
[14]	ARKAN A, ATUKEREN P, IKITIMUR B, et al. The importance of circulating levels of salusin-α,salusin-β,and heregulin-β1 in atherosclerotic coronary arterial disease[J]. Clin Biochem, 2021, 87(1):19-25. doi:10.1016/j.clinbiochem.2020.10.003.
[15]	WANG W J, JIANG X, GAO C C, et al. Salusin-β participates in high glucose-induced HK-2 cell ferroptosis in a Nrf-2-dependent manner[J]. Mol Med Rep, 2021, 24(3):674-685. doi:10.3892/mmr.2021.12313.
[16]	WANG T, ZHU H, HOU Y, et al. Ketamine attenuates high-glucose-mediated endothelial inflammation in human umbilical vein endothelial cells[J]. Can J Physiol Pharmacol, 2020, 98(3):156-161. doi:10.1139/cjpp-2019-0185.
[17]	VENU V K P, SAIFEDDINE M, MIHARA K, et al. Metformin prevents hyperglycemia-associated,oxidative stress-induced vascular endothelial dysfunction:essential role for the orphan nuclear receptor human nuclear receptor 4A1(Nur77)[J]. Mol Pharmacol, 2021, 100(5):428-455. doi:10.1124/molpharm.120.000148.
[18]	SUN H J, ZHAO M X, REN X S, et al. Salusin-β promotes vascular smooth muscle cell migration and intimal hyperplasia after vascular injury via ROS/NFκB/MMP-9 pathway[J]. Antioxid Redox Signal, 2016, 24(18):1045-1057. doi:10.1089/ars.2015.6475.
[19]	阳创, 薛莱, 吴阳, 等. NF-κB-iNOS/COX-2信号通路在高糖损伤血管内皮依赖性舒张中的作用[J]. 中国病理生理杂志, 2020, 36(12):2159-2165.
	YANG C, XUE L, WU Y, et al. Activation of NF-κB-iNOS/COX-2 signaling pathways is involved in impaired endothelium-dependent relaxation under high glucose condition[J]. Chinese Journal of Pathophysiology, 2020, 36(12):2159-2165. doi:10.3969/j.issn.1000-4718.2020.12.006.
[20]	CHEN H, JIN G. Downregulation of Salusin-β protects renal tubular epithelial cells against high glucose-induced inflammation, oxidative stress, apoptosis and lipid accumulation via suppressing miR-155-5p[J]. Bioengineered, 2021, 12(1):6155-6165. doi:10.1080/21655979.2021.1972900.
[21]	GAO J, LIANG Z, ZHAO F, et al. Triptolide inhibits oxidative stress and inflammation via the microRNA-155-5p/brain-derived neurotrophic factor to reduce podocyte injury in mice with diabetic nephropathy[J]. Bioengineered, 2022, 13(5):12275-12288. doi:10.1080/21655979.2022.2067293.

基因名称	引物序列（5′→3′）	产物大小（bp）
Salusin-β	上游：TCACTTCTCTCCTATCATCCACTCC	238
	下游：GGCAGCTTGTCCATCTCATCG	238
NOX2	上游：TTTCCGATCCTATCAAAGTGCC	216
	下游：GTACACGTGCGTGTGTCTGTTC	216
GAPDH	上游：GTGGAGTCTACTGGCGTCTT	195
	下游：TGCTGACAATCTTGAGGGA	195

基因名称	引物序列（5′→3′）	产物大小（bp）
Salusin-β	上游：TCACTTCTCTCCTATCATCCACTCC	238
	下游：GGCAGCTTGTCCATCTCATCG	238
NOX2	上游：TTTCCGATCCTATCAAAGTGCC	216
	下游：GTACACGTGCGTGTGTCTGTTC	216
GAPDH	上游：GTGGAGTCTACTGGCGTCTT	195
	下游：TGCTGACAATCTTGAGGGA	195

