Fibrotic effect of monoculture, conditioned media and coculture of fibroblast NIH3T3 cells on collagen secretion and mRNA TGF-beta expressions

Abstract

Fibrosis is common profound in diabetes condition, they characterized by accumulation of extracellular matrix in tissue then cause tissue injury. The study aim to investigate effect of monoculture, conditioned media, and coculture fibroblast of NIH3T3 cells (NIH3T3 cells) in LPS-high glucose media (HG-LPS media) conditions on mRNA TGF-β and collagen secretion. The fibroblast NIH3T3 were cultured with various culture type there is mono culture NIH3T3 cells, conditioned media from macrophage RAW264.7 cells culture media, and coculture with RAW264.7 cells (ratio 1:1 and 1:2) in normal media (media containing 4500 µg/mL of glucose) and HG-LPS media (media containing LPS 0.25 µg/mL and D-glucose 5400 µg/mL). Our findings shows that the TGF-β mRNA expression in RAW 264.7 and NIH 3T3 cell coculture media with a culture ratio of 1:1 (Coculture 1:1) were the highest (p<0.05) compared than monoculture of NIH3T3 cells (cultured in normal media or HG-LPS Media), conditioned media from RAW264.7 cells in normal media (CM-M0 Media), and the RAW 264.7 and NIH 3T3 co-culture cells with 1:2 cultured ratio in HG-LPS Media (Coculture 1:2). On other hand, the the relative ratio of collagen secretion on Coculture 1:1 were the highest compared to CM-M0 media (p<0.05). Our study conclude that RAW 264.7 and NIH 3T3 coculture cells with a (1:1) cultured ratio in LPS and high glucose media (Coculture 1:1) is appropriate for diabetic fibrosis in vitro models.