Molecular mechanism underlying the hypolipidemic effect of Shanmei Capsule based on network pharmacology and molecular docking

1.Introduction

Hyperlipidemia is one of the most prevalent global chronic metabolic diseases, which is closely related to many high-incidence cardiovascular diseases, such as hypertension, diabetes, coronary heart disease and atherosclerosis [1, 2, 3]. It is characterized as elevated contents of total cholesterol (TC), triglyceride (TG), low-density-lipoprotein cholesterol (LDL-C), as well as decreased high-density-lipoprotein cholesterol (HDL-C) levels [4]. With the elevating prevalence and morbidity, hyperlipidemia has become a critical global issue for public health. Regulating lipid metabolism disorders may be a potential approach to decelerating or preventing the progression of hyperlipidemia. At present, statins, such as atorvastatin, lovastatin and simvastatin, are among the most commonly used lipid-lowering drugs to reduce plasma lipids. Statins are generally regarded as safe and tolerable drugs, however, certain controversies remain. A survey analyzed all case reports from the FDA AE Reporting System database linking muscle-related adverse events to statin use. The results show that dose-dependent side effects occur across the statin class [5, 6, 7, 8]. Thus, it is necessary to develop and identify an alternative drug that may be valuable in regulating lipid metabolism. Compound formulae of traditional Chinese medicine (TCM) emphasizes emphasizes the synergism among active ingredients, with proper herbs and relevant dosage to synergize the desirable effects and minimize side effects integrally. Compared with Western medicine, TCM are considered relatively safe and produces fewer adverse reactions [9, 10].

TCM has been used for thousands of years for the treatment of hyperlipidemia in China, with advantages of multitarget mechanisms, remarkable curative effect and safety. They are featured by global regulation of glucose and lipid metabolism, as well as energy homeostasis via active ingredients in a prescription [11]. Shanmei Capsule, a famous preparation in China, is made of a combination of Crataegi Folium (called Shanzhaye in Chinese, the leaves of Grataegus pinnatifida Bge. var. major N.E. Br. or Grataegus pinnatifida Bge.) and Dahurian rose fruit (called Cimeiguo in Chinese, the fruit of Rosa davurica Pall. var. davurica) ethanol extract in proportion [12]. Crataegi Folium and Dahurian rose fruit both are well-known traditional medicinal plants, which have been confirmed to possess various biological and pharmacological activities, such as anti-inflammatory, antioxidant, hypocholesterolaemic and hypolipidaemic effects [13, 14, 15]. In clinical application, they are included in remedies on their own or in combination with other herbs to regulate lipid metabolism disorders induced by cardiovascular diseases. The above evidence suggests that Shanmei Capsule may be an alternative approach for treating hyperlipidemia. However, the hypolipidemic effect and related activity mechanisms of Shanmei Capsule against hyperlipidemia have yet to be revealed. Due to the complexity of active ingredients and the unknown synergistic effects of multiple components, it remains a laborious task to clarify the molecular mechanism of the Shanmei Capsule.

With the booming development of systems biology and bioinformatics, network pharmacology approach has been used successfully for the discovery of the key targets and their potential molecular mechanisms [16, 17]. Contrary to “one drug, one target, one disease” principle of traditional drug design, network pharmacology based on the “drug-disease-target” interaction network, which comprehensively and systematically evaluates the intervention or effect of drugs on diseases. Network pharmacology combines the multi-level information of “ingredient-disease-target-pathway”, which corresponded with the characteristics of TCM. Molecular docking is an important computer-aided drug discovery method, which can be used to predict the interactions between drug ingredients and target proteins, and the possible binding sites of drugs [18, 19]. Interactions between drug molecules and target proteins provide important information on drug discovery, which will help towards the development of drugs to treat hyperlipidemia.

In this study, network pharmacology approach was applied to investigate the relationships between active ingredients, core targets and pathways. Based on the results of network analysis and molecular docking, multi-ingredient, multi-target, and multi-pathway potential mechanisms related to Shanmei Capsule against hyperlipidemia were predicted.

