Impact of 4-methylsculetin on inflammation and membrane integrity in Caco-2 Cells

Coumarins are a group of promising natural compounds from a biotechnological standpoint. They possess a wide range of biological activities, including anti-inflammatory, antioxidant, and antimicrobial properties, making them potential candidates for theranostic drug applications. These compounds are commonly found in various plant species, particularly in aromatic, food, and medicinal plants. The derivative 4-methylsculetin (4-ME) has demonstrated significant efficacy in in vivo models of intestinal inflammation, indicating its potential use in treating inflammatory bowel diseases. In this study, we utilized an in vitro model of Caco-2 cells to simulate both healthy and controlled inflamed states of the intestine, investigating the effects of 4-methylsculetin (4-ME) on maintaining cell barrier integrity. The inflamed state was induced in Caco-2 cells by stimulating them with lipopolysaccharide (LPS), both with and without the presence of 4-ME, followed by immunocytochemical staining. Enzyme-linked immunosorbent assay (ELISA) was conducted to evaluate cytokine production in the Caco-2 cell cultures. Additionally, cytotoxicity tests were performed to assess the potential toxicity of 4-ME, with the highest concentration tested (100 µM/mL) showing over 85% cell viability. The in vitro assays indicated that 4-ME inhibited the production of IL-1β and TNF-α in the Caco-2 cell cultures. Microscopic analysis confirmed the integrity of tight junction (TJ) membranes in untreated Caco-2 cells, as shown by staining for nuclei (DAPI), the cytoskeleton (Phalloidin), and the TJ protein zonula occludens (ZO)-1. In inflamed cultures, the TJ network and cytoskeleton appeared irregular and less organized compared to the control, with nuclear fragmentation also observed. Confocal microscopy revealed that 4-ME was absorbed and localized primarily in the mitochondrial region of Caco-2 cells. The intestinal anti-inflammatory activity of 4-ME was demonstrated by its ability to mitigate morphological damage in the Caco-2 cells.

This work was supported by FAPESP (Process nº: 2023/00997-7, 2020/15185-0 and 2018/22214-6).