Evaluation of Angelicae sinensis radix as a promising treatment option for hyperlipidemia based on network pharmacology

Background:Angelicae sinensis radix has been widely applied in traditional Chinese medicine while little is explored in its potential mechanism.This study aims to elucidate the effective components and defatting mechanism based on network pharmacology.Methods:Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was screened to collect the possible active ingredients and their CAS and SMILES was searched in Pubchem,which further used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets.Hyperlipidemia-related molecules were obtained from GeneCards database,and the predicted targets of Angelicae sinensis radix for hyperlipidemia treatment were selected by Wayne diagram.For mechanism analysis,the protein-protein interactions were constructed with String,the Gene Oncology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes analysis were conducted in DAVID.Results:Using network-based systems biology analysis,we predicted that 5 active ingredients in Angelicae sinensis radix has antilipemic effects with 71 potential targets.Through Gene Oncology and Kyoto Encyclopedia of Genes and Genomes analysis,we found that the related signaling pathways mainly involved in arachidonic acid metabolism,and regulation of lipolysis in adipocytes.The related genes are ALOX5,CYP2C19,EPHX2,PTGS1,PTGS2,ADRB1,and ADRB3.Conclusion:Angelicae sinensis radix may alleviate hyperlipidemia through arachidonic acid metabolism,and regulation of lipolysis in adipocytes.ALOX5,CYP2C19,EPHX2,PTGS1,PTGS2,ADRB1,and ADRB3 may be new targets for treatment.

Keywords :Angelicae sinensis radix,Hyperlipidemia,Network pharmacology,Arachidonic acid metabolism

Background

Hypercholesterolemia is a common disorder primarily characterized by high plasma levels of low-density lipoprotein cholesterol (LDL-C),due to its reduced catabolism [1].LDL-C and high-density lipoprotein cholesterol (HDL-C) in the blood are involved in the cholesterol metabolism.LDL-C and HDL-C regulate the level of cholesterol,the imbalance of LDL-C and HDL-C can increase the risk of atherosclerotic plaque development and premature cardiovascular disease risk[2].

Cholesterol was an essential component of cell barrier formation and cell signaling transduction that regulated membrane fluidity and interacted with other lipids and proteins [3].The movement of cholesterol through plasma was mediated by lipoprotein particles that carry hydrophobic cholesteryl esters.It is now known that cellular cholesterol levels are determined by the interplay between de novo biosynthesis,uptake,export,and storage [4].In brief,cholesterol synthesis starts from acetyl-CoA and involves concerted actions of more than 20 enzymes,most of which localize in the membrane of the endoplasmic reticulum.The liver—the main site of cholesterol biosynthesis—delivers both endogenously synthesized and exogenously acquired cholesterol to the bloodstream as very-low-density lipoproteins (VLDLs).After processing in the bloodstream,the VLDLs generate circulating low-density lipoproteins (LDLs),which can be taken up by peripheral cells via receptor-mediated endocytosis [5].Cholesterol homeostasis at systemic levels requires collaboration between various tissues,which ensures a balance between cholesterol absorption and cholesterol biosynthesis with its release into the bloodstream and subsequent uptake by cells in the body.In the bloodstream,cholesterol is transported as various lipoproteins,mostly LDLs.As low-density lipoprotein receptor (LDLR) is nearly ubiquitously expressed,the liver and extrahepatic tissues can take up LDLs,including the small intestine,via the LDLR pathway.Elevated cholesterol can cause some physiological abnormalities.How to treat hyperlipidemia safely is our clinical focus.

Recently,physicians in the USA were strongly recommended to treat-to-targets regarding LDL cholesterol reduction.The treatment for hyperlipidemia is mainly statins.Inflammation had a fundamental role in all stages of the atherothrombotic process.Statins,in addition to reducing LDL cholesterol,also had anti-inflammatory properties [6].Lomitapide,was an inhibitor of microsomal triglyceride transport protein,also reduced circulating LDL cholesterol by targeting hepatic VLDL production[7].Proprotein convertase subtilisin kexin type 9(PCSK9) was the most promising novel target for additional LDL-cholesterol reduction [8],a protein secreted by hepatocytes that binds to the LDL receptor,leading to its cellular internalization and subsequent lysosomal degradation [9].While statins had greater side-effect to the liver and kidneys,and somewhat higher risks of diabetes have been observed [10].Toxicity issues related to PCSK9 inhibition form part of the ongoing concerned among clinicians about the lowest safe concentration for LDL cholesterol.Considering the disadvantages,we are supposed to find new suitable drugs for hyperlipidemia.