2.Methods

3.Results

3.1Screening of Shanmei Capsule active ingredients

A total of 59 candidate active ingredients were screened from the TCMSP, including 25 from Crataegi Folium, 29 from Dahurian rose fruit, and 5 shared ingredients. Details are shown in Table 1. The obtained candidate ingredients and all of the potential targets were applied to construct a network of ingredient-target interactions, including 330 nodes (59 ingredients and 271 targets) and 928 edges, as shown in Fig. 1. The degree of targets was adopted as a characteristic topological parameter to define the significance of candidate ingredients. The interactions indicated that one ingredient could regulate numerous targets, such as quercetin, kaempferol, β-sitosterol, isorhamnetin, ursolic acid, chlorogenic acid and so on, suggesting that Shanmei Capsule has multi-ingredients and multi-target characteristics for the treatment of hyperlipidemia.

Table 1

A list of the selected compounds in Shanmei Capsule for network analysis

Source	Compound	Molecular formula	CAS number
Crataegi Folium	Tyramine	C8H11NO	51-67-2
Crataegi Folium	Vitamin c	C6H8O6	50-81-7
Crataegi Folium	Cyanidol	C15H11ClO6	528-58-5
Crataegi Folium	Heriguard	C16H18O9	327-97-9
Crataegi Folium	Caffeic acid	C9H8O4	331-39-5
Crataegi Folium	Vitamin-G	C6H8O6	83-88-5
Crataegi Folium	Isorhamnetin	C16H12O7	480-19-3
Crataegi Folium	Vitexin	C21H20O10	3681-93-4
Crataegi Folium	Caffeate	C9H8O4	3843-74-1
Crataegi Folium	Nicotinic acid	C6H5NO2	59-67-6
Crataegi Folium	Hyperin	C21H20O12	482-36-0
Crataegi Folium	Stigmasterol	C29H50O	68555-08-8
Crataegi Folium	Orientin	C21H20O11	28608-75-5
Crataegi Folium	Maslinic acid	C30H48O4	4373-41-5
Crataegi Folium	Octacosanol	C28H58O	557-61-9
Crataegi Folium	Thiamine	C12H18Cl2N4OS	70-16-6
Crataegi Folium	Orbitol	C6H14O6	50-70-4
Crataegi Folium	Ent-Epicatechin	C15H14O6	35323-91-2
Crataegi Folium	p-coumaric acid	C9H8O3	501-98-4
Crataegi Folium	Isoorientin	C21H20O11	4261-42-1
Crataegi Folium	2-methoxyphenethylamine	C9H13NO	2045-79-6
Crataegi Folium	(1S)-1-phenylethanamine	C8H11N	2627-86-3
Crataegi Folium	Vitexin-2-O-rhamnoside	C27H30O14	64820-99-1
Crataegi Folium	Diethylamine	C4H11N	109-89-7
Crataegi Folium	Valamine	C4H11N	78-81-9
Crataegi Folium/Dahurian rose fruit	Sitosterol	C29H50O	83-46-5
Crataegi Folium/Dahurian rose fruit	Rutin	C27H30O16	153-18-4
Crataegi Folium/Dahurian rose fruit	Kaempferol	C15H10O6	520-18-3
Crataegi Folium/Dahurian rose fruit	Ursolic acid	C30H48O3	77-52-1
Crataegi Folium/Dahurian rose fruit	Quercetin	C15H10O7	117-39-5
Dahurian rose fruit	luteolin-7-O-rutinoside	C27H30O15	25694-72-8
Dahurian rose fruit	Hyperoside	C21H20O12	482-36-0
Dahurian rose fruit	Isoliquiritigenin	C15H12O4	961-29-5
Dahurian rose fruit	β-sitosterol	C29H50O	64997-52-0
Dahurian rose fruit	Tiliroside	C30H26O13	20316-62-5
Dahurian rose fruit	Hesperidin	C28H34O15	520-26-3
Dahurian rose fruit	Oleanolic acid	C30H48O3	508-02-1
Dahurian rose fruit	Stearic acid	C18H36O2	57-11-4
Dahurian rose fruit	Palmitic acid	C18H36O2	57-10-3
Dahurian rose fruit	Oleic acid	C18H34O2	112-80-1
Dahurian rose fruit	Linoleic acid	C18H32O2	60-33-3
Dahurian rose fruit	Linolenic acid	C18H30O2	463-40-1
Dahurian rose fruit	Eicosadienoic acid	C20H36O2	2091-39-6
Dahurian rose fruit	Strictinin	C27H22O18	517-46-4
Dahurian rose fruit	Heptacosane	C27H56	593-49-7
Dahurian rose fruit	Cerotic acid	C26H52O2	506-46-7
Dahurian rose fruit	Daucosterol	C35H60O6	474-58-8
Dahurian rose fruit	Betulinic acid	C30H48O3	472-15-1
Dahurian rose fruit	Gallic acid	C7H6O5	149-91-7
Dahurian rose fruit	Furfuryl acetate	C7H8O3	623-17-6
Dahurian rose fruit	n-hexyl furan	C10H16O	3777-70-6
Dahurian rose fruit	2-furan-2-yl-pentanal	C9H12O2	31681-26-2
Dahurian rose fruit	β-sitosterol acetate	C31H52O2	915-05-9

