Figure 4 | Scientific Reports

Figure 4

From: Palmitate-induced lipotoxicity is crucial for the pathogenesis of nonalcoholic fatty liver disease in cooperation with gut-derived endotoxin

Figure 4

Long-term palmitate injection induced liver fibrosis. (AC) We intraperitoneally injected 3 μmol/g palmitate, vehicle, 0.025 mg/kg LPS or PBS twice a day for 2 weeks into 8-week-old ad libitum BD-fed WT mice, 28-week-old ad libitum HFD-fed WT mice and 8-week-old ad libitum BD-fed TLR4 KO mice. (A) Collagen deposition was evaluated by sirius red staining. Immunohistochemical detection of sirius red (×200) in representative liver and quantification of sirius red staining (×100) in liver were analyzed in long term palmitate or vehicle injected 10-week-old BD-fed WT mice, 10-week-old BD-fed TLR4 KO mice and 30-week-old HFD-fed WT mice. Scale bars, 100 μm. n = 5 mice per group. (B) αSMA expression in whole liver was determined by western blot analysis in long term palmitate or vehicle injected BD-fed 10-week-old WT mice. n = 3 mice per group. (C) Acta2, tissue inhibitor of metalloproteinases-1 (Timp1), collagen1a1 and transforming growth factor beta 1 (Tgfβ1) mRNA expression levels in whole liver were analyzed by qPCR in long term palmitate or vehicle injected BD-fed 10-week-old WT mice. n = 5 mice per group. (DF) A single palmitate injection induced HSC activation. A single intraperitoneal injection of 3 μmol/g palmitate, vehicle, pamitate plus 0.025 mg/kg LPS, vehicle plus 0.025 mg/kg LPS was administered to the mice. Scale bars, 50 μm. n = 5 mice per group. (D) Immunohistochemical detection of αSMA (×400) in representative liver samples and average % of αSMA-positive area (×100) 24 h after palmitate, vehicle, vehicle-0.025 mg/kg LPS or palmitate-0.025 mg/kg LPS injection were examined in 8-week-old BD-fed WT mice, 8-week-old BD-fed TLR4 KO mice and 28-week-old HFD-fed mice that were gut-sterilized for 4 weeks or untreated. (E) Ly-6G or control IgG was intraperitoneally injected. Immunohistochemical detection of αSMA (×400) in representative liver samples and average % of αSMA-positive area (×100) 24 h after palmitate or vehicle injection were examined in 8-week-old BD-fed and 28-week-old HFD-fed mice. (F) Clodronate or control liposomes were injected into the tail vein. Immunohistochemical detection of αSMA (×400) in representative liver samples and average % of αSMA-positive area (×100) 24 h after palmitate or vehicle injection were examined in 8-week-old BD-fed and 28-week-old HFD-fed mice. Data represent the mean ± SD. *P < 0.05. NS, not significant.

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