Angelicae sinensis radixis a widely used Chinese herb with bioactivities including antimicrobial,anticancer,analgesic,anti-inflammatory,hepatoprotective.A research has been implemented that high-fat diet-induced obese rats were treated orally with the polyphenolic-rich extract of Angelica acutiloba root once daily for 8 weeks.Extract of Angelica acutiloba root increases metabolism by down-regulating the lipid metabolism of SREBPs and increasing the expression of ACO and CYP4A1 in the liver,thereby eliminating the accumulation of internal organs in high-fat diet-induced obesity and improving hyperlipidemia [11].

In traditional Chinese medicine,hyperlipidemia is often classified as “Blood Stasis Syndrome”.The classic ancient prescription of Chinese medicine Xue-Fu-Zhu-Yu decoction,Dang-Gui-Shao-Yao decoction,Ge-Xia-Zhu-Yu decoction et al.are all commonly used,andAngelicae sinensis radixis the most significant medicine among them obtaining a supreme lipid-lowering effects [12].However,the mechanism for its treatment of hyperlipidemia was unclear.We aimed to discover the pharmacological treatment through a network of possible mechanisms,which can indicate the experimental direction for our later research.This also provided us with innovations for finding new drugs to treat hyperlipidemia.

Materials and methods

Screening of active components of Angelicae sinensis radix

The main components ofAngelicae sinensis radixwere identified by searching the pharmacological database and analysis platform of traditional Chinese medcine system (TCMSP:https://tcmspw.com/tcmsp.php).The drug components were screened by combining the oral absorption and type drugindex.CAS obtained from the TCMSP,while the drug “SMILES” was found in PubChem.(PubChem:https://pubchem.ncbi.nlm.nih.gov/).

Prediction of potential targets for active components

Log on to Swiss Target Prediction(http://www.swisstargetprediction.ch),SEA(http://sea.bkslab.org),and TargetNet(http://targetnet.scbdd.com) and enter “SMILES” of monomer drugs with species limited to “homo sapiens”.The obtained protein targets and corresponding Uniprot IDs were imported into excel.

Screening of hyperlipidemia-related-targets

By inserting the keyword “hyperlipidemia” into the GeneCards database (https://www.genecards.org),we searched for the reported hperlipidemia-related-targets genes,removed the false positive genes,and the common target ofAngelicae sinensis radixand hyperlipidemia was screened by Wayne diagram.

Acquisition of uniprot ID

According to the Wayne diagram,common targets are obtained and their corresponding Uniprot IDs were searched.Uniprot KB (https://www.uniprot.org/) was used to identify the gene target ID.

Protein interaction network construction

String database (https://string-db.org/) is a database containing known and predicted protein-protein interactions (PPIs).The common targets were input into String,and the species were defined as “homo sapiens” to obtain a PPI relationship.The results were saved in the TSV format.

Biological process and pathway analysis

DAVID (https://david.ncifcrf.gov/) can provide large-scale functional annotations of genes or proteins and can identify the most significant enrichment of biological annotations.The common target obtained was imported into DAVID database,and the species was limited to “homo sapiens”.Gene Oncology (GO)analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out on the target of hyperlipidemia.

Results

The active components of Angelicae sinensis radix

Using TCMSP platform,125 main components ofAngelicae sinensis radixwere screened.There were only 2 monomers meeting the criteria of OB (> 30%)and DL (> 0.18).Besides,we also counted in several high content chemicals namely ferulic acid (CIS),cis-ligustilide and sitogluside.SMILES were further obtained from PubChem.We can see the “CAS” and“SMILES” in Table 1.

Drug target prediction

The “SMILES” of 5 components were input into Swiss Target Prediction,SEA,and TargetNet databases.We obtained 249 targets after removing repetitive targets.Uniprot IDs of 249 target proteins were identified by Uniprot KB to confirm that these genes belong to human.

Screening of hyperlipidemia targets

By inserting the keywords “hyperlipidemia” into the GeneCards database,we searched 1,164 hyperlipidemia genes.As well,we selected all genes and imported them into Webgestalt for KEGG enrichment (http://www.webgestalt.org/).We selected the top 20 channels to make a chart (Table 2,Figure 1).Moreover,inserting the keywords “hyperlipidemia” in KEGG,and there were no pathways which were closely related to hyperlipidemia.