Table 1, continued
Source	Compound	Molecular formula	CAS number
Dahurian rose fruit	2-amino-propionic acid ethyl ester	C5H11NO2	17344-99-9
Dahurian rose fruit	Isobutyl formate	C5H10O2	542-55-2
Dahurian rose fruit	Isoamyl acetate	C7H14O2	123-92-2
Dahurian rose fruit	Ethyl hexanoate	C8H16O2	123-66-0
Dahurian rose fruit	Ethyl 1-acetylcyclopropane-1-carboxylate	C8H12O3	32933-03-2
Dahurian rose fruit	Ethyl palmitoleate	C18H34O2	56219-10-4

Figure 1.

Establishment of component-target network. Components are shown as red dovetails nodes and target are marked as purple circular nodes.

3.2Prediction of candidate targets of hyperlipidemia

A total of 1018 potential targets to have correlations with hyperlipidemia were found via the GeneCards database. After merging hyperlipidemia-related targets and active ingredient targets, 34 overlapping targets were considered as candidate targets (Table 2, Fig. 2). Further linking of target ingredients with candidate targets based on Cytoscap 3.3.0 resulted in a network of disease-ingredient-target, with 51 nodes and 301 edges (Fig. 3). Among them, 17 red dovetail nodes represent active ingredients of Shanmei Capsule, and 34 purple ellipse nodes represent candidate targets of hyperlipidemia. After analyzing the topological characteristics of each node in the interaction network, it shows that the node degrees of eight targets, HMGCR, MMP9, MAPK8, PPARα, PPARγ, FABP4, ALOX5 and NOS2 are more than 13, which implied that these targets might be key targets for hyperlipidemia. Previous study found that these targets were primarily involved in inflammatory reactions, lipid metabolism, etc.

Table 2

Related parameters of potential targets of Shanmei Capsule

No.	Uniprot ID	Target gene	Target protein
1	P15121	AKR1B1	Aldo-keto reductase family 1 member B1
2	O60218	AKR1B10	Aldo-keto reductase family 1 member B10
3	P08253	MMP2	72 kDa type IV collagenase
4	P14780	MMP9	Matrix metalloproteinase-9
5	P03956	MMP1	Interstitial collagenase
6	P08254	MMP3	Stromelysin-1
7	P10636	MAPT	Microtubule-associated protein tau
8	P10275	AR	Androgen receptor
9	P04035	HMGCR	3-hydroxy-3-methylglutaryl-coenzyme A reductase
10	P11511	CYP19A1	Aromatase
11	P01130	LDLR	Low-density lipoprotein receptor
12	P03372	ESR1	Estrogen receptor
13	Q92731	ESR2	Estrogen receptor beta
14	P09917	ALOX5	Arachidonate 5-lipoxygenase
15	P08183	ABCB1	ATP-dependent translocase ABCB1
16	P35869	AHR	Aryl hydrocarbon receptor
17	P04626	ERBB2	Receptor tyrosine-protein kinase erbB-2
18	P00533	EGFR	Epidermal growth factor receptor
19	P05164	MPO	Myeloperoxidase
20	Q9UNQ0	ABCG2	ATP-binding cassette sub-family G member 2
21	P15090	FABP4	Fatty acid-binding protein
22	P07148	FABP1	Fatty acid-binding protein
23	P37231	PPARγ	Peroxisome proliferator-activated receptor gamma
24	Q07869	PPARα	Peroxisome proliferator-activated receptor alpha
25	P21554	CNR1	Cannabinoid receptor 1
26	P35354	PTGS2	Prostaglandin G/H synthase 2
27	P00734	F2	Prothrombin
28	Q96RI1	NR1H4	Bile acid receptor
29	P08246	ELANE	Neutrophil elastase
30	P07900	HSP90AA1	Heat shock protein HSP 90-alpha
31	P45983	MAPK8	Mitogen-activated protein kinase 8
32	Q16539	MAPK14	Mitogen-activated protein kinase 14
33	P29474	NOS3	Nitric oxide synthase
34	P35228	NOS2	Nitric oxide synthase

