Research Of Intervention Of Luteolin On Inorganic Mercury-Induced Acute Liver Injury In Mice

Mercury?Hg?and its compounds,as critical environmental toxicants,are harmful to organic nervous system,liver,kidney,and other tissues and systems.In all forms of mercury,inorganic mercury?I-Hg?and methyl mercury are the most harmful to organisms.Though measures have been taken to reduce anthropogenic Hg emissions,China is still one of the countries with the largest atmospheric Hg emission worldwide.Excessive Hg enters the biogeochemical cycle seriously threatening the health of poultry and livestocks and the security of animal food.As thimerosal is a kind of preservative in some vaccines and mericuric chloride is the catalyst of vinyl chloride synthesis reaction,I-Hg has irreplaceable applications in daily life and industrial production greatly increasing the risk of poulty and livestocks exposed to Hg.As the main metabolic organ of organisms,liver is one of the main target tissues of the toxicity and accumulation of I-Hg.However,there has still lacked clinical therapies which could effectively reduce the hepatotoxicity of I-Hg.Luteolin?3',4',5,7-tetrahydroxyflavone?,a natural flavone derived from many traditional Chinese medicinal plants,has numerous health benefits.Luteolin has been of great interest because of its bioactivities on enhancing immune system function,scavenging free radicals and antitumor.In this study,mouse models of acute mercuric chloride?HgCl₂?exposure were established to explore the intervention of luteolin on acute liver injury induced by I-Hg and its molecular mechanisms.In vivo experiments,28 health male Kunming mice?6-8 weeks?were randomly and equally divided into 4 groups.The groups were:Control,Luteolin,HgCl₂,and HgCl₂+luteolin.HgCl₂ was intraperitoneal injected once at a dose of 4 mg/kg to establish mouse models of acute liver injury.Luteolin,as detoxification,was given orally 24 h after administration of HgCl₂.Blood and serum were collected 24 h after the last administration for complete blood cell count and biochemical determination.Liver samples were collected for histological observation,and oxidative stress markers including glutathione?GSH?and malondialdehyde?MDA?in livers were determined by kits.Apoptosis rate,protein levels and miR-146a level in livers were detected by terminal dexynucleotidyl transferase?TdT?-mediated dUTP nick end labeling?TUNEL?,western blot analysis,and realtime quantitative polymerase chain reaction?qPCR?,respectively.In vitro experiments,primary hepatocytes from Kunming mice were cultured and identified.Primary hepatocytes were treated with HgCl₂ and luteolin,respectively.The groups and treatments were as follow:Control,Luteolin?20?M?,HgCl₂?5?M?,and HgCl₂?5?M?+luteolin?20?M?.Hepatocytes were treated with HgCl₂ for 24 h with or without pre-treatment of luteolin for 2 h.Hepatocyte viability and reactive oxidative species?ROS?production were determined according to manufacturer's instructions.The results showed:?1?The results of complete blood