Mechanisms of Cynarine for treatment of non-alcoholic fatty liver disease based on the integration of network pharmacology, molecular docking and cell experiment

Background

Nonalcoholic Fatty Liver Disease (NAFLD) is a chronic Liver Disease prevalent all over the world. It has become more and more common in Japan, China and most western developed countries. The global prevalence rate is 25.24%, and the trend is increasing year by year. Related studies have shown that Cynarine has certain liver protection, lipid lowering and immune intervention effects. So, this study to systematically predict and analyze the mechanism of Cynarine in the treatment of non-alcoholic fatty liver disease (NAFLD) based on the integration of network pharmacology, molecular docking, and cell experiment.

Methods

We performed Heatmap and Venn diagram analyses to identify genes and targets in Cynarine treat NAFLD. The network of Cynarine-therapeutic targets and the protein-protein interaction network (PPI) was constructed. We used gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to visualize associated functional pathways. The Sybyl tool was used to dock the Cynarine with key therapeutic targets molecularly. Finally, cell experiments were applied to validate the role of Cynarine in the treatment of NAFLD.

Results

The Cynarine could act on 48 targets of NAFLD, and the role of CASP3, TP53, MMP9, ELANE, NOTCH1 were more important. The PPI network showed that immune and inflammation-related targets played a pivotal role. The KEGG analysis found that the PI3K-Akt signaling pathway, cell cycle and MAPK signaling pathway may be the main pathways for Cynarine to prevent and treat NAFLD. Molecular docking studies confirmed that Cynarine has good binding activity with therapeutic targets. Cynarine reduced the fat deposition ability of NAFLD model cells, and effectively reduced the levels of ALT and AST released by liver cells due to excessive lipid accumulation. We also found that Cynarine inhibited the expression of AKT1 and MAPK1.

Conclusions

This study revealed that Cynarine could significantly reduce the fat deposition ability of NAFLD model cells, which may be closely related to the effective regulation of AKT1 and MAPK1 expression by Cynarine.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases, which affects approximately 25% of adults worldwide. It is characterized by fatty degeneration of liver parenchymal cells with the absence of heavy drinking and no other clear liver damage factors [1]. In recent years, the prevalence of NAFLD in China has been increasing and with a trend of patients getting younger. Studies have shown that the prevalence of NAFLD in urban areas and rural areas in China is 21.83 and 20.43% [2]. At first, NAFLD was considered to be a kind of benign disease. With the continuous in-depth study of the disease, it has been discovered that NAFLD will possibly develop into liver fibrosis and is closely related to end-stage liver diseases such as liver cirrhosis, liver cancer, and liver failure, which poses a serious threat to public health [3, 4]. At present, it is not very clear about the pathogenesis of NAFLD, and its treatment measures are mainly to change bad living habits and control metabolic factors [1]. A single treatment method is difficult to achieve the desired effect. However, blindly multi-drug intervention may increase the burden on the liver. Therefore, looking for an effective and diversified approach to prevention and treatment is of great significance for treating NAFLD and could reduce the incidence of end-stage liver disease caused by it. Based on dialectical treatment, the Traditional Chinese Medicine (TCM) compound that has been clinically verified for a long time and has the advantages of multiple action pathways is undoubtedly worthy of further research.

Cynarine is one of the major caffeoylquinic acid compounds extracted from the TCM artichoke (Cynara scolymus L.), which has anti-obesity and anti-oxidant liver effects in high fat diet-induced obese rats [5, 6]. It has also been reported that Artichoke leaf extract has liver-protective effects and causes downregulation of oxidative stress in acute diazinon-induced liver injury in rats [7]. Therefore, it is concluded that Cynarine may play a role in treating NAFLD.

However, the current research is mostly limited to a certain target or a certain pathway to discuss the action mechanism of Cynarine. The characteristics of its multi-target and multi-pathway couldn’t be reflected, which failed to explain the mechanism of Cynarine in protecting liver. Network pharmacology combines multi-disciplinary research methods and content, including hierarchical network construction and analysis, bioinformatics, computational biology, and multi-directional pharmacological analysis. It can transform from a single target and single approach research form to a holistic and systematic form on exploring the relationship between drugs, targets, and diseases, consistent with the overall view of Chinese Medicine [8].

In this study, a method of the integration of network pharmacology, molecular docking and cell experiments was used to discover cynarine’s multiple targets and multiple pathways for the treatment of NAFLD, which provided a scientific basis and research direction for the systematic development of Cynarine.