Figure 2.

Target-target interaction network between active ingredients of Shanmei Capsule against hyperlipidemia.

Figure 3.

Component-target-diseas interactive network of Shanmei Capsule. Red dovetails nodes are the main active ingredients of Shanmei Capsule, and purple circular nodes are the potential targets for treating hyperlipidemia.

3.3GO and KEGG pathway enrichment analysis

To further clarify relevant functions and pathways, GO functional analyses and KEGG pathway enrichment were performed for above 34 core targets using DAVID database. GO enrichment analysis was annotated in the following three categories, including biological process, cellular component and molecular function, with P< 0.01 serving as the threshold. There were 40 biological processes involved, including oxidation-reduction processes, intracellular receptor signaling pathway, regulation of inflammatory response, positive regulation of MAP kinase activity, etc. (Fig. 4A). In the cellular component group, GO terms were mainly included nucleus, nucleoplasm, extracellular space, perinuclear region of cytoplasm and basolateral plasma membrane (Fig. 4B). Figure 4C shows that the enriched molecular functions of targets are mainly associated with RNA polymerase II transcription factor activity, enzyme binding, steroid hormone receptor activity, etc. These results indicated that the potential mechanism of Shanmei Capsule against hyperlipidemia was related to oxidation-reduction processes, intracellular receptor signaling, and inflammatory response. A total of 15 signal pathways were enriched by KEGG pathway enrichment analysis (Table 3), including PPAR signaling pathway, Bile secretion and TNF signaling pathway, et al. Among them, Pathway in cancer is a cancer-related signaling pathway. Recent studies have reported that a carcinogenic role of the metabolic syndrome in variety of cancers [24]. Based on these results, Shanmei Capsule may attenuate hyperlipidemia partially by regulating inflammation or apoptosis, lipid metabolism and decrease oxidative stress during hyperlipidemia progression.

Table 3

Pathway enrichment of potential targets based on KEGG pathway analysis

No.	Pathway description	P value	Nr. Genes	Ratio
1	Pathways in cancer	0.00001	11	32.4
2	Estrogen signaling pathway	0.00001	7	20.6
3	Bladder cancer	0.00004	5	14.7
4	PPAR signaling pathway	0.00024	5	14.7
5	Bile secretion	0.00027	5	14.7
6	TNF signaling pathway	0.00140	5	14.7
7	Ovarian steroidogenesis	0.00150	4	11.8
8	Toxoplasmosis	0.00160	5	14.7
9	Proteoglycans in cancer	0.00210	6	17.6
10	Hepatitis C	0.00320	5	14.7
11	Prolactin signaling pathway	0.00420	4	11.8
12	Transcriptional misregulation in cancer	0.00710	5	14.7
13	Prostate cancer	0.00770	4	11.8
14	GnRH signaling pathway	0.00840	4	11.8
15	HIF-1 signaling pathway	0.00970	4	11.8

Figure 4.

Go and KEGG pathway enrichment bubble diagrams for candidate targets (P⩽ 0.01). (A) GO analysis of Biological processes, (B) GO analysis of cellular components, (C) GO analysis of molecular functions, (D) KEGG analysis.

Figure 5.

The component-target-pathway network for Shanmei Capsule. Purple circular nodes represent the candidate targets, red dovetails nodes represent the active ingredients and rose red parallelogram nodes represent the KEGG pathways. The size of nodes was proportional with the degree and larger sizes represent greater associations with hyperlipidemia treatment.

