Journal: Cell Death & Disease

Article Title: MicroRNA-146a-5p attenuates irradiation-induced and LPS-induced hepatic stellate cell activation and hepatocyte apoptosis through inhibition of TLR4 pathway

doi: 10.1038/s41419-017-0038-z

Figure Lengend Snippet: miR-146a-5p negatively regulates TLR4 signaling in primary HSCs. a qRT-PCR analysis showed the expression levels of pro-inflammatory cytokines, TLR4, IRAK1, TRAF6, and α-SMA in primary HSCs of mice in each group. Data are presented as the mean ± SD ( n = 3 mice). * P < 0.05 vs. Ctr group; # P < 0.05 vs. RT group; ## P < 0.05 vs. miR-NC group. b Representative immunofluorescence staining of desmin and α-SMA for cultured primary HSCs at the first day and 7 days after isolation. Scale bar: 100 μm. c Quiescent HSCs (24 h after isolation) were transfected with miR-146a-5p mimics or negative control and then subjected to 8 Gy X-ray irradiation and 50 ng/ml LPS treatment for 24 h. The expression levels of TLR4, IRAK1, TRAF6, pro-inflammatory cytokines, and α-SMA were detected by qRT-PCR. Data are presented as the mean ± S.E.M. of three independent experiments. * P < 0.05 vs. miR-NC group. d Apoptosis analysis of primary hepatocytes at 72 h after 8 Gy X-ray irradiation and co-culture with the supernatants from irradiated and LPS-stimulated primary HSCs transfected with miR-146a-5p mimics or negative control. Data are presented as the mean ± S.E.M. of three independent experiments. * P < 0.05 vs. miR-NC group. miR-NC miRNA negative control, miR-M miRNA mimics

Article Snippet: After the indicated treatment, cells were fixed with 4% paraformalde-hyde for 15 min, permeabilized with 0.2% Triton X-100 in PBS for 5 min, and then blocked with 5% bovine serum albumin in PBS for 1 h. After incubation with primary antibodies against NF-κB p65(1:100, NB100-82088, Novus Biologicals, Littleton, USA), α-SMA(1:100, NBP2-33006, Novus Biologicals), or desmin (1:100, NB120-15200, Novus Biologicals) overnight at 4 °C, cells were incubated with Cy3-labeled Goat Anti-Rabbit IgG(1:500, A0516, Beyotime, China) or FITC-labeled Goat Anti-Rabbit IgG (1:500, A0562, Beyotime) for another 1 h. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and the images were captured using fluorescence microscopy (FV300, Olympus, Japan).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Cell Culture, Isolation, Transfection, Negative Control, Irradiation, Co-Culture Assay