Aquaporins (AQPs) as marker in the Physiology of inflammation and its interaction studies with Garcinol

Abstract

Aquaporins mainly AQP1, AQP3 and AQP4 have been known to be involved in the pathophysiology of inflammation based on earlier reports. This in vitro study using both RAW264.7 and THP-1 cells aimed to evaluate the involvement of Aquaporins as potential target of inflammation and the efficacy of methanolic extract of Garcinia (GME) and its potent phytocompound (Garcinol) against the different markers of inflammation including the Aquaporins involved. siRNA silencing of AQP3 was carried out in RAW264.7 cells followed by LPS stimulation (1µg/ml) and assessment of important markers of inflammation including NO, PGE2, TNF-α, IL-6, IL-1β, CCL20, iNOS and COX-2. To assess the anti-inflammatory potential of Garcinia extract and its active component Garcinol, cells were treated with 1µg/ml LPS in the absence and presence of increasing concentrations of GME and Garcinol. During the experimental period, extract concentrations (115µg/ml and 230 µg/ml for RAW264.7; 118 µg/ml and 236 µg/ml for THP-1) and Garcinol concentrations (6µM and 12µM for RAW264; 3µM and 6µM for THP-1) based on the IC50 determined by MTT assay were used. The anti-inflammatory effects were assessed by measuring the levels of secretory proteins of TNF-α, IL-1β, IL-6, and CCL20 in LPS-activated cells. qPCR and Western blot analysis were performed to measure the AQP expression both at transcriptional and translational levels respectively. AQP3 knock down significantly decreased the NO, PGE2, TNF-α, IL-1β levels as well as iNOS and COX-2 mRNA expression. LPS stimulation led to significant increase in the mRNA and protein level expression AQP1, AQP3 and AQP4 in RAW264.7 cells; AQP1 and AQP3 in THP-1 cells indicating their role as markers of inflammation. GME and Garcinol effectively suppressed the LPS-induced proinflammatory cytokine release in dose-dependent manner in both the cell lines. These results indicate that AQP1, AQP3 and AQP4 could play a crucial role as markers of inflammation. Anti-inflammatory agents like Garcinia could potentially decrease the expression of such AQPs, thus inhibiting the process of inflammation.