At last, in order to elucidate the connection between the core targets and corresponding pathways, a component-target-pathway interaction network map was generated by Cytoscape 3.3.0 (Fig. 5). The 17 red dovetails nodes represent the active pharmaceutical ingredient, the 34 purple circular nodes denote the potential targets, and the 15 rose red parallelogram nodes represent the pathways. And the node sizes depend on degree value. The larger the node is, the larger the degree value is. There were 66 nodes and 340 edges were involved in the association between these targets, which reflected the biological process of the key targets. These results were revealed that Shanmei Capsule take effect in hyperlipidemia treatment by multi-components, multi-targets and multi-pathways.

3.4Docking results analysis

Molecular docking was performed among 17 ingredients and 34 potential targets using the Autodock Vina software, and detailed results are presented in Fig. 6. Affinity was the binding score for the molecular docking, and when the docking score value was smaller, the binding affinity to target protein was stronger. The docking score <-7 indicated a high binding activity [25]. The molecular docking results revealed that the docking scores of 47% active ingredients were lower than -7, and the ingredients with the lowest scores among these targets were quercetin and chlorogenic acid (heriguard) (Fig. 6). PPAR-α is a key target of lifibrate (positive drug), which belongs to the nuclear hormone receptor family, which is linked to dyslipidemia and diabetes. The docking results showed that the binding activity of quercetin, kaempferol, isorhamsin and chlorogenic acid for PPAR-α are equivalent to, and even better than that of the positive drug lifibrate. The molecular docking models are illustrated in Fig. 7. These findings indirectly verified that Shanmei Capsule had a regulatory effect on hyperlipidemia targets. At the same time, the above molecular docking results were consistent with those of previous network screening, which verified the reliability of network pharmacology.

Figure 6.

The heat map of molecule docking scores.

3.5Results of drug ADME and toxicity estimations

The ADME properties of 17 active ingredients were calculated by SwissADME web tool. As is can be observed in Table 4, all the ingredients, besides heriguard, have bioavailability score above the threshold of 0.5, which might suggest good oral bioavailability. The majority of the tested ingredients have no ability to penetrate the blood-brain barrier, thus presenting a low risk for central nervous system side effects. Based on toxicity tests, it appears that all the ingredients have no nongenetic carcinogen properties, with only vitaminG was predicted to be genetic carcinogen. The results of SwissADME calculations and toxicity tests provided useful information about the selected ingredients and found to be free from high risks of undesired effects.

Table 4

ADME and toxicity prediction results of 17 key ingredients in Shanmei Capsule

No.	Compound	GI absorption	BBB permeant	Bioavailability score	Genetic carcinogen	Nongenetic carcinogen	Ames
1	β-Sitosterolacetate	Low	No	0.55	-	-	-
2	Eicosadienoicacid	High	No	0.56	-	-	-
3	Ergosterol	Low	No	0.55	-	-	-
4	2-amino-propionic acid ethyl ester	High	No	0.55	-	-	-
5	Ethylpalmitoleate	High	Yes	0.55	-	-	-
6	Heriguard	Low	No	0.11	-	-	-
7	Isoliquiritigenin	High	Yes	0.55	-	-	+
8	Isorhamnetin	High	No	0.55	-	-	+
9	Kaempferol	High	No	0.55	-	-	-
10	Linoleicacid	High	Yes	0.56	-	-	-
11	Linolenicacid	High	Yes	0.56	-	-	-
12	Oleicacid	High	No	0.56	-	-	-
13	Quercetin	High	No	0.55	-	-	+
14	Sitosterol	Low	No	0.55	-	-	-
15	Stigmasterol	Low	No	0.55	-	-	-
16	Ursolicacid	Low	No	0.56	-	-	-
17	VitaminG	Low	No	0.55	+	-	+

Notes: (-) negative; (+) positive.

Figure 7.

Molecular models of bioactive ingredients and positive drug binding to the predicted PPARα target. (A) Quercetin, (B) Heriguard, (C) Lifibrate.

