Oxidized Low-Density Lipoproteins Trigger Hepatocellular Oxidative Stress with the Formation of Cholesteryl Ester Hydroperoxide-Enriched Lipid Droplets

Figures

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  Figure 1. Cell toxicity test of C3A cells at 24 h after adding nLDL or oxLDL. Cell toxicity in the control group phosphate-buffered saline (PBS) was set as 100%. Results are expressed as mean ± standard deviation. n = 6. One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test, n.s. not significant.

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  Figure 2. Evaluation of accumulation of neutral lipids and lipid hydroperoxides using fluorescence imaging. (A–C) Lipid droplets with neutral lipids and lipid hydroperoxides formed by C3A cells supplemented with (A) PBS, (B) 2.0 ng protein/µL nLDL, and (C) 2.0 ng protein/µL oxLDL (100×). (D) Number of non-oxidized lipid droplets (non-oxLDs) per cell analyzed using fluorescence imaging. (E) Number of oxidized lipid droplets (oxLDs) per cell analyzed using fluorescence imaging. Results were expressed as a box plot (n = 53–84). One-way ANOVA with Kruskal–Wallis test, * p < 0.05, *** p < 0.001, **** p < 0.0001.

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  Figure 3. Comparison of CE and CE-OOH species in the LDL-supplemented C3A cells detected using Orbitrap LC-MS/MS. (A) CE species. (B) The sum of CEs detected in this experiment. (C) The CE-OOH species. (D) The sum of CE-OOHs detected in this experiment. Results are shown as mean ± standard deviation. n = 6. One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 4. Comparison of TG and TG-OOH species in the LDL-supplemented C3A cells detected using Orbitrap LC-MS/MS. (A) TG species. (B) The sum of TGs detected in this experiment. (C) TG-OOH species. (D) The sum of TG-OOHs detected in this experiment. Results are shown as mean ± standard deviation. n = 6. One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

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  Figure 5. The molar ratio of PC-OOH/PC. Results are shown as mean ± standard deviation. n = 5–6. One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05.

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  Figure 6. Expression of genes related to lipid metabolism in the LDL-supplemented C3A cells detected using real-time PCR. (A) Biosynthesis and degradation of CE: SOAT1 and LIPE; (B) Biosynthesis and degradation of TG: DGAT1 and ATGL; (C) Biosynthesis marker of fatty acids: SREBP1, FASN, and SCD1; (D) Antioxidants: CAT. Results are shown as the relative expression levels compared to the control (PBS), which was set to 1.0. n = 6–8. One-way ANOVA with Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

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  Figure 7. The hypothesized mechanism of lipid metabolism changes in the LDL-supplemented C3A cells. (A) Changes in lipid metabolism in the nLDL-supplemented C3A cells. nLDL decreased the expression of genes involved in CE lipolysis (LIPE), which may be associated with accumulated CE and increased lipid droplets. nLDL increased the expression of genes involved in fatty acid synthesis (FASN and SCD1) and TG hydrolysis (ATGL). Additionally, nLDL decreased some TG molecular species. Moreover, nLDL increased the expression of an antioxidative enzyme, CAT. Taken together, nLDL might exert protective effects on the hepatocytes by increasing TG hydrolysis and antioxidant enzyme expression. (B) Changes in lipid metabolism in the oxLDL-supplemented C3A cells. oxLDL caused downregulation of genes involved in CE metabolism (LIPE and SOAT1) and increased lipid hydroperoxide-containing lipid droplets, CE-OOH and PC-OOH/PC. oxLDL also decreased the expression of genes involved in fatty acid synthesis (SREBP1, FASN, and SCD1) and some TG.