Figure 1.
Experimental design and biochemical analysis.
A) UV-VIS spectra of gold nanoparticle, 20 µl of ADP355-gold nanoparticle conjugate were injected intraperitoneally into mice. Following 2 h treatment, mice were euthanized, and serum samples and homogenized liver tissues were collected for UV-VIS spectra. Control mice received an equal volume of saline per injection. Saline and ADP355-gold nanoparticles were also included for analysis. B) Experimental design of the schedule for CCl4 and nanoparticle injections. C) Percent change in liver weight divided by body weight of mice. D–E) Serum ALT and AST levels markers of chronic liver injury. Goldn-ADP355 showed significant reduction in CCl4-induced serum ALT and AST. F) Liver hydroxyproline was determined by acid hydrolysis; hydroxyproline content was significantly increased in CCl4-gavaged mice compared to normal control mice, while hydroxyproline content was significantly reduced in CCl4–ADN treated mice (*p<0.05) (N = 5 mice/cohort).
Figure 2.
Histological analysis of CCl4-induced chronic liver injury.
A) Representative H&E stained liver section are shown. B) Representative Sirius Red staining liver sections obtained from control (saline group) revealed normal lobular structure while in CCl4-gavaged mice liver sections reveal extensive collagen deposition and bridging fibrosis. The degree of collagen deposition was significantly decreased in CCl4–ADN treated mice (N = 5 mice/cohort).
Figure 3.
Effect of ADP355 on CCl4-induced liver fibrosis in mice.
A) Representative immunohistochemical (α-SMA) stained liver sections obtained for various treatments outlined (left panel); antibody control were presented in right panel. The original magnification was 4X. B) Representative Western blot analysis and densitometry for α-SMA liver lysates obtained from saline, CCl4, CCl4–CTN and CCl4–ADN treated mice. C) Hepatic α-SMA mRNA expression was assessed by qRT-PCR compared to housekeeping gene 18 s. *p<0.05 (N = 5 mice/cohort).
Figure 4.
Figure 4. ADP355 attenuates CCl4-induced desmin, CTGF and TIMP1 expression in mice.
A) Representative Western blots analysis of liver lysates: Desmin (Top panel); CTGF (middle); β-actin expression (lowest panel). Bar graph represents densitometric quantification compared to saline groups. B) mRNA expression of desmin from liver tissues. C) Western blot of TIMP1 in lysates obtained from liver tissues. D) Hepatic mRNA expression of TIMP1 (N = 5 mice/cohort).
Figure 5.
ADP355 induced MMP13 expression in CCl4-induced liver tissues of mice.
A) Representative Western blot of MMP13 from liver lysates under four experimental conditions described previously. B) mRNA expression of MMP13 outlined in (A). C) In situ collagen zymography; collagen I-degrading activity in cryopreserved liver sections. Degraded collagen was detected by FITC–labeled DQ–collagen I. The original magnification was 4X (N = 5 mice/cohort).
Figure 6.
ADP355 reduced TGF-β1 expression.
A) Representative of Western blot analysis for TGF-β1 (Top panel), densitometric analysis presented in bottom panel. B) mRNA expression of TGF-β1 total RNA obtained from whole liver tissue lysates, data are represented as the TGF-β1 expression relative to 18 s housekeeping gene, *p<0.05 (N = 5 mice/cohort).
Figure 7.
ADP355 modulates eNOS, AMPK and Akt phosphorylation in vivo.
A–B) Western blot analysis showed that CCl4 reduced the phosphorylation of eNOS and AMPK, while ADP355 treatment significantly increased the phosphorylation of eNOS ser1177 and AMPK tyr172 residue. C) Western blot and densitometric ratio of p-AKT ser473/AKT, *p<0.05 (N = 5 mice/cohort).