4.Discussion

Hyperlipidemia is one of the most prevalent global chronic metabolic diseases, which usually involved in a disorder of lipid metabolism and accompanied by elevated contents of TC, TG, LDL-C, as well as decreased HDL-C levels. During this process, blood lipid level, oxidative stress, inflammation, and other factors might be the key link of the development of dyslipidemia-induced hyperlipidemia. Shanmei Capsule, a famous preparation in China, is made of a combination of Crataegi Folium and Dahurian rose fruit ethanol extract in proportion. Crataegi Folium and Dahurian rose fruit both are well-known traditional medicinal plants, which have been confirmed to possess various biological and pharmacological activities, such as anti-inflammatory, antioxidant and hypolipidaemic effects. Therefore, in this study, network pharmacology approach and molecular docking were applied to investigate the potential mechanism of multi-ingredient, multi-targets, and multi-pathway on Shanmei Capsule against hyperlipidemia (Fig. 8) [26, 27, 28, 29, 30].

Figure 8.

Detailed signaling pathway of Shanmei Capsule in the treatment of hyperlipidemia.

Via network pharmacology analysis, a total of 17 key active ingredients were regarded to be effective on hyperlipidemia, including quercetin, kaempferol, isorhamnetin, chlorogenic acid, ursolic acid and β-sitosterol, etc. Quercetin, one of antioxidant substances, has been reported that it could upregulate the expression of PPARα. Quercetin and its metabolites induced eNOS activity by AMPK phosphorylation, which resulted in an improvement in vessel function. They were also inhibited the formation of foam cell through activating PPARγ-ABCA1 pathway, and thereby improving lipid levels [31, 32]. Foam cell accumulation in atherosclerotic lesions is a critical early stage process of atherosclerosis. Treatment with kaempferol markedly suppresses oxidized low-density lipoprotein-induced macrophage foam cell formation, which promotes an increase in cholesterol efflux and a decrease in lipid accumulation in foam cells [33]. Zhang et al. [34] indicated that isorhamnetin is a dietary source of PPARγ antagonist. Isorhamnetin treatment inhibited the adipocyte differentiation induced by the PPARγ agonist rosiglitazone, and ameliorated insulin resistance, which may be beneficial to prevent obesity development and hepatic steatosis. Several researchers reported that anti-lipidemic and Anti-diabetic effects of chlorogenic acid are mediated through the activation of AMPK (AMP-activated protein kinase). Chronic administration of chlorogenic acid inhibited hepatic glucose-6-phosphatase expression and activity, improved lipid homeostasis and skeletal muscle glucose uptake, attenuated hepatic steatosis, which in turn improved glucose tolerance, fasting glucose level, dyslipidemia and insulin sensitivity in Leprdb/db mice [35, 36]. Ursolic acid is a triterpenoid compound commonly present in fruits, vegetables and TCM, and that have a wide range of biological functions, such as anti-inflammatory, antihyperlipidemic, hypoglycemic and antioxidant properties [37]. He et al. [38] showed that ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis via the LKB1/AMPK pathway, so as to suppress 3T3-L1 preadipocyte differentiation and lipid accumulation by regulating the transcriptional factors and their downstream lipogenic targets. β-sitosterol is a plant sterol. Experimental and clinical studies have shown that β-sitosterol has hypolipidemic, anti-diabetic, hepatoprotective, anti-cancer, and anti-arthritic role [39]. In summary, the main active ingredients (e.g., quercetin, kaempferol, isorhamnetin, chlorogenic acid, ursolic acid and β-sitosterol) of Shanmei Capsule in the treatment of hyperlipidemia could regulate multiple cellular signaling pathways, thereby involving in the regulation of lipid synthesis and metabolism, lowering cholesterol levels, and preventing or delaying the development of dyslipidemia.

