Determination of the Underlying Mechanisms for the Anti-fibrogenic Effect of n-3 Polyunsaturated Fatty Acids in Hepatic Stellate Cells (P08-105-19)

We previously reported that n-3 polyunsaturated fatty acids (PUFAs) exhibit potent anti-fibrogenic effects independent of the SMA- and MAD-related protein (SMAD3) pathway in primary human hepatic stellate cells (HSCs), the major cell type responsible for extracellular matrix production for the development of liver fibrosis. The objective of this study was to further elucidate the underlying mechanisms for the anti-fibrogenic effect of n-3 PUFAs in HSCs with a primary focus on peroxisome proliferator-activated receptor γ (PPARγ) as it is known to inhibit HSC activation. n-3 PUFAs, including alpha linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were complexed with bovine serum albumin (BSA) at a BSA to fatty acid molar ratio of 1:2.5. Primary human HSCs were treated either with BSA vehicle control or 50 μM of a fatty acid for 24 h, after which they were activated with transforming growth factor β1 (TGFβ1; 4 ng/ml), a potent fibrogenic cytokine, for additional 24 h. Also, in another experiment, primary human HSCs were pre-treated with 10 μM of GW9662, a PPARγ antagonist, 18 h prior to the fatty acid and TGFβ1 treatments. ALA, EPA and DHA significantly decreased TGFβ1-induced mRNA expression of fibrogenic genes such as α-smooth muscle actin and collagen type I alpha 1 chain with EPA and DHA being more potent than ALA. The similar trend was also observed in their protein levels. In the absence of TGFβ1 treatment, EPA and DHA markedly increased PPARγ mRNA abundance. TGFβ1 significantly decreased PPARγ mRNA, which was attenuated by EPA and DHA, but not by ALA. Inhibition of PPARγ by GW9662 did not alter either the basal expression of PPARγ or the basal expression of fibrogenic genes. However, the inhibitory effect of EPA and DHA on the induction of fibrogenic gene expression by TGFβ1 was not diminished by GW9662. n-3 PUFAs showed a potent inhibitory effect on TGFβ1-induced fibrogenic gene expression in primary human HSCs. Inhibition of PPARγ activity did not alter the anti-fibrogenic effect of n-3 PUFAs. The results suggest that n-3 PUFAs, particularly EPA and DHA to a similar degree, exert their anti-fibrogenic effect in primary human HSCs independent of the activation of PPARγ pathway. This study was supported by USDA Multistate Hatch and USDA Hatch.