Salvianolic acid B protects hepatocytes from H2O2 injury by stabilizing the lysosomal membrane

Figure 1 Effect of Sal B on apoptosis and death. A: The viability of hepatocytes treated with different concentrations of Sal B for 24 h; B: The viability of hepatocytes treated with different concentrations of Sal B for 22 h following induction by H₂O₂ for 2 h; C: The Annexin V/PI assay with FACS. All data are presented as mean ± SD. Statistical analysis was performed using Student’s t-test (^aP < 0.05 vs Con; ^dP < 0.01 vs H₂O₂).

Figure 2 Effect of Sal B on cell proliferation. A: BrdU incorporation assay, with 4000 cells seeded in a 96-well plate being treated with 10 μmol/L of Sal B for 24 h and with 500 μmol/L H₂O₂ for an additional 2 h. A final concentration of 10 μmol/L BrdU was supplemented in the medium for 24 h (scale bar = 50 μmol/L); B: Quantification of BrdU incorporation assay. All data are presented as mean ± SD. Statistical analysis was performed using Student’s t-test (^aP < 0.05 vs Con; ^cP < 0.05 vs H₂O₂).

Figure 3 Effect of Sal B on protein carbonyl content. A: The expressions of protein carbonyl content in hepatocytes detected by western blot; B: Quantitative analysis of optical densities of protein carbonyl content and normalized to the expression of β-actin (^aP < 0.05, ^bP < 0.01 vs Con; ^dP < 0.01 vs H₂O₂).

Figure 4 Effect of Sal B on lysosomal membrane permeability. A: The expressions of LAMP1 in hepatocytes detected by western blot; B: Quantitative analysis of optical densities of LAMP1 and normalized to the expression of β-actin; C: Levels of LAMP1 mRNA were measured in cells by qRT-PCR; D: LysoTracker green staining; E: Quantification of the LysoTracker green staining (^bP < 0.01 vs Con; ^aP < 0.05, ^dP < 0.01 vs H₂O₂).

Figure 5 Effect of Sal B on the leakage of CatB/D into the cytosol. A and B: CatB/D levels in the cytosol/whole lysate were measured by western blot; C and D: Quantitative analysis of optical densities of CatB/D in the cytosol/whole lysate and normalized to the expression of β-actin; E: CatB activity analysis was performed with chemiluminescence detection. (^bP < 0.01 vs Con; ^dP < 0.01 vs H₂O₂).

Figure 6 Effect of Sal B on lysosomal PH. A: AO staining; B: Quantification of the AO staining (^aP < 0.05, ^bP < 0.01 vs Con; ^cP < 0.05, ^dP < 0.01 vs H₂O₂).

Figure 7 Effect of Sal B on CCl₄-induced liver injury. A: Hematoxylin-eosin staining is shown (scale bar =100 μm). Con:control group (n = 6), CCl₄: CCl₄ injection group (n = 6), Sal B: CCl₄ injection and Sal B administration group (n = 6), NAC: CCl₄ injection and NAC administration group (n = 6); B: Serum ALT activity in mice; C: MDA content of liver tissues in mice. Statistical analysis was performed using Student’s t-test (^bP < 0.01 vs Con; ^dP < 0.01 vs CCl₄). ALT: Alanine aminotransferase; MDA: Malondialdehyde.

Figure 8 Effect of Sal B on LAMP1 and leakage of CatB to the cytosol. A: Expressions of LAMP1 in liver detected by western blot; B, C: CatB/D levels in the cytosol/whole lysate were measured by western blot; D: Quantitative analysis of optical densities of LAMP1 and normalized to the expression of β-actin. E, F: Quantitative analysis of optical densities of CatB/D in the cytosol/whole lysate and normalized to the expression of β-actin. All data are presented as mean ± SD. Statistical analysis was performed using Student’s t-test (^bP < 0.01 vs Con; ^dP < 0.01 vs CCl₄).