Thirty-four potential targets consist of HMGCR, MMP9, MAPK, PPARα, PPARγ, FABP4, NOS2, etc., were screened as effective targets of Shanmei Capsule against hyperlipidemia. HMGCR (HMG-CoA reductase), is a rate-limiting enzyme of cholesterol biosynthesis. HMGCR inhibitors (e.g., statins) are currently the mainstay of treatment for dyslipidemia, which can reduce the levels of cholesterol via blocking mevalonate synthesis [40]. MMP9, belongs to matrix metalloproteinase (MMP) molecular family, has been considered as a key target for treating metabolic diseases. Studies have shown that MMP inhibitor was closely associated with calcific aortic valve disease (CAVD) among high fat induced mice, and that these enzymes might be important targets for preventing CAVD development [41]. Intracellular mitogen-activated protein kinases (MAPK) are an important class of proline-dependent protein kinases, which involved in the regulation of various biological functions, such as inflammation, cell proliferation and differentiation. Aouadi et al. [42] demonstrated that p38MAPK increased adipogenesis via inhibition of C/EBPβ and PPARγ activations. PPAR, a member of the nuclear receptor transcription factor family, has 3 isoforms: PPARα, PPARβ and PPARγ. It is known that lipid metabolism could be regulated by PPAR, which were shown to decrease lipoprotein secretion, accelerate triglyceride catabolism, and influences serum total- and LDL-cholesterol levels. On the other hand, PPARγ stimulates the fatty acids storage in adipocytes and regulates glucose metabolism, thereby improving insulin sensitivity [43]. FABP4, also known as Adipocyte fatty acid–binding protein (A-FABP), is a major cytoplasmic protein in adipocytes. It is shown that FABP4 is expressed at high levels in adipose tissues, especially in adipocyte differentiation [44, 45, 46]. NO is generated from L-arginine catalyzed by a class of nitric oxide synthases (NOS), including neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). All three NOS isoforms were expressed in atherosclerotic plaque. Once the enzyme is expressed, iNOS is regulated by the transcription fact and catalyzes the high expression of NO. On the contrary, eNOS and nNOS produce a small number of NO in a highly regulated way [47]. The above results indicated that Shanmei Capsule treatment may play its role of hypolipidemic via multi-targets.

The KEGG enrichment analysis indicated that the main pathways involved in the hyperlipidemia treatment process are PPAR signaling pathway, Bile secretion and TNF signaling pathway. PPAR signaling pathway participate in many physiological processes implicated in cardiovascular diseases associated with hyperlipidemia and diabetes, including lipid metabolisms, energy metabolisms, and inflammatory reactions, etc. PPARα is an essential transcription factor involved in lipid metabolism and inflammation, and there is predominantly expressed in β-oxidation activities and tissues with high mitochondrial (e.g., liver) [48]. While PPARγ is mainly expressed in immune system and adipose tissue, where it modulates genes involved in cell differentiation, cell cycle regulation, insulin sensitivity and glucose uptake [49]. Bile acids are famous for their effects on lipid digestion and cholesterol homeostasis. The conversion of cholesterol to bile acids (i.e., bile acid synthesis) in the liver is the major pathway of cholesterol removal from the body [50], which itself is endogenous ligands of the farnesoid X receptor (FXR). FXR, together with small heterodimer partner (SHP), plays an important role in linking bile acids regulation with lipid, lipoprotein, and glucose metabolism [51, 52]. As for TNF signaling pathway, it has direct effect on inflammation, whereas many cytokines such as TNF-α are able to increase lipolysis, which in turn contributes to a chronic state of insulin resistance and increases circulating free fatty acids. It has been reported that expanding adipose tissue results in chronic low-grade inflammation that leads to the development of metabolic disorders, including insulin resistance, dyslipidemia, and type 2 diabetes [53, 54].

The potential binding sites and intermolecular interaction of active ingredients for target proteins were further elaborated by docking analysis. PPARα is commonly considered as a key target of lifibrate, and at the same time it is also an important target screened from the network pharmacology of Shanmei Capsule. The melocule docking results confirmed the potential hypolipidemic mechanism of Shanmei Capsule, showing excellent affinity towards PPARα target protein. The above findings also support the network pharmacology analysis results of Shanmei Capsule. Therefore, such compounds could provide a molecular foundation for the subsequent development of novel pharmacological inhibitors against hyperlipidemia.

5.Conclusion

In the present study, a network pharmacology approach in combination with molecular docking was proposed to explore the underlying mechanism of action of Shanmei Capsule against hyperlipidemia. The active ingredients of Shanmei Capsule in hyperlipidemia treatment are composed of 59 ingredients. Among them, quercetin, kaempferol, isorhamsin and chlorogenic acid are main active ingredients. The possible molecular mechanism mainly involved PPAR signaling pathway, Bile secretion and TNF signaling pathway via acting on MAPK8, PPARγ, MMP9, PPARα, FABP4 and NOS2 targets. In conclusion, Shanmei Capsule, as a traditional Chinese medicine preparation, has multiple constituents, multiple targets and multiple pathways properties, which could serve as a promising therapeutic drug for hyperlipidemia.