Mechanism Of Fe₃O₄ Nanoparticles Aggravating Liver Injury In Mice With Nonalcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease（NAFLD）is considered to be a chronic liver disease with steatosis in the hepatocytes and lipid accumulation of neutral lipid droplets such as triglycerides as the main clinical manifestations.The disease Accompanied by various degrees of hepatic steatosis,iron deposition and other physiological phenomena and clinical pathological characteristics,it affects about 30%of men and 15%of women in the world.It is one of the most common liver diseases worldwide.Current clinical studies have shown that about 25%of NAFLD patients will further develop non-alcoholic steatohepatitis,liver fibrosis,cirrhosis,and even hepatocellular carcinoma on this basis.Recent reports have demonstrated that hepatic iron overload is associated with the development and progression of NAFLD.The iron oxide nanoparticles（IONPs）have already been widely used in magnetic resonance imaging（MRI）,drug delivery,cancer therapy mainly due to their unique physicochemical properties and excellent magnetic features.Liver is the main target organ for sequestering the administered targeted nanoparticles.Recently,IONPs have been utilized for MRI in the imaging and diagnosis of chronic liver disease,including NAFLD.Therefore,the increasing biomedical applications of IONPs have raised public concerns and comprehensive understanding of their interactions with liver,especially in NAFLD.In this study,we explored the in vitro and in vivo biological effects of two types of surface functionalized IONPs including carboxyl-modified（COOH-IONPs）and amino-modified IONPs（NH₂-IONPs）in NAFLD.Firstly,IONPs were prepared using chemical co-precipitation of Fe（II）and Fe（III）chlorides in an alkaline solution,and then characterized by transmission electron microscope（TEM）,dynamic light scattering（DLS）,fourier transform infrared（FTIR）spectroscopy and ultraviolet-visible（UV-vis）spectroscopy.The monodisperse spherical of IONPs were well-dispersed in Milli-Q water,and the average diameters of COOH-IONPs and NH₂-IONPs were 24.3±3.8 and 28.8±4.2 nm with a narrow size uniformity,respectively.Secondly,we observed that hepatic iron content was significantly higher in HFD-fed mice after treatment with IONPs than in mice with NAFLD,indicating that IONP exposure aggravated hepatic iron overload in HFD-fed mice.We also found that IONP treatment aggravated SREBP-1c-mediated de novo lipogenesis and liver inflammation obtained by H&E staining,serum index,RT-q PCR,indicating that IONPs might promote the progression of hepatic steatosis to steatohepatitis in HFD-induced NAFLD mice.Lastly,we confirmed that IONP injection aggravated hepatic iron overload-induced liver inflammation in mice with in part through disrupting hepatic BMP-SMAD pathway and then inducing hepatic pro-inflammatory response.In conclusion,BMP-SMAD-mediated hepatic iron overload aggravated lipid accumulation in the liver and hepatic inflammatory responses,implying that effective measures in addition to hepatic iron overload are needed for individuals at the risk of IONPs in NAFLD.