*2.3. YA Pre-Administration Attenuated Apoptotic Signals in ALF Model*

Apoptotic signals were analyzed in liver tissues obtained from the LPS/D-GalNinjected mice. The number of apoptotic cells exhibiting green fluorescence was increased in the LPS/D-GalN group. In contrast, the number of these cells was decreased in the YA and silymarin pre-administered groups, according to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, a method detecting DNA fragmentation of apoptotic cells (Figure 3A). In the LPS/D-GalN group, the B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax)/Bcl2 ratio was increased; poly ADP-ribose polymerase (PARP) and caspase 3 (Cas 3) were cleaved, and mitochondrial cytochrome C was secreted into the cytoplasm. In comparison to the LPS/D-GalN group, the apoptotic signals were significantly reduced in the YA and silymarin pre-administered groups (Figure 3B, *p* < 0.05, n = 4). The inhibitory effect on LPS/D-GalN-induced apoptotic signals was higher in the 50 mg/kg YA pre-administration group than in the 10 mg/kg YA pre-administration group.

**Figure 3.** Anti-apoptotic effect of YA on liver tissues obtained from LPS/D-GalN-injected mice. (**A**) TUNEL staining. Representative fluorescence images of hepatocyte apoptosis in LPS/D-GalN group. Positive control (PC) treated with DNase I is displayed as a comparison. The cells showing green fluorescence in the nucleus are apoptotic. Scale bar, 200 μm. (**B**) Western blotting assay for detection of apoptotic signals. Pro-apoptotic Bax and anti-apoptotic Bcl2 expression levels, cleaved (CL) PARP and caspase 3 (Cas 3), and translocation of cytochrome C (Cyt C) into cytoplasm were analyzed. Data are shown as the mean ± SD (n = 4 in each group). \* *p* < 0.05 compared to vehicle group. † *p* < 0.05 compared to the LPS/D-GalN group. The plus sign (+) represents a combination of LPS/D-GalN and each substance.
