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, 56 (10), 1936-46

Docosahexaenoic Acid Attenuates Western Diet-Induced Hepatic Fibrosis in Ldlr-/- Mice by Targeting the TGFβ-Smad3 Pathway

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Docosahexaenoic Acid Attenuates Western Diet-Induced Hepatic Fibrosis in Ldlr-/- Mice by Targeting the TGFβ-Smad3 Pathway

Kelli A Lytle et al. J Lipid Res.

Abstract

DHA (22:6,ω3), but not EPA (20:5,ω3), attenuates Western diet (WD)-induced hepatic fibrosis in a Ldlr(-/-) mouse model of nonalcoholic steatohepatitis. We examined the molecular basis for the differential effect of dietary EPA and DHA on WD-induced hepatic fibrosis. DHA was more effective than EPA at preventing WD-induced effects on hepatic transcripts linked to fibrosis, including collagen 1A1 (Col1A1), transforming growth factor-β (TGFβ) signaling and proteins involved in remodeling the extracellular matrix, including metalloproteases, tissue inhibitors of metalloproteases, and lysyl oxidase subtypes. Examination of the TGFβ pathway showed that mice fed the WD supplemented with either olive oil or EPA had a significant (≥2.5-fold) increase in hepatic nuclear abundance of phospho-mothers against decapentaplegic homolog (Smad)3 when compared with mice fed the reference diet (RD); Smad3 is a key regulator of Col1A1 expression in stellate cells. In contrast, mice fed the WD supplemented with DHA had no increase in phospho-Smad3 when compared with mice fed the RD. Changes in hepatic phospho-Smad3 nuclear content correlated with proCol1A1 mRNA and protein abundance. Pretreatment of human LX2 stellate cells with DHA, but not other unsaturated fatty acids, blocked TGFβ1-mediated induction of Col1A1. In conclusion, DHA attenuates WD-induced fibrosis by targeting the TGFβ-Smad3-Col1A1 pathway in stellate cells.

Keywords: collagen1A1; inflammation; nonalcoholic steatohepatitis; stellate cells.

Figures

Fig. 1.
Fig. 1.
Inflammatory and endocrine factors affecting liver fibrosis. Plasma levels of TLR2 (A) and TLR4 (B) activators were quantified using a cell-based assay as described in Materials and Methods. Results are represented as TLR activation units (U)/ml (n = 8; mean ± SD). The feeding groups were RD, WD + O, WD + E, and WD + D. Plasma TNFα (C) and leptin (D) were quantified as described in Materials and Methods. Results are expressed as pg/ml and ng/ml, respectively (mean ± SD; n = 8). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O (ANOVA).
Fig. 2.
Fig. 2.
Diet effects on the expression of genes involved in fibrosis. The mouse fibrosis array was used to quantify expression of 84 transcripts linked to fibrosis. The method uses a qRT-PCR approach. The heat map was generated using Qiagen online software and represents the relative abundance of transcripts. Each group had eight mice per group, and the groups were RD, WD + O, WD + E, and WD + D. The magnitude of gene expression was represented by green and red bars, indicating decreased and increased expression, respectively. The pie plots represent the number of transcripts affected by diet and the relative change in transcript abundance (fold change). Detailed analysis of transcripts is provided in Figs. 3–6 and supplementary Fig. 3.
Fig. 3.
Fig. 3.
Diet effects on hepatic collagen subtype expression. Expression of hepatic collagen subtypes used qRT-PCR and primers listed in supplementary Table 1. The upper panels represent relative mRNA abundance of the collagen subtypes in mice maintained on the RD; cyclophilin was the reference transcript. The lower panel represents the fold change in hepatic expression of the collagen subtypes of mice fed the RD or the WD + O, WD + E, or WD + D diets. Results are expressed as fold change (mean ± SD; n = 8). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O.
Fig. 4.
Fig. 4.
Diet effects on expression of remodeling enzymes. Transcript abundance of proteins involved in extracellular matrix remodeling was quantified using data from the qRT-PCR array described in Fig. 2. Results are represented as mRNA abundance-fold change (mean ± SD; n = 8). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O.
Fig. 5.
Fig. 5.
Diet effects on hepatic expression of Lox and LoxL subtypes and hydroxyproline abundance. A: The relative expression of hepatic LOX and LOXL subtypes was quantified by qRT-PCR using liver RNA from mice fed the RD; cyclophilin was the reference transcript. The qRT-PCR primers are listed in supplementary Table 1. B: Effect of diet on LOX and LOXL subtype mRNA abundance. Results are expressed as mRNA abundance-fold change (mean ± SD; n = 8). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O. C: Hepatic hydroxyproline abundance was quantified as described in Materials and Methods and represented as μg hydroxyproline/mg protein (mean ± SD; n = 8). P values were calculated using Student’s t-test.
Fig. 6.
Fig. 6.
Diet effects on expression of the TGFβ superfamily. Transcript abundance of proteins involved in TGFβ signaling was quantified using data from the qRT-PCR fibrosis array (Fig. 2). Results are represented as mRNA abundance-fold change (mean ± SD; n = 8). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O.
Fig. 7.
Fig. 7.
Diet effects on Smad3 phosphorylation and Col1A1 precursor protein. Mouse liver cytosolic and nuclear extracts were prepared for immunoblotting as described (17). Antibodies used to detect total and phosphorylated Smad2 and -3 are listed in the Materials and Methods section. A: The pathway for TGFβ regulation of Col1A1 gene transcription (–59). B: Representative immunoblot of total Smad 2, Smad3, phospho-Smad3, and proCol1A1. The loading control for phospho-Smad3 was total Smad3; while the loading control for proCol1A1 was GAPDH. The number of independent samples for each group was: RD, 2; WD + O, 3; WD + E, 3; WD + D, 3. C and D: Results are quantified for phospho-Smad3 (C) and proCol1A1 (D) and expressed as fold change (mean ± SD). *P ≤ 0.05 versus RD; #P ≤ 0.05 versus WD + O.
Fig. 8.
Fig. 8.
TGFβ1 and DHA regulation of proCol1A1 expression in human LX2 stellate cells. LX2 cells were plated at ∼10% confluence on plastic petri dishes in DMEM + 5% FCS and antibiotics. The next day, cells were treated with vehicle (Veh: fatty acid- and endotoxin-free BSA at 10 μM) or 25 μM fatty acids in BSA-containing media. Media was change every third day. This treatment enriches membrane lipids with exogenous fatty acids. A: Cells were harvested for fatty acid extraction and gas chromatographic quantitation. The results are presented as fatty acid, Mol% (mean ± range of two separate studies). B: Cells were treated with oleic acid (OA), arachidonic acid (ARA), or DHA as described above. After fatty acid pretreatment, cells were treated without or with 100 pM TGFβ1 overnight in the absence of fatty acids. Cells were harvested for RNA for qRT-PCR quantitation of proCol1A1 mRNA; hydroxymethylbilane synthase was the reference RNA. Results are presented as Col1A1 mRNA-fold change (mean ± SD; n = 3). *P ≤ 0.05 versus Veh; #P ≤ 0.05 versus 18:1,ω9 + TGFβ.

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