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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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....

Results

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 (

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30% (29:1) Acrylamide/Bis Solution	Beijing Solarbio Science & Technology Co., Ltd ,China	A1010
4% Paraformaldehyde Fix Solution	Beyotime Biotech Inc (Beyotime) , China	P0099
5*loading buffer	Beijing Solarbio Science & Technology Co., Ltd ,China	P1040
Alkaline Phosphatase Assay Kit	Beyotime Biotech Inc (Beyotime) , China	P0321S
AlphaView Software	Proteinsimple Inc.USA	AlphaView SA
BCA Protein Assay Kit	Beyotime Biotech Inc (Beyotime) , China	P0012
Bluing Solution	Beijing Solarbio Science & Technology Co., Ltd ,China	G1866
Calcium Colorimetric Assay Kit	Beyotime Biotech Inc (Beyotime) , China	S1063S
Collagen Fiber And Elastic Fiber Staining Kit(EVG-Verh eff Method)	Beijing Solarbio Science & Technology Co., Ltd ,China	G1597
Dewatering machine	Diapath Biosciences  Ltd, Italy	Donatello
Embedding machine	Wuhan Junjie Electronics Co., Ltd,China	JB-P5
Enzyme-labeled instrument	Biotek Co., Ltd,USA	Epoch
Ethanol absolute	GHTECH  Co., Ltd, China	64-17-5
Goat Anti-Mouse IgG (H+L) HRP	Bioworld technology, co, Ltd.,China	BS20242-Y
GraphPad Prism Software	GraphPad Software.,USA	GraphPad Prism 9.0
Hematoxylin-Eosin Stain Kit	Beijing Solarbio Science & Technology Co., Ltd ,China	G1120
High-density lipoprotein cholesterol assay kit	Nanjing Jiancheng Bioengineering Research Institute Co., Ltd,China	A112
HRP-labeled Goat Anti-Rabbit IgG(H+L)	Guangzhou saiguo biotech Co.,LTD	A0208
Image J Software	National Institutes of Health(NIH),USA	Image J
IκB Alpha Polyclonal antibody	Proteintech Group, Inc.A,USA	10268-1-AP
JAK2 Antibody	Affinity Biosciences Co., Ltd,China	AF6022
Low-density lipoprotein cholesterol assay kit	Nanjing Jiancheng Bioengineering Research Institute Co., Ltd,China	A113
NF-κB p65 Antibody	Proteintech Group, Inc.A,USA	10745-1-AP
Pathological microtome	Leica Biosystems,USA	RM2016
Phosphatase Inhibitor Cocktail Tables	F. Hoffmann-La Roche, Ltd,Switzerland	04906845001
Phospho-JAK2 (Tyr931) Antibody	Affinity Biosciences Co., Ltd,China	AF3024
Phospho-NF-κB p65(Ser276) Antibody	Affinity Biosciences Co., Ltd,China	AF2006
Phospho-STAT3(S727) Antibody	Abways  Science & Technology Co., Ltd ,China	CY5291
Protease Inhibitor Cocktail	F. Hoffmann-La Roche, Ltd,Switzerland	11873580001
PVDF membrane	F. Hoffmann-La Roche, Ltd,Switzerland	3010040001
Rat IL-1β ELISA Kit	Beyotime Biotech Inc (Beyotime) , China	PI303
Rat IL-6 ELISA Kit	Beyotime Biotech Inc (Beyotime) , China	PI328
Rat TNF-α ELISA Kit	Beyotime Biotech Inc (Beyotime) , China	PT516
RIPA Lysis Buffer	Beyotime Biotech Inc (Beyotime) , China	P0013B
Salisoroside	Shanghai yuanye Bio-Technology Co., Ltd,China	S25475
SDS	Guangzhou saiguo biotech Co.,LTD,China	3250KG001
Sodium carbonate	China National Pharmaceutical Group Co., Ltd. , China	1001921933
Sodium hydrogen carbonate	China National Pharmaceutical Group Co., Ltd. , China	10018960
Sodium thiosulfate	China National Pharmaceutical Group Co., Ltd. , China	20042518
STAT3 Antibody	Proteintech Group, Inc.A,USA	10253-2-AP
TBST (10×)	Beyotime Biotech Inc (Beyotime) , China	ST673
Total cholesterol assay kit	Nanjing Jiancheng Bioengineering Research Institute Co., Ltd,China	A111
Triglyceride assay kit	Nanjing Jiancheng Bioengineering Research Institute Co., Ltd,China	A110
Tris Base	Guangzhou saiguo biotech Co.,LTD	1115GR500
Upright optical microscope	Nikon Corporation,Japan	Eclipse E100
Von Kossa Solution	Wuhan servicebio technology CO.,LTD,China	G1043
Western Blotting Luminol Reagent	Santa Cruz Biotechnology, Inc. ,USA	SC-2048
β-Actin antibody	Cell Signaling Technology, Inc.,USA	E4967