Investigating the Hypolipidemic Mechanism of Anthocyanins Combined with Allicin in Rats Using Network Pharmacology

: Objectives: Using network pharmacology methods, investigate the potential lipid-lowering mechanism of the combination of anthocyanins and allicin in hyperlipidemic rats. Methods: Apply databases such as Pubchem, SwissTargetPrediction, TCMSP, DrugBank and BATMAN-TCM to predict potential targets for anthocyanins and allicin. Additionally, targets connected to hyperlipidemia were found in multiple databases (GeneCards, OMIM, Drugbank, and TTD). Upload the discovered drug-disease intersection targets into the database of STRING in order to construct a common target protein-protein interactions network (PPI). To find important targets, a PPI network analysis was built using Cytoscape 3.9.1. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enriched and analyzed these common drug-disease targets. In order to further confirm the key targets of anthocyanins combined with allicin in hyperlipidemia, animal experiments were conducted. Results: There are 63 potential targets for the combined effect of anthocyanins and allicin on hyperlipidemia. The PPI topology analysis results found that TNF, IL-6, AKT1, PTGS2, GSK3B, EGFR, etc. are the main key targets. The main pathways included PI3K-Akt, JAK-STAT, Fluid shear stress and atherosclerosis, HIF-1and MAPK signaling pathway. The animal experiments results revealed that anthocyanins combined with allicin can improve blood lipid levels in hyperlipidemic rats and decrease the serum levels of inflammatory factors. Conclusion: Anthocyanins combined with allicin can intervene in hyperlipidemia through a variety of targets and pathways. This research offers a theoretical reference for the investigation of the pathogenesis of hyperlipidemia and the production of functional foods.


Introduction
Hyperlipidemia (HLP) is a type of disease caused by abnormal lipid metabolism.With the improvement of the living standards of Chinese residents, the proportion of the three major productive nutrients in the dietary structure is gradually changing.Among them, the proportion of lipid energy supply has reached 32.9%, exceeding the upper limit recommended by the dietary guidelines [1] .With the increase of fat intake, the overweight rate, obesity rate and the incidence rate of dyslipidemia of Chinese residents are on the rise, and dyslipidemia is an important risk factor leading to atherosclerosis, hypertension and other cardiovascular diseases [1] .Dyslipidemia is an independent risk factor of multiple disorders, therefore, research on hyperlipidemia is receiving increasing attention.
At present, statins are a group of essential medications used in dyslipidemia, but they are dose dependent and have many side effects.In recent years, research has revealed that many chemical components in natural products are crucial for controlling lipid metabolism.Therefore, there is mounting interest in functional foods from plant sources which can prevent diet-related diseases.
Anthocyanins are a family of polyphenolic substances that are abundant in natural plants and are mostly present in their flowers, fruits, and seeds.Allicin is an organic sulfur compound extracted from garlic.Research has found that both anthocyanins and allicin have various biological effects, such as combating free radical attacks, anti-inflammatory, regulating lipid metabolism, and regulating blood sugar [2][3] .Our early experimental research showed that the combination of allicin and anthocyanins can significantly ameliorate blood lipid levels, but the research on its mechanism is still unclear.Network pharmacology builds and analyzes networks based on biological theories, combined with multidisciplinary technologies including pharmaceutical science, biological information technology, and computational science.The analysis results are combined with traditional experiments to investigate the relationship among medicines and diseases, offering new methods to the research of new drugs.This study predicts the key targets of anthocyanin combined with allicin in the intervention of hyperlipidemia, and analyzes the signal pathway.Meanwhile, the key targets are validated through animal experiments, providing theoretical basis and pioneering ideas for multi-level research on the mechanism of anthocyanin combined with allicin in regulating blood lipids and its application in the field of dual use of medicine and food.

Experimental drugs and reagents
High-fat and high-cholesterol (HFC) feed was purchased from Guangdong Medical Experimental Animal Center, Yue Si No. (2019) 05073, batch number: X2106250007; Anthocyanins were purchased from Tianjin Jianfeng Natural Product Research and Development Co., Ltd., batch number 002-2008055-24; Allicin was purchased from Shaanxi Jinkangtai Biotechnology Co., Ltd., batch number JKT20210423; Simvastatin was purchased from Zhejiang Jingxin Pharmaceutical Co., Ltd.