Screening of hyperlipidemia therapeutic targets of Angelicae sinensis radix

The therapeutic target and hyperlipidemia related targets were imported into Venny 2.1 to obtain the therapeutic target of hyperlipidemia.We got 71 therapeutic targets ofAngelicae sinensis radix.(Table 3,Figure 2).

Construction of PPI network and target analysis

Targets ofAngelicae sinensis radixfor hyperlipidemia treatment were input into String database with species limited to “homo sapiens”.We got the figure (Figure 3)from this website.

Table 1 Main active ingredients of Angelicae sinensis radix

Table 2 Kyoto Encyclopedia of Genes and Genomes pathway analysis of hyperlipidemia-related genes

Figure 1 Kyoto Encyclopedia of Genes and Genomes pathway analysis of hyperlipidemia-related genes

GO classification and enrichment analysis

The common targets were imported into DAVID database with species limited to “homo sapiens”.The targets ofAngelicae sinensis radixfor hyperlipidemia treatment were used to conduct GO analysis.GO analysis found 161 biological processes,27 cellular components and 76 kinds of molecular function were involved in the mechanism of treatment of hyperlipidemia ofAngelicae sinensis radix.We found that cholesterol homeostasis,regulation of cholesterol homeostasis,lipid metabolic process,cholesterol biosynthetic process,cholesterol metabolic process,negative regulation of cholesterol storage,and positive regulation of cholesterol efflux from biological processes were related to the treatment of hyperlipidemia.

KEGG analysis

Common targets were imported into DAVID database with species limited to “homo sapiens”.KEGG enrichment analysis was carried out forAngelicae sinensis radix.KEGG enrichment analysis involves 22 pathways.All 22 pathways are selected and plotted.Also we combined compounds,targets and pathways to draw an alluvial diagram (Table 4,Figure 4,Figure 5).Possible mechanisms for treating hyperlipidemia are involved in 2 pathways,including arachidonic acid(AA) metabolism,and regulation of lipolysis in adipocytes.Moreover,the related genes areALOX5,CYP2C19,EPHX2,PTGS1,PTGS2,ADRB1,andADRB3(Table 5).

Table 3 Therapeutic targets of Angelicae sinensis radix

Figure 2 Targets of Angelicae sinensis radix for hyperlipidemia treatment.A,represents the targets of Angelicae sinensis radix; B,represents the targets of hyperlipidemia.

Figure 3 Interaction diagram of target proteins of Angelicae sinensis radix.*Line thickness indicates the strength of data support.

Table 4 Kyoto Encyclopedia of Genes and Genomes pathway analysis of treatment targets of Angelicae sinensis radix

Figure 4 Kyoto Encyclopedia of Genes and Genomes pathway analysis of target genes of Angelicae sinensis radix

Figure5 Compound-target-pathway alluvial diagram

Table 5 Hyperlipidemia-related pathway and corresponding gene of Angelicae sinensis radix

Discussion

Network pharmacology can predict the targets and pathways of drugs to treat diseases at the molecular level.Our study uses this method to study the mechanism ofAngelicaesinensisradixin hyperlipidemia treatment.There were 5 monomers ofAngelicae sinensis radixincluding β-sitosterol,stigmasterol,sitogluside,ferulic acid (CIS),cis-ligustilide.249 targets were obtained by removing repetitive targets.Therapeutic targets ofAngelicae sinensis radixwere 71.Through GO classification and enrichment analysis of common targets,we found that cholesterol homeostasis,lipid metabolic process,cholesterol biosynthetic process,cholesterol metabolic process,negative regulation of cholesterol storage and positive regulation of cholesterol efflux from biological processes were closely related to the hyperlipidemia treatment.Cholesterol is of vital importance for vertebrate cell membrane structure and function [13].Metabolites of cholesterol,such as bile salts,steroid hormones and oxysterols,fulfill important biological functions [14].LDLR,ubiquitously expressed and a key receptor for maintaining cholesterol homeostasis in mammals,is a cell membrane glycoprotein that functions in the binding and internalizing of circulating cholesterol-containing lipoprotein particles.PCSK9 is secreted from the hepatocytes,which undergo PPI with LDLR,while inhibition of PCSK9 effectively increases hepatic LDLR expression and reduces LDL cholesterol in plasma in mice [15].As a result,LDLR is a pivotal receptor for endocytic machinery and plays a pivotal role in maintaining cholesterol homeostasis [16].Cholesterol homeostasis is achieved by means of a fine balance between cholesterol intake,absorption/excretion and synthesis.The liver has been considered the major site of control in maintenance of cholesterol homeostasis.The liver facilitates clearance of LDL particles and cholesterol-containing chylomicron remnants,synthesizes cholesterol,synthesizes and secretes (nascent) HDL particles,secretes cholesterol and BS to bile and is involved in reverse cholesterol transport [17].Lipid metabolism is a complex physiological process that is involved in nutrient adjustment,hormone regulation,and homeostasis.Current studies suggest that the transcription factor forkhead box protein O1 is involved in lipid metabolism and plays a critical role in the development of these lipid-related diseases [18].Cholesterol synthesis in the liver is mediated by an extensive series of reactions.The liver accounts for approximately 50% of elimination of cholesterol through the formation of bile acids.It excretes both bile acids and cholesterol into the intestine,partially accounting for the remainder of cholesterol elimination[19].Liver X receptors α and peroxisome proliferator-activated receptor γ might play a significant role in adipocyte cholesterol metabolism through mediation of cholesterol efflux [20].The process known as reverse cholesterol transport promotes excess cholesterol efflux from peripheral cells and returns it to the liver by HDL particles [21].

