Asiatic Acid Modulates Il6 and Tgfb1 mRNA Expression and Soluble Collagen Levels in RAW 264.7–NIH 3T3 Co-culture Cells under High-Glucose and LPS Stimulation

Abstract

BACKGROUND: Chronic inflammation contributes to diabetes-associated fibrosis, through persistent hyperglycemia, leading to increased interleukin-6 (IL-6), transforming growth factor-β (TGF-β), and collagen levels. While metformin effectively controls blood glucose, it may not adequately suppress these pathogenic pathways. Asiatic acid has demonstrated anti-inflammatory and antifibrotic properties, although its effects within a direct-contact macrophage–fibroblast microenvironment such as in co-culture models remain unclear. This study was conducted to evaluate the effects of asiatic acid on Il6, Tgfb1, and soluble collagen levels in a RAW 264.7–NIH 3T3 co-culture under high-glucose and lipopolysaccharide (HG–LPS) stimulation.

METHODS: RAW 264.7 and NIH 3T3 cells were co-cultured at ratios of 1:1–1:6, and the selected co-culture was treated with asiatic acid (2.5, 5.0, and 10.0 µg/mL) for 24 h while HG–LPS stimulation was maintained. Unstimulated NIH 3T3 monocultures and untreated HG–LPS co-cultures served as controls. The inflammatory and profibrotic markers such as Il6 and Tgfb1 mRNA expression were quantified by polymerase chain reaction (PCR), and soluble collagen was measured using a modified Sirius Red assay.

RESULTS: Among the tested ratios, the 1:1 co-culture exhibited the highest Tgfb1 mRNA expression (0.35±0.02) and soluble collagen level (1.30±0.00) and was selected for subsequent experiments, whereas Il6 mRNA expression did not differ significantly among co-culture ratios. Under HG–LPS stimulation, Il6 and Tgfb1 mRNA expression increased 3.5-fold and 10.3-fold, respectively, compared with unstimulated NIH 3T3 monocultures. Asiatic acid significantly reduced Il6 mRNA expression at 5.0 and 10.0 µg/mL, Tgfb1 mRNA expression at 2.5 µg/mL, and soluble collagen levels at all tested concentrations, indicating a non-monotonic concentration–response pattern.

CONCLUSION: The 1:1 RAW 264.7–NIH 3T3 co-culture was the most responsive under HG–LPS stimulation and could be in vitro platform for investigating macrophage–fibroblast interactions under hyperglycemic inflammatory conditions. Asiatic acid modulated Il6 and Tgfb1 mRNA expression and soluble collagen levels in RAW 264.7–NIH 3T3 co-culture, which suggest that Asiatic acid may be potential for the management of fibrotic inflammation.

KEYWORDS: co-culture, collagen, fibroblast dysfunction, NIH 3T3 fibroblast, RAW 264.7 macrophage