Saikosaponin D Inhibits the Proliferation and Promotes the Apoptosis of Rat Hepatic Stellate Cells by Inducing Autophagosome Formation

Figure 4

SSd inhibits the proliferation and promotes the apoptosis of HSC-T6 cells via autophagosome formation. (a) LC3B expression level in acetaldehyde-activated HSC-T6 cells was detected by western blot analysis after 6 h of SSd treatment. (b) HSC-T6 cells were transfected using GFP-LC3 adenovirus. GFP-LC3 puncta formation was mediated by SSd in acetaldehyde-activated HSC-T6 cells. Bar: 10 μm. (c) Acetaldehyde-activated HSC-T6 cells were incubated with 10 μmol/L SSd, 10 μmol/L rapamycin, and 5 mmol/L 3MA alone or in combination for 48 h. Cell proliferation was detected using the EdU kit and flow cytometry. (d) The percentage of proliferating cells based on EdU flow cytometry results. , , versus control group; versus SSd treatment group; versus rapamycin treatment group. The ns means no significant difference versus control group. (e) Acetaldehyde-activated HSC-T6 cells were incubated with 10 μmol/L SSd, 10 μmol/L rapamycin, and 5 mmol/L 3MA alone or in combination for 48 h. Cell apoptosis was detected using the Annexin V-FITC/PI double-stained kit and flow cytometry. (f) The percentage of apoptotic cells based on Annexin V-FITC/PI double-stained flow cytometry results. , versus control group; versus SSd treatment group; versus rapamycin treatment group. The ns means no significant difference versus control group. (d, f) Data are presented as mean ± SD (n = 3). One-way ANOVA and then Tukey’s multiple comparisons test were used for statistical analysis. (g) The expression levels of LC3B, Ki67, cleaved caspase 3, Bcl2, and Bax were detected by western blot analysis.

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