Fig. 4

Fluda reduced LPS-mediated abnormal autophagic responses in BAL cells at 24 h after LPS administration in GFP-LC3 transgenic mice. A Representative images of immunofluorescence staining for GFP-tagged LC3B. Confocal microscopy (× 1200) was used to monitor LC3-dependent autophagy. Scale bar, 10 μm. B The average number of LC3-GFP puncta per cell was counted. C In BAL cell lysate, the mRNA expression of beclin-1 was determined using real-time PCR. D LC3B, p62, and β-actin expression in BAL cells was determined using western blotting (left). Densitometric ratio of LC3B-II in BAL cells against β-actin (middle) and that of p62 in BAL cell lysates against β-actin (right). E The protein expression of LC3B and p62 was detected in RAW264.7 cells. Densitometric ratio of LC3B-II in RAW264.7 against β-actin (middle) and that of p62 in RAW264.7 β-actin (right). F LC3B and β-actin expression in whole-lung lysates was determined using western blotting (left). Densitometric ratio of LC3B-II in whole-lung lysates against β-actin (right) (n = 3 for CTL, n = 2 for Fluda, n = 4 for LPS, and n = 3 for LPS + Fluda). The data are presented as mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group. †p < 0.05, ††p < 0.01, and †††p < 0.001 compared with the LPS group