Animals
SPF-grade SD rats were obtained from Zhuhai Bestone Biotechnology Co., Ltd. with an experimental animal license number of SCXK (Guangdong) 2020-0051.

Drug-disease intersection genes
The targets of anthocyanins combined with allicin and hyperlipidemia were respectively imported into the Venny2.1.0to get drug-disease common targets and these targets presenting in the form of a Venn diagram.

Construction of PPI and screening of important targets
To create a common target protein-protein interaction network (PPI), the drug-disease intersection targets were imported into the STRING database.To identify key targets, PPI network analysis was built and examined using Cytoscape 3.9.1 software.Then, using the CytoNCA plugin and the betweenness centrality (BC) technique, 10 important targets were chosen.

GO and KEGG enrichment analyses
These common targets of anthocyanins combined with allicin and hyperlipidemia were imported into the Metascape platform (http://metascape.org) for enrichment analysis.It is statistically significant because the enrichment factor is larger than 1.5 and P< 0.01.The first ten GO and KEGG pathways with the greatest number of enriched genes are chosen for visualization, and the findings are shown using bar graphs and bubble charts.

Grouping and administration of rats
Forty SPF grade SD male rats were adaptive fed for one week and divided into five groups: control, model, positive control, high dose and low dose groups of anthocyanin combined with allicin, with 8 rats in each group.The normal group was fed with basic feed, while the other four groups were fed with high-fat feed to replicate the HLP model.Nine weeks later, the control and model group were given double distilled water by gavage.The high and low-dose groups of anthocyanin combined with allicin were given 180mg and 45mg/kg/d of anthocyanin, 90mg and 30mg/kg/d of allicin.The positive control group was given 4mg/kg/d of simvastatin by gavage.Each group was gavage for 4 weeks, once a day, with a gavage volume of 4ml each time.Except for the normal group receiving SPF level foundation feed, the other four groups continued to receive high-fat feed.

Detection of serum lipid levels in rats
The supernatant of rats was sent to Zhuhai Baishitong Biotechnology Co., Ltd. for blood lipid testing.

Detection of inflammatory factors in rat serum
Use ELISA kits to test the content of tumor necrosis factor -α (TNF-α) and interleukin-6 (IL-6) in at serum.

Statistical analysis of experimental data
The experimental results were represented as mean± S.D. using SPSS 27.0 statistical program for statistical analysis.Univariate analysis of variance was performed for inter group comparison, while LSD was used for pairwise comparisons between groups.P<0.05 represents a statistically significant difference.

Collection of Drug-Disease Targets
165 anthocyanin targets and 27 allicin targets have been assembled using the following six databases: Drugbank, BATMANTCM, Chemb, CTD, Swisstargetprediction and TCMSP.Merge the obtained allicin and anthocyanin targets, and remove duplicate data to obtain a total of 182 drug targets.After retrieving multiple databases, 2156 HLP-related genes were obtained.

Common targets for drugs and diseases
The anthocyanins combined with allicin targets and HLP targets were intersected using the Venny2.1.0platform.Figure 1 and Table 1 show that there are 63 common targets for drug-diseases.These common targets were used as the potential targets of anthocyanins combined with allicin in treating HLP.

PPI network construction and analysis of key target
To create the PPI diagram (Figure 2), upload the 63 common targets from the Venn diagram to the STRING platform.Upload the interaction information of 63 common targets to Cytoscape 3.9.1 software for visualization, obtain the PPI network diagram (Figure 3) of the common targets, and screen out key genes.According to the Betweenness (BC) algorithm, the first ten targets are TNF, IL-6, AKT1, PTGS2, GSK3B, EGFR, HSP90AA1, CASP3, AMAOA and APP.The larger the circle where the target is located in Figure 3, the higher the score of BC.Ten key targets are represented in red and yellow, and the specific topology analysis data of key targets in the network is shown in Table 2.

Enrichment pathway analysis
63 potential anti-hyperlipidemic targets were subjected to KEGG enrichment pathway analysis, which produced 130 KEGG signaling pathways.Sort by the number of enriched genes from most to least, select the top 15 pathways and draw a bubble chart (Figure 5). Figure 5 shows that targets are mainly involved in PI3K-Akt signaling, JAK-STAT signaling , Fluid shear stress and atherosclerosis, and MAPK signaling pathway, etc. Select the signal pathways involved in key gene targets and draw a chord diagram (Figure 6).From Figure 6, it can be seen that TNF and IL-6 have a strong connection to the pathways that are involved in the development of hyperlipidemia.