组别	FBG（mmol/L）	Salusin-β（μg/L）
NC组	5.74±1.08	0.62±0.11
DM组	26.03±3.15^a	1.35±0.20^a
Ad-Scr shRNA组	27.85±3.24^a	1.44±0.18^a
Ad-Salusin-β shRNA组	15.29±2.36^abc	0.73±0.12^bc
F	186.794^＊＊	85.501^＊＊

组别	FBG（mmol/L）	Salusin-β（μg/L）
NC组	5.74±1.08	0.62±0.11
DM组	26.03±3.15^a	1.35±0.20^a
Ad-Scr shRNA组	27.85±3.24^a	1.44±0.18^a
Ad-Salusin-β shRNA组	15.29±2.36^abc	0.73±0.12^bc
F	186.794^＊＊	85.501^＊＊

组别	Ach浓度
组别	1×10^-8 mol/L	1×10^-7 mol/L	1×10^-6 mol/L	1×10^-5 mol/L	1×10^-4 mol/L
NC组	5.15±0.60	24.38±2.59	61.42±7.30	85.09±9.62	90.41±9.75
DM组	5.09±0.55	12.57±2.06^a	40.29±5.42^a	53.18±7.15^a	57.62±8.31^a
Ad-Scr shRNA组	5.11±0.57	11.48±2.15	37.68±6.04	51.36±6.90	54.53±7.42
Ad-Salusin-β shRNA组	5.14±0.52	19.25±2.32^bc	51.33±7.15^bc	68.45±8.74^bc	73.86±9.50^bc
F	0.029	83.675^＊＊	33.542^＊＊	44.256^＊＊	42.445^＊＊

沉默Salusin-β对糖尿病大鼠内皮功能障碍的影响及机制研究

Effect and mechanism of silencing Salusin-β on endothelial dysfunction in diabetes rats

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 11

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

组别	SNP浓度
组别	1×10^-9 mol/L	1×10^-8 mol/L	1×10^-7 mol/L	1×10^-6 mol/L	1×10^-5 mol/L
NC组	10.20±1.14	24.16±2.74	64.48±8.20	87.50±10.31	94.25±12.18
DM组	10.15±1.29	21.82±2.53	59.31±7.45	82.36±9.24	89.82±10.56
Ad-Scr shRNA组	9.87±1.05	22.30±2.46	60.27±8.16	84.02±11.37	91.40±10.29
Ad-Salusin-β shRNA组	10.13±1.21	24.11±2.80	62.54±7.93	85.75±9.68	93.37±11.44
F	0.190	2.545	1.027	0.568	0.383

组别	TNF-α（ng/g）	IL-6（ng/g）	IL-1β（ng/g）	MDA（μmol/g）	SOD（kU/g）
NC组	82.45±11.03	34.76±5.62	15.22±2.41	3.24±0.50	54.06±6.39
DM组	275.26±32.58^a	156.35±20.13^a	61.68±7.53^a	9.57±0.76^a	21.83±3.65^a
Ad-Scr shRNA组	280.31±35.27	164.49±21.58	63.19±6.82	9.63±0.81	22.12±4.08
Ad-Salusin-β shRNA组	143.19±24.65^bc	82.51±14.07^bc	34.04±5.39^bc	5.80±0.64^bc	40.35±5.54^bc
F	153.271^＊＊	167.687^＊＊	186.272^＊＊	245.793^＊＊	115.173^＊＊

组别	ROS	胸主动脉内膜-中膜厚度（μm）
NC组	1.00±0.00	45.18±5.26
DM组	3.58±0.37^a	82.37±9.41^a
Ad-Scr shRNA组	3.64±0.42	84.20±9.33
Ad-Salusin-β shRNA组	1.76±0.23^bc	63.56±7.68^bc
F	229.973^＊＊	61.418^＊＊

