BRAZILIAN ORGANIC PROPOLIS MODULATES HEPATIC GENE EXPRESSION AND INFLAMMATORY PATHWAYS

Propolis is a resinous substance produced by bees and widely recognized for its remarkable biological properties, including antioxidant, antimicrobial, and anti-inflammatory activities. This study investigated the bioactive potential of organic propolis-type 1 (OP1), collected from permanent preservation forests in southern Paraná, Brazil. Previous research has demonstrated that OP1 maintains its biological properties and chemical stability after simulated gastrointestinal digestion and epithelial transport of its bioactive compounds (SALIBA et al., 2023). Building upon these findings, the present work aimed to elucidate how OP1-derived compounds modulate hepatic gene expression using an in vitro co-culture model that mimics the intestinal-liver axis. An ethanolic extract of OP1 was subjected to simulated gastrointestinal digestion according to the standardized INFOGEST protocol (BRODKORB et al., 2019). The digested extract was then applied to a Caco-2/HepG2 co-culture system, in which Caco-2 cells represented enterocytes and HepG2 cells simulated hepatocytes. This setup allowed the basolateral fraction obtained after epithelial transport to interact with liver cells, reflecting post-absorptive metabolism of propolis compounds. A control group without treatment was used as the reference for normal gene expression. To identify phenolic compounds, present in the basolateral fraction, liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QToF MS) analysis was performed. The results revealed the presence of compounds belonging to several bioactive classes, including terpenes, flavonoids, lignans, and phenolic acids, all of which are widely described in the literature for their anti-inflammatory potential. Following treatment, total RNA was extracted from HepG2 cells, and sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (Illumina, USA) and sequenced on an Illumina NovaSeq S4 platform. Differential gene expression analysis was conducted using the DESeq2 package in R, and functional enrichment analysis using ClueGO v.2.5.10 in Cytoscape v.3.10.1 identified several significantly enriched Gene Ontology (GO) terms. Among these, GO:0006954, associated with the inflammatory response, showed negative modulation. Notably, down-regulated genes (p < 0.05) included CXCL5, CX3CL1, and IL-34, all linked to inflammation, angiogenesis, and disease progression, as well as CD36, whose reduced expression is associated with decreased lipid accumulation and lower risk of non-alcoholic fatty liver disease (NAFLD). In conclusion, OP1-derived compounds appear to modulate key genes involved in inflammation and lipid metabolism, suggesting potential hepatoprotective and anti-inflammatory effects. These findings support the promising role of OP1 as a natural bioactive source, encouraging further in vivo studies to explore its pharmacokinetics and therapeutic potential in metabolic and inflammatory disorders.