方法文章

Evaluating Salidroside as a Therapeutic Agent for Vascular Calcification Using Network Pharmacology and Experimental Rat Models

DOI：

10.3791/67728

⸱

2025年1月31日

 ,  ,  ,  ,  ,  , 
1Department of Encephalopathy, The Affiliated Hospital of Changchun University of Chinese Medicine, 2Research Center of Traditional Chinese Medicine, The Affiliated Hospital to Changchun University of Chinese Medicine, 3Department of Nephrology, The Affiliated Hospital to Changchun University of Chinese Medicine, 4Scientific Research Office, The Affiliated Hospital to Changchun University of Chinese Medicine, 5Rehabilitation Department, The Affiliated hospital to Changchun University of Chinese Medicine

* These authors contributed equally

本文内容

摘要

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This study establishes a rat model of vascular calcification induced by a high-fat diet (HFD) combined with vitamin D3 (VD3). The model was used to evaluate the therapeutic efficacy of salidroside in preventing and treating vascular calcification, providing insights into its potential mechanisms of action through network pharmacology and in vivo experiments.

摘要

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Vascular calcification (VC) is a critical pathological condition associated with significant morbidity and mortality. This study employs a hybrid approach of network pharmacology and molecular biology to delineate the therapeutic mechanisms of salidroside (SAL), an active compound from Rhodiola crenulata, against VC. Through database mining and network analysis, 388 SAL targets intersecting with 2871 VC-associated targets were identified, resulting in 208 common targets. A protein-protein interaction (PPI) network constructed via the String database and topological analysis in Cytoscape 3.9.1 pinpointed 10 key targets, including IL6, TNF, TP53, IL1B, HIF1A, CASP3, and STAT3, among others. The identified genes were concentrated in the lipid and atherosclerosis pathways, indicating that the improvement of VC by SAL may occur through the regulation of abnormal expression of lipid and inflammatory factors. It was also found that SAL inhibits the abnormal expression of inflammatory factors, thereby activating the JAK2/STAT3 pathway to intervene in the progression of VC. The JAK2/STAT3 pathway is a key molecular mechanism by which SAL prevents further deterioration of VC. Functional enrichment analyses revealed the involvement of these targets in inflammatory responses and lipid metabolism, pivotal pathways in VC. In vivo studies in rats demonstrated SAL's efficacy in mitigating dyslipidemia and vascular inflammation, with improved serum lipid profiles and reduced vascular calcium deposition. The mechanistic exploration, grounded in Western blot analysis, demonstrated salidroside's ability to regulate the JAK2/STAT3 signaling pathway, highlighting its potential as a modulator in this critical molecular mechanism and offering a potential therapeutic target for VC. The strength of this research lies in its methodological rigor, integrating computational predictions with in vivo validations. This comprehensive approach establishes a robust framework for exploring the therapeutic mechanisms of natural compounds in combating VC.

引言

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Vascular calcification (VC) refers to the abnormal deposition of calcium within the vessel walls, which leads to arterial stiffening and decreased elasticity, ultimately impairing vascular function. Traditionally, VC has been divided into two types: intimal calcification, linked to lipid buildup, and medial calcification. The former is closely associated with inflammatory infiltration, triggering an osteogenic transformation in the vascular wall, characterized by the migration, proliferation, and differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells1.

The ability of VSMCs to undergo osteo....

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方案

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The protocol was approved by the Experimental Animals Committee of Changchun University of Chinese Medicine (Approval No. 2023091). This study adheres to international guidelines, including the European Community Guidelines and the EEC Directive of 1986, ensuring the ethical treatment of animals throughout the study. Male Wistar rats (8-10 weeks, weight 200-220 g) were used for the study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Network pharmacology prediction of potential salidroside-VC targets

NOTE: Network pharmacol....

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结果

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Network pharmacology analysis

Using databases such as HERB, TCMSP, Pubmed, SwissTargetPrediction, CTD, PharmMapper, SEA, and STITCH, 388 potential target genes for salidroside were identified. Additionally, 2871 potential target genes related to VC were retrieved from databases like GeneCards, OMIM, PharmGkb, and DrugBank. Intersection analysis via VENN diagrams revealed 208 overlapping targets, considered key targets for salidroside's intervention in VC (

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讨论

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VC is characterized by degenerative changes in vascular cells and tissues, with pathological mineral deposits within blood vessels leading to stiffening of the vessel walls or the formation of atherosclerotic plaques, which can result in obstructive vascular diseases25. Studies show that about 85% of VC plaques may evolve into thrombosis, which can trigger acute cardiovascular episodes. Additionally, VC is a crucial indicator of potential acute cardiovascular events, strokes, and peripheral vascul.......

