Figure 2.

Lycorine inhibits the SREBF activity and decreases the cellular lipids without inducing ER stress and NR1H3 transactivation. (A) Lycorine downregulates SREBF activity. HL-7702/SRE-Luc cells were depleted of incubating in medium D for 16 h. The cells then were treated with different compounds as indicated. After incubation of another 16 h, cells were lysed with reporter lysis buffer and luciferase activity was measured (n = 4). (B) HL-7702 cells were treated with increasing concentrations of lycorine for 24 h, and cell viability was detected by MTT (n = 6). (C-F) HL-7702 cells were treated with cholesterol (20 μM) or lycorine (10, 20 μM) for 12 h (C and E), or lycorine (20 μM) for increasing time (D and F), whole-cell extracts underwent western blotting (WB) with indicated antibodies (left). Statistical analysis of expression of each protein was adjusted to ACTB (right) (n = 3). pSREBF1 or pSREBF2 represents precursor SREBF1 or precursor SREBF2; mSREBF1 or mSREBF2 represents mature SREBF1 or mature SREBF2. (G) HL-7702 cells were treated with 10 or 20 µM lycorine for 12 h, RNAs were extracted from these cells. The expression of various genes was analyzed by qRT-PCR (n = 3). (H) The cellular TG and TC contents were measured in HL-7702 hepatocytes treated with lycorine (5, 10 and 20 µM) for 16 h (n = 3). (I) HL-7702 cells were treated with indicated concentrations of lycorine for 16 h. 1–¹⁴ C-labeled acetic acid sodium salt was directly added into the medium and incubated for an additional 2 h. The fatty acid and total cholesterol were extracted and resolved by thin-layer chromatography. Radioactive products were visualized by phosphoimager and densitometric quantification is shown accordingly (n = 3). (J) The treated HL-7702 cells were fixed and stained with Filipin or Nile-Red. Quantification of the cellular cholesterol or neutral lipids was analyzed by image-pro plus (n = 3). (K) HL-7702 cells were treated with lycorine or sterol for 16 h, then RNAs were extracted. The expression of ER stress-related genes was analyzed by qRT-PCR (n = 3). (L) HL-7702 cells transfected with NR1H3 reporter and beta-gal plasmid were incubated with lycorine or sterol for 16 h, luciferase activity was then measured and normalized by the value of beta-gal (n = 6). (M) HL-7702 cells were treated with lycorine or sterol for 16 h, RNAs were extracted. The expression of NR1H3 target genes were analyzed by qRT-PCR (n = 3). Error bars are represented as mean ± SEM. Statistical analysis was done with one-way ANOVA (Dunnett’s posttest). *p < 0.05, **p < 0.01, ***p < 0.001 vs control