Effect of anthocyanins combined with allicin on blood lipid levels in HLP rats
Table 3 shows that the model group's blood levels of TC, TG, and LDL-C dramatically when compared to the normal group, whereas HDL-C levels declined significantly (P< 0.01), demonstrating the replication of the hyperlipidemic rat model.The levels of TC, TG, and LDL-C in the serum of the rats treated with anthocyanins and allicin were lower than those of the model group.
Additionally, following dosing, rats had greater blood HDL-C levels than the model group, with statistically significant changes in the high-dose group and no appreciable effects in the low-dose group.The combination of anthocyanins and allicin has a regulatory effect on blood lipids in hyperlipidemic rats.Note: The results are represented by Mean±S.D, and P<0.05 represents a statistically significant difference.

Effects of anthocyanins combined with allicin on serum TNF-α and IL-6 levels in HLP rats
Table 4 shows that the model group's TNF-αand IL-6 content rose statistically significantly as compared to the normal group (P<0.01).TNF-αand IL-6 levels were lower in the high and low dosage anthocyanins+allicin groups compared to the model group.The high dose group was statistically significant (P<0.05),while the low dose group was not significant, indicating that anthocyanins combined with allicin can reduce the levels of TNF-α and IL-6 and have a protective effect on vascular endothel.

Discussion
Hyperlipidemia is a major risk factor leading to diabetes, gout, hypertension, and atherosclerotic cardiovascular disease, which causes serious harm to human health.Currently, statins are commonly used drugs in clinical practice to regulate blood lipids, but long-term medication has many side effects.Due to the fact that hyperlipidemia is a type of disease closely related to diet, it is of great significance to search for drugs from natural foods that have the same medicinal and dietary origins in regulating blood lipids, in order to prevent chronic diseases caused by hyperlipidemia.Research has found a reasonable combination of natural drugs can provide patients with mild dyslipidemia and statin intolerance additional medication therapy choices [4] .Anthocyanins are common flavonoids that are widely present in human diets.According to research, adding anthocyanins to one's diet helps prevent the onset of obesity, dyslipidemia, insulin resistance, and steatosis in rats that are fed a high-fat diet [5] .A diet high in fat may increase oxidative damage, resistance to insulin, and tinflammation in tissues.Supplementing a mixture rich in anthocyanins can effectively reduce obesity, dyslipidemia, and insulin resistance.Therefore, the negative consequences of a diet rich in fat can be controlled through diet or supplementation with anthocyanins [6] .According to Nawaka N et al., allicin increases the absorption of LDL by HepG2 cells by upregulating LDLR and downregulating PCSK9 expression.This suggests that using allicin as a supplement to treat hyperlipidemia is possible [7] .According to studies, allicin can reduce the production of lipid rafts and boost LXRαactivity to treat staphylococcus aureus mastitis [8] .The above research results suggest that both anthocyanins and allicin have a certain effect on regulating blood lipids, but the mechanism of their combined use in intervening hyperlipidemia is not clear.Thus, this study discusses the targets and pathways of anthocyanins combined with allicin action on hyperlipidemia.
This study analyzed the intersection of targets between anthocyanin combined with allicin and hyperlipidemia, and found 63 common targets, indicating that anthocyanin combined with allicin may have a potential intervention effect on hyperlipidemia.In further protein interaction network analysis, it was found that TNF, IL-6, AKT1, PTGS2, GSK3B, EGFR, etc. are the main key targets in the network.According to the BC score, TNF and IL-6 rank in the top two, and in enrichment analysis of 10 key targets, it was found that they are also most closely related to the signaling pathways involved in hyperlipidemia.Research has found that hyperlipidemia stimulates inflammatory cell infiltration, promots the release of inflammatory cytokines, and results in aberrant lipid deposition on the inner wall of blood vessels, all of which hasten the development of atherosclerosis [9] .In order to further explore the relationship between TNF, IL-6 and hyperlipidemia, this study was validated through animal experiments.Results indicated that anthocyanins combined with allicin significantly reduced the expression of TNF and IL-6 in hyperlipidemic rats, thus confirming the accuracy of network pharmacology prediction.All of the aforementioned findings imply that TNF and IL-6 may be crucial in the process by which allicin and anthocyanins improve blood lipids.
In the GO enrichment analysis, it was found that anthocyanins combined with allicin mainly intervene in hyperlipidemia by regulating the activity of various enzymes, hormone reactions, phospholipid biosynthesis, platelet function, etc.The main areas of action of each target are concentrated in the membrane area, membrane raft, and axon area.The lipid-lowering effect of drugs is mainly related to ligand receptor activity and signal factor activation.The above results suggest that the combination of anthocyanins and allicin may participate in different cellular reaction processes through the action of multicellular components, exerting the anti hyperlipidemic effect.
In the analysis of KEGG enrichment pathway, it was found that anthocyanin combined with allicin against hyperlipidemia may be related to multiple signaling pathways.PI3K/AKT signaling is an important pathway closely related to lipid metabolism, insulin resistance, atherosclerosis, etc. Research has found that AKT1, TNFα, IL6, etc. are main cell signaling factors in the PI3K/AKT signaling pathway and are currently widely regarded therapeutic targets.Research has found that supplementing pueraria flavonoids can intervene in nonalcoholic fatty liver by inhibiting the PI3K/Akt/mTOR signaling pathway, reducing intracellular lipid accumulation and inflammation levels [10] .Yang Qin et al. found that exogenous H2S can cause atherosclerosis by regulating Toll-like receptor 4 (TLR4), TNF-αand IL-6 expression and its mechanism is related to the activation of PI3K/AKT signaling pathway [11] .The JAK/STAT signaling pathway primarily affects immunoregulation, cell differentiation, proliferation, and death.The activation of this signaling pathway is related to various receptors, including growth factors, interferon, or interleukin [12] .Research has found that exercise intervention can downregulate the production of TNF and IL-6 in obese mice, thereby inhibiting the JAK/STAT pathway and intervening in the occurrence and development of obesity [13] .It has been found that the activation of HIF-1 signaling pathway will aggravate the progression of atherosclerosis, which is mainly related to the aggravation of endothelial cell damage, the promotion of inflammatory factor release and angiogenesis [14] .The MAPK pathway is crucial for adipocyte differentiation and is crucial for the treatment and prevention of obesity.Fluid shear stress refers to the frictional force caused by flowing blood on the vascular wall, which can directly affect vascular endothelial cells.The injury of vascular endothelial cells directly affects the formation of atherosclerotic thrombus.Therefore, the fluid shear stress signaling pathway has regulatory effects on various cardiovascular diseases.