组别	NOX2/ GAPDH	细胞核NF-κB p65/Lamin B1	细胞质NF-κB p65/GAPDH
NC组	0.34±0.05	0.45±0.07	0.86±0.09
DM组	0.82±0.09^a	0.90±0.10^a	0.41±0.05^a
Ad-Scr shRNA组	0.85±0.07	0.92±0.11	0.39±0.06
Ad-Salusin-β shRNA组	0.49±0.06^bc	0.63±0.08^bc	0.72±0.08^bc
F	157.571^＊＊	73.437^＊＊	125.903^＊＊

[1]	范慧慧, 任玉梅, 田新磊, 张凯, 李晓丽. 止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响[J]. 天津医药, 2024, 52(9): 924-929.
[2]	向近杰, 吕懋鑫, 王梦悦, 张坤, 李颢. Ca²⁺诱导HK-2细胞焦亡及黏附性变化对含钙肾结石形成的分子机制研究[J]. 天津医药, 2024, 52(3): 250-255.
[3]	易娜, 肖雯, 田源, 袁李礼. BMAL1减轻H₂O₂诱导的心肌细胞损伤机制研究[J]. 天津医药, 2024, 52(2): 119-123.
[4]	陈玺龙, 王宏君, 宋征宇, 王静. 牛蒡苷元通过抑制HMGB1/TLR4/NF-κB通路减轻急性脑梗死大鼠的神经元损伤[J]. 天津医药, 2023, 51(8): 825-829.
[5]	刘艳文, 刘水清, 林少伟, 刘协红. 毛蕊花糖苷调节HMGB1/RAGE/NF-κB信号通路对动脉粥样硬化大鼠内皮功能障碍的影响[J]. 天津医药, 2023, 51(12): 1339-1343.
[6]	陈黎旭, 熊佳, 谢昆, 朱挺德, 钟志英, 官亮, 潘永平. 扶正化瘀方含药血清对肝星状细胞activinA/smad信号通路活化的影响[J]. 天津医药, 2022, 50(7): 707-712.
[7]	贺静, 孙晓慧, 杨莉, 乌宇亮 . 分泌型丛生蛋白通过抑制线粒体自噬缓解H2O2诱导的心肌细胞损伤 #br#[J]. 天津医药, 2022, 50(2): 136-142.
[8]	马治, 王欣爽, 刘玥, 魏丽萍, 齐新. 甘草次酸的差向异构体对顺铂诱导H9c2心肌细胞损伤的保护机制[J]. 天津医药, 2022, 50(12): 1264-1269.
[9]	董超, 李华宇, 区豪杰, 孙嘉, 张陆勇, 刘冰△. 非诺贝特改善脂多糖诱导的急性肺损伤及其机制研究[J]. 天津医药, 2022, 50(1): 59-66.
[10]	周平, 裴文迪, 孙异凡, 于洋, 金丹△. 人参皂苷Rg3对H2O2诱导人卵巢颗粒细胞氧化应激损伤的保护作用#br#[J]. 天津医药, 2021, 49(9): 916-920.
[11]	区豪杰, 孙嘉, 李华宇, 董超, 刘冰. RITA通过ROS/Src/Stat3通路诱导肺鳞癌H226细胞凋亡[J]. 天津医药, 2021, 49(8): 785-790.
[12]	李丹, 高珊 . NLRP3炎性体在心肌缺血再灌注损伤中作用的研究进展 #br#[J]. 天津医药, 2021, 49(6): 656-662.
[13]	李旭东, 马勇, 邓润鹏, 苍雪玉, 吴飞燕, 李永涛, 金海峰△. 黄芪总皂苷对缺氧性肺动脉高压作用探讨[J]. 天津医药, 2021, 49(3): 264-268.
[14]	刘琳琳. 百里醌对辐射暴露小鼠造血系统的保护作用[J]. 天津医药, 2021, 49(3): 268-270.
[15]	李涛, 王玉, 陈伟, 夏春辉, 伦志强 . 酞菁锌光动力治疗诱导大肠癌Lovo细胞产生活性氧 #br#[J]. 天津医药, 2020, 48(4): 253-257.