AgNPs Aggravated Hepatic Steatosis, Inflammation, Oxidative Stress, and Epigenetic Changes in Mice With NAFLD Induced by HFD

Abstract

The recent development of silver nanoparticles (AgNPs) has sparked increased interest in biomedical and pharmaceutical applications, leading to the possibility of human exposure. The liver is the primary target organ in the metabolism and transport of nanoparticles. Non-alcoholic fatty liver disease (NAFLD) is the most common and leading cause of hepatic metabolic syndrome with approximately 15% of patients will develop into non-alcoholic steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Thus, the potential hepatotoxicity of AgNPs on NAFLD development and progression should be of great concern. Herein, we explored the potential hepatic effect of a single intravenously injected dose of 0.5, 2.5, and 12.5 mg/kg BW on the liver function of high-fat-diet (HFD)-fed mice for 7 days. AgNP treatment increased serum levels of alanine aminotransferase, aspartate transaminase, triglycerides and cholesterols, the number of lipid droplets, and the contents of triglycerides and cholesterols in NAFLD mice livers compared to HFD-fed mice. The mechanism of AgNP-induced worsen hepatotoxicity in mice is associated with hyperactivation of SREBP-1c-mediated de novo lipogenesis and liver inflammation. Additionally, HFD-fed mice treated with AgNPs had significantly higher oxidative damage and lower global DNA methylation and DNA hydroxymethylation than NAFLD mice. This study suggests that AgNP treatment exacerbated HFD-induced hepatic steatosis, liver inflammation, oxidative stress, and epigenetic changes in mice, which is relevant to the risk of AgNP exposure on NAFLD development and progression.

Keywords: global DNA methylation; hepatic steatosis; hepatotoxicity; liver inflammation; non-alcoholic fatty liver disease; silver nanoparticles.

FIGURE 1

Physicochemical characterization of silver nanoparticles (AgNPs). (A) Representative TEM images of AgNPs. (B) Size distribution of AgNPs measured by TEM. (C) The detailed information on physicochemical characterization of AgNPs includes size, zeta potential, hydrodynamic size, and polydispersity index (PDI). (D) Dissolution of AgNPs in original stock suspensions and simulated biological conditions after incubation for 5, 10, 60 min, and 24 h. Results for ionic silver are expressed as a percentage of total silver measured in the stock suspensions.

FIGURE 2

AgNPs aggravated HFD-induced hepatic dysfunction and liver injury in mice. (A) Schematic illustration of the murine model of NAFLD induced by HFD along with AgNP treatment once at the concentration of 0.5, 2.5, and 12.5 mg/kg for 1 week. (B)The daily changes in body weight of the mice during the 8-weeks feeding and 1-week treatment course. The statistical analysis of the body weight (C) and liver weight (D) were obtained from the NAFLD mice after 1-week treatment. (E–F) The liver function of the NAFLD without and with AgNPs was analyzed by the levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST). ICP-MS analysis of the biodistribution of AgNPs at the liver (G), heart (H), spleen (I), lung (J), and kidney (K) of HFD-fed mice after intravenous injection at the concentration of 0.5, 2.5, and 12.5 mg/kg for 1 week. Representative images of H&E staining (L) and statistical analysis of the pathological changes (M) of the liver in ND-fed mice and HFD-fed mice treated with AgNPs. Scale bars, 50 μm *p < 0.05 vs. ND-fed group; ^# p < 0.05 vs. HFD-treated group.

FIGURE 3

AgNPs exacerbated HFD-induced hepatic steatosis in a manner involving altered expression of the genes for de novo lipogenesis (DNL) in mice. Representative images of ORO staining (A) and quantitative analysis (B) of the positive ORO-stained sections in the liver in ND-fed mice and HFD-fed mice treated with AgNPs. Scale bars, 50 μm. (C) The level of plasma total triglyceride (TG). (D) The level of plasma total cholesterol (TC). (E) The level of plasma low-density lipoprotein (LDL). (F) The level of plasma high-density lipoprotein (HDL). (G) The level of plasma very low-density lipoprotein (VLDL). (H) The content of hepatic TG. (I) The content of hepatic TC. The expression of genes related to hepatic de novo lipogenesis, such as Srebp-1c (J) and its target genes including Fasn (K), Acc1 (L), Elovl6 (M), and Scd1 (N). Gene expression was normalized to Gapdh. ^* p < 0.05 vs. ND-fed group; ^# p < 0.05 vs. HFD-treated group.

FIGURE 4

AgNPs worsen liver inflammation in mice with NAFLD induced by HFD. The expression of hepatic proinflammatory genes including Tnf-α (A), Il-6 (B), and Il-1β (C) in ND-fed mice and HFD-fed mice treated with AgNPs. Gene expression was normalized to Gapdh. The contents of proinflammatory cytokines including TNF-α (D), IL-6 (E), and IL-1β (F) in the liver of ND-fed mice and HFD-fed mice treated with AgNPs were measured by ELISA. ^* p < 0.05 vs. ND-fed group; ^# p < 0.05 vs. HFD-treated group.

FIGURE 5

AgNPs increased oxidative stress markers in the liver of NAFLD mice induced by HFD. Hepatic levels of ROS production (A), GSH content (B), MDA content (C), and MPO (D) in ND- and HFD-fed mice following AgNP exposure for 1 w at the concentration of 0.5, 2.5, and 12.5 mg/kg *p < 0.05 vs. ND-fed group; ^# p < 0.05 vs. HFD-treated group.

FIGURE 6

Epigenetic changes induced by AgNPs in the liver of HFD-fed mice AgNPs furtherly decreased the percentage of global DNA methylation (A) and DNA hydroxymethylation (B) in the liver of NAFLD mice induced by HFD. *p < 0.05 vs. ND-fed group; ^# p < 0.05 vs. HFD-treated group.

FIGURE 7

Schema of the possible mechanisms of intravenously injected AgNPs on NAFLD development and progression. The potential mechanism of AgNP-induced hepatotoxicity in NAFLD mice is associated with hyperactivation of SREBP-1c-mediated de novo lipogenesis and liver inflammation. Additionally, significantly higher oxidative damage and lower global DNA methylation and DNA hydroxymethylation were observed in HFD-fed mice treated with AgNPs compared with NAFLD group. This study suggests that AgNP treatment exacerbated HFD-induced hepatic steatosis, liver inflammation, oxidative stress, and epigenetic changes in mice, which is relevant to the risk of AgNP exposure on NAFLD development and progression.