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披露

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Ensure that all authors have disclosed any and all conflicts of interest.

致谢

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This work was financially supported by the Jilin Provincial Department of Science and Technology Project (YDZJ202301ZYTS460), and Jilin Provincial Department of Education Project (JJKH20230991KJ).

....

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材料

本文使用的材料清单
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4% 多聚甲醛固定液Beyotime Biotech Inc （Beyotime） ，中国P0099
5*加载缓冲液北京阳光生物科技科技有限公司，中国P1040
碱性磷酸酶检测试剂盒Beyotime Biotech Inc （Beyotime） ， 中国P0321S
AlphaView SoftwareProteinsimple Inc.美国AlphaView SA
BCA 蛋白检测试剂盒Beyotime Biotech Inc （Beyotime） ， 中国P0012
Bluing Solution北京 Solarbio Science &中国G1866
钙比色测定试剂盒Beyotime Biotech Inc （Beyotime） ， 中国S1063S
胶原纤维和弹性纤维染色试剂盒（EVG-Verh eff 法）北京阳光生物科技中国G1597
脱水机Diapath Biosciences Ltd， 意大利Donatello
包埋机武汉骏杰电子有限公司，中国JB-P5
酶标仪 Biotek Co.， Ltd，美国Epoch
Ethanol absoluteGHTECH  Co.， Ltd， China64-17-5
山羊抗小鼠 IgG （H+L） HRPBioworld technology， co.， Ltd.，中国BS20242-Y
GraphPad Prism 软件GraphPad Software.，美国GraphPad Prism 9.0
苏木精-伊红染色试剂盒Beijing Solarbio Science &科技有限公司，中国G1120
高密度脂蛋白胆固醇测定试剂盒南京建成生物工程研究院有限公司，中国A112
HRP 标记的山羊抗兔 IgG（H+L）广州赛果生物科技有限公司A0208
Image J Software美国国立卫生研究院 （NIH）Image J 
我κB α 多克隆抗体Proteintech Group， Inc.A，美国10268-1-AP
JAK2 抗体亲和生物科学有限公司，中国AF6022
低密度脂蛋白胆固醇检测试剂盒南京建成生物工程研究院有限公司，中国A113
NF-κB p65 抗体Proteintech Group， Inc.A，USA10745-1-AP
病理切片机Leica Biosystems，USARM2016
磷酸酶抑制剂鸡尾酒表F. Hoffmann-La Roche， Ltd，瑞士04906845001
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磷酸化-NF-κB p65（Ser276） 抗体Affinity Biosciences Co.， Ltd，ChinaAF2006
Phospho-STAT3（S727） 抗体抗体 Abways 科学 &中国CY5291
蛋白酶抑制剂混合物F. Hoffmann-La Roche， Ltd，瑞士11873580001
PVDF 膜F. Hoffmann-La Roche， Ltd，瑞士3010040001
大鼠 IL-1&β;ELISA 试剂盒Beyotime Biotech Inc （Beyotime） ，中国PI303
大鼠 IL-6 ELISA 试剂盒Beyotime Biotech Inc （Beyotime），中国PI328
大鼠 TNF-α;ELISA 试剂盒Beyotime Biotech Inc （Beyotime） ， 中国PT516
RIPA 裂解缓冲液Beyotime Biotech Inc （Beyotime） ， 中国P0013B
水利索苷上海元业生物科技有限公司，中国S25475
SDS广州赛果生物科技有限公司，中国3250KG001
碳酸钠中国医药集团有限公司，Ltd. ， 中国1001921933
碳酸氢钠中国医药集团有限公司 ， 中国10018960
硫代硫酸钠中国医药集团有限公司 ， 中国20042518
STAT3 抗体Proteintech Group， Inc.A，美国10253-2-AP
TBST （10×）Beyotime Biotech Inc （Beyotime） ， 中国ST673
总胆固醇检测试剂盒南京建成生物工程研究院有限公司A111
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Tris Base广州赛果生物科技有限公司1115GR500
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