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

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, PPAR α , PPAR γ , 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.

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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 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.

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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.

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.

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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.

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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].

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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 PPAR α . 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 PPAR γ -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 PPAR γ antagonist. Isorhamnetin treatment inhibited the adipocyte differentiation induced by the PPAR γ 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, PPAR α , PPAR γ , 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/EBP β and PPAR γ activations. PPAR, a member of the nuclear receptor transcription factor family, has 3 isoforms: PPAR α , PPAR β and PPAR γ . 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, PPAR γ 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. PPAR α 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 PPAR γ 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. PPAR α 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 PPAR α 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.

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, PPAR γ , MMP9, PPAR α , 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.