Figure 1.
AZT-mediated hepatic fat accumulation, inflammation, and injury.
(A, B) Representative photomicrographs of H&E stained liver sections from the indicated mouse livers are presented: (A) vehicle-control; (B) AZT–treated mice (outside inset: inflammatory foci as they are scattered). Accumulated fat are indicated by circular lines and arrows. (C) Hepatic triglyceride levels, (D) Serum ALT levels, and (E) inflammation foci (per 5 high power fields) are presented for the different groups. F) Equal amounts of total liver homogenates (50 µg/lane) from different groups were used to evaluate the amounts of full-length OPN, cleaved (active) OPN and β-actin protein (as a loading control). (G, H) Densities of full-length OPN and cleaved OPN (cOPN) protein bands were normalized to that of the β-actin. *Significantly different from the control group (n = 6/group). Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 2.
Effect of AZT on the levels of CYP3A4, CYP4A and CYP2E1 proteins.
(A) Equal amounts of whole liver homogenates (50 µg/lane) from both groups were used to evaluate the levels of CYP3A4, CYP4A and CYP2E1 (upper, middle and lower panel), respectively. (B, C, D) The densities of each immunoreactive band were normalized to that of β-actin as the loading control. *Significantly different from the control group. Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 3.
AZT-mediated increments of oxidative stress markers.
(A) Equal amounts of mitochondrial proteins (50 µg/lane) were used to evaluate the levels of mitochondrial iNOS (upper panel) and ATP synthase (lower panel) from both groups. (B) The densities of iNOS bands were normalized to that of ATP synthase, the loading control. (C) The rate of production of hydrogen peroxide and (D) the level of MDA+HAE as a marker of lipid peroxidation was measured as described in the Materials and Methods. *Significantly different from the control group. Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 4.
AZT-mediated protein modifications.
(A) Equal amounts of mitochondrial proteins (50 µg/lane) from the different groups were used to evaluate protein oxidation (upper panel) using Oxyblot analysis or negative control (lower panel). (B) Densities of mitochondrial oxidized proteins were normalized with that of the controls set at 1. (C) Equal amounts of mitochondrial proteins were used to evaluate protein nitration as reflected by 3-NT bands (upper panel) and ATP synthase loading control (lower panel) from different groups. (D) The densities of mitochondrial 3-NT bands were normalized to that of ATP synthase and plotted as the value of controls being 1. (E) Total liver homogenates (50 µg/lane) were used to evaluate the levels of AGE (upper panel) and p38 (lower panel) as a loading control. (F) The densities of AGE bands were normalized to that of p38 and plotted as the value of controls being 1. *Significantly different from the control group. Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 5.
AZT-induced increases in the ER stress markers.
(A) Equal amounts of total liver homogenates (50 µg/lane) from different groups were used to evaluate the levels of GRP78, p-PERK, p-eIf 2α, IRE-1α and p38 (as a loading control). (B, C, D, E) The densities of each protein were normalized to that of p38 and presented. *Significantly different from the control group. Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 6.
AZT-induced alterations of key proteins involved in fat metabolism.
Equal amounts of nuclear, total liver homogenates and mitochondria from different groups were used to measure the levels of (A) n-SREBP-1, PPARα and Histone H3 (as loading control for nuclear proteins) (B) p-ACC, p-AMPK and β-actin (as a loading control for total liver homogenates), and (C) thiolase and ATP synthase (as a loading control for mitochondrial proteins), respectively. (D–H) The density of each protein was normalized to that of the indicated loading control, respectively. (I) Thiolase activity was measured as described in the Materials and Methods. *Significantly different from the control group. Data indicate mean±SE, p<0.05. All experiments have been conducted three times.
Figure 7.
Schematic diagram describing potential mediators of AZT-mediated steatosis and injury.
AZT-induced hepatic fat accumulation and injury is mediated through increased oxidative and nitrative stress, lipid peroxidation, protein modifications and inflammation.