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

doi:10.11958/20221272

Abstract

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

CLC Number:

R587.1

Figures/Tables 11

References 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^＊＊