The advantages of TCM in the treatment of NAFLD have gradually emerged that single medicine or a combination of various medicines can regulate liver lipid metabolism, promote absorption and excretion, and avoid adverse reactions caused by long-term use of western medicine.
In this study, the targets of Cynarine were obtained by predicting and database searching. Then 48 targets for Cynarine in treatment of NAFLD were obtained by cross-mapping them with NAFLD -related targets in GeneCards, GEO, OMIM, and CTD databases. With the help of network pharmacology and network analysis, the " Cynarine target- NAFLD target" network topology analysis was carried out. The PPI network was constructed, which showed that these target proteins did not act alone but had complex and interlocking interactions. The targets with high degree value had better connectivity which was essential for the entire regulatory network . Therefore, they may play a central role in the treatment of NAFLD by Cynarine, which deserves our attention. The core targets include CASP3, TP53, VEGFA, MMP9, ELANE etc. CASP3 (caspase-3), which has the highest degree value, has been reported to a prominent role in hepatocyte apoptosis, fibrogenesis and fibrosis in a mouse model of nonalcoholic steatohepatitis . It has been reported that p53 (TP53), another target p53 (TP53) with a high degree value, regulated various aspects of NAFLD development which was linked to insulin resistance, inflammation, lipid metabolism, and oxidative stress . Also, MMP9, ELANE, NOTCH1, with fewer connected targets, have all been proven to have important regulatory effects in reducing liver lipid accumulation, inflammatory responses, fibrosis, and improving insulin resistance . Pivotal targets in the PPI network were closely related to immunity and inflammation. Inflammation in liver cells led to the release of pro-inflammatory cytokines and the increase of apoptosis which was seen as a driving force for NAFLD progress . The activation of immune cells could also trigger liver cell apoptosis, leading to tissue damage and laying the foundation for advanced fibrosis and liver cirrhosis . Therefore, exploring inflammation and immune-related targets can improve the understanding of the underlying mechanisms of NAFLD and help the development of new therapies. The above research results indicate that the multiple core targets predicted in this study have an important role in regulating NAFLD, which is valuable for further research.
NAFLD is a complex disease, with lipid accumulation and inflammation being two important steps in its pathogenesis and progression, as well as two important links in the "second hit" theory. Therefore, reducing liver fat accumulation and reducing inflammation is the key to treat NAFLD.
The results of GO functional enrichment analysis in this study show that NAFLD's therapeutic targets were mainly involved in the regulation of biological processes such as transcription, translation, and inflammation. These processes have the effects of improving metabolism, promoting tissue repair and reducing inflammation, and participating in the repair process of NAFLD diseased tissues from many aspects, which are consistent with the pathogenesis of NAFLD.
Analysis of target-pathway integration network based on the KEGG pathway enrichment showed that PI3K-Akt signaling pathway, cell cycle and MAPK signaling pathway were enriched by more targets, indicating that these three pathways played an important role in the treatment of NAFLD by Cynarine. The three pathways are closely related to the onset and progression of NAFLD. PI3K-Akt signaling pathway can be abnormally activated by various toxic insults or cellular stimuli related to NAFLD . Cell cycle was closely related to the metabolic regulation of NAFLD . A study demonstrated that the inhibition of MAPK signaling pathway decreased hepatocyte apoptosis and improved hepatic fibrosis in rats with NAFLD . In addition, Proteoglycans, Thyroid hormone signaling pathway, and Human cytomegalovirus infection have also been shown to be closely related to NAFLD in the results of KEGG pathway enrichment analysis . They were all enriched with more Cynarine therapeutic targets, suggesting Cynarine may interfere with inflammation and immunity, metabolic homeostasis, lipotoxicity, liver cell death and other pathogenesis and progression mechanisms in NAFLD by regulating these pathways.
Cell experiment results showed that Cynarine could reduce the fat deposition ability of NAFLD model cells, and effectively reduce the levels of ALT and AST released by liver cells due to excessive lipid accumulation. We also found that Cynarine can also inhibit the expression of AKT1 and MAPK1.