Fig 2: Adenovirus-endothelial nitric oxide synthase (Ad-eNOS) inhibited apoptosis in liver tissues after small-for-size liver transplantation. Rats in the experimental group were intraperitoneally injected with Ad-eNOS (4.0×109 PFU), whereas rats in the control and sham groups were injected with Ad-null (4.0×109 PFU each). After 36 h, rats in the experimental and control groups underwent small-for-size liver transplantation. (A) Rats pretreated with Ad-eNOS displayed significantly fewer TUNEL-positive apoptotic cells in liver tissues at 6 h after ischemic/reperfusion (I/R; magnification, ×400). (B) Flow cytometric analysis revealed a lower percentage of apoptotic cells in liver tissues of rats pretreated with eNOS at 6 h after I/R (3.9±0.9 vs. 11.8±1.3% in the control group; P<0.05), although this number was still higher than that in the sham group (1.2±0.4%; P<0.05).

Fig 3: In vitro experiments. (A) Western blot analysis showed increased eNOS protein expression in Ad-eNOS-transfected L02 cells, as compared with L02 cells and Ad-null-treated L02 cells. (B) Quantification of protein levels of eNOS, normalized by GAPDH expression, P<0.05. (C) Ad-eNOS exerted a protective effect against apoptosis in L02 cells according to flow cytometric analysis. The rates of against in the experimental, control and normal groups were 10.10±2.91, 20.53±2.8 and 5.21±1.41%, respectively (P<0.05, experimental vs. control group). eNOS, endothelial nitric oxide synthase; Ad, adinovirus.

Fig 4: Histological findings. (A) Adenovirus-endothelial nitric oxide synthase (Ad-eNOS)-pretreated rats exhibited improvement in liver histological abnormalities, with normal hepatocyte morphology, complete vascular walls and reduced inflammatory cell infiltration in the periportal region at 6 h after ischemia/reperfusion (I/R), as compared to controls (magnification, ×200). (B) Immunohistochemical staining showed fewer tumor necrosis factor-α-positive cells in liver tissues of Ad-eNOS-pretreated rats than in Ad-null-pretreated rats at 6 h after I/R (magnification, ×200). (C) Rats pretreated with Ad-eNOS showed significantly fewer CD68-positive immunoreactive macrophages in vascular walls at 6 h after reperfusion than the control group (magnification, ×200).

Fig 5: Elderberries (EB) prevents eNOS uncoupling via Akt pathway (a) HUVECs pretreated with wortmannin (W) (30 nM) for 30 min and stimulated with EB (50 µg/mL) for 1 h. (b) HUVECs were pretreated with EB 50 µg/mL for 1 h and stimulated with TNF-a for an additional 4 h. eNOS activity was probed using western blots with its representative graph. Data are presented as mean and standard error of the mean (SEM) (n = 3). Significant value was set at #p < .05, ##p < .01; *p < .05, compared with TNF-a; $p < .05 compared with EB 50 µg/mL.