Conclusions
In summary, this study identified 63 potential targets for the intervention of anthocyanins combined with allicin in hyperlipidemia through network pharmacology analysis.
Anthocyanins combined with allicin play a role mainly by participating in PI3K-Akt signaling, fluid shear stress and atherosclerosis, MAPK signaling, JAK-STAT signaling and HIF-1 signaling pathways.It is revealed that the combination of anthocyanins and allicin may intervene in hyperlipidemia through various pathways such as reducing inflammatory response and combating endothelial damage.Through the use of bioinformatics technology and animal tests, this study investigates the main targets and associated pathways of anthocyanin combined with allicin in the treatment of hyperlipidemia, offering a theoretical reference for the study of plant medicines.

Figure 3
Figure 3 PPI network

4. 4
GO functional annotationGO enrichment analysis was conducted on 63 potential targets of anthocyanins combined with allicin in treating HLP, resulting 1194 entries (P<0.05),including 1034 biological processes (BP), 101molecular function(MF) and 59 Cellular component (CC).BP mainly involves regulation of phosphatidylinositol biosynthetic process, ovulation from ovarian follicle, ovulation from ovarian follicle, negative regulation of platelet activation and regulation of phospholipid biosynthetic process.MF mainly involves nitric-oxide synthase regulator activity, platelet-derived growth factor receptor binding, transcription regulator activator activity, ABC-type xenobiotic transporter activity, oxidoreductase activity and protein tyrosine kinase activator activity.CC mainly involves nuclear envelope lumen, plasma membrane raft, platelet alpha granule lumen, brush border membrane, main axon and membrane raft.The results show the top ten pathways with enriched gene numbers (Figure4), with BP represented by green columns, MF represented by purple columns, and CC represented by red columns.

Figure 4
Figure4GO functional enrichment analysis

Figure 5 Figure 6
Figure 5 Main KEGG Pathways of Drug-Disease Common Targets

Table 1
Common targets of drug -diseases Figure 1 Venn diagram of potential targets

Table 2
Top ten targets with BC values in the drug-disease common target interaction network