Concerning the KEGG analysis results,AA metabolism and regulation of lipolysis in adipocytes demonstrates vivid connection withAngelicae sinensis radix’s treatment of hyperlipidemia.Metabolites and enzymes involved in AA metabolism and their biological functions in metabolic and cardiovascular diseases.Many AA metabolites are highly bioactive and involved in various crucial vital processes,namely COX1,COX2,LOX,PGE2,PGI2,CYP and so on[22].AA and its derivatives can enter numerous metabolic pathways that interconnect lipid metabolism with immunity.Adipose tissue constitutes a major location for cholesterol storage,and it may play a role in the regulation of circulating cholesterol levels.A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high-density lipoprotein,HDL,was investigated in 3T3-L1 adipocytes by Philip B Verghese [23].In the presence of HDL,composed of human apoA-I and phosphatidylcholine,adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion.β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles [23].Hypercholesterolemia and lipoprotein imbalances,such as increases in LDL-C and enhanced production of oxidized LDL-C contribute to vascular wall inflammation via interaction with pattern recognition receptors of accumulating macrophages[22].It is noteworthy that cholesterol and AA metabolism are interrelated.The latter was demonstrated by the inhibition of the 5-lipoxygenase pathway by the hydroxymethylglutaryl-CoA reductase antagonist atorvastatin,and the alteration of cholesterol homeostasis by the COX inhibitor aspirin.Specifically,aspirin induces the bile salt export pump Abcb11 and supports reverse cholesterol transport from atherosclerotic lesions to the liver for their subsequent biliary excretion [24].5-lipoxygenase catalyzes 2 steps in the biosynthesis of leukotrienes,lipid mediators of inflammation derived from AA [25].AA is metabolized to epoxyeicosatrienoic acids (EETs)in endothelial cells by various cytochromes P450.The EETs possess potent vasodilatory anti-inflammatory and fibrinolytic effects and are considered one of the primary endothelial-derived hyperpolarizing factors[26].EPHX2 exerts pro-inflammatory activity by metabolizing anti-inflammatory EETs into the less active dihydroxyeicosatrienoic acids [27].Together with cyclooxygenase and lipoxygenase,EPHX2 is involved in the AA metabolic cascade [28].One strategy for increasing the action of EETs involves decreasing the activity of EPHX2.The regulation of important oxylipin metabolic genes in peripheral blood mononuclear cells varied with the extent of change in ARA concentrations in the case of PTGS1 and ALOX12 regulation [29].Defining from above,the AA metabolome can be conserved regulator of cholesterol metabolism,and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.TheADRB1is a candidate gene for obesity due to its role in catecholamine mediated energy homeostasis.TheADRB3gene is expressed in adipose tissues and stimulates the mobilization of lipids from the white adipose tissue and increases thermo-genesis in brown adipose tissue.Mutation ofADRB3in white adipose tissue could slow lipolysis and thereby cause the retention of lipids in adipocytes and may contribute to visceral obesity in humans [30].

Conclusion

This study showed that 5 active components ofAngelicae sinensis radixcould act on 71 targets to treat hyperlipidemia,mainly involving AA metabolism,and regulation of lipolysis in adipocytes.ALOX5,CYP2C19,EPHX2,PTGS1,PTGS2,ADRB1,and ADRB3 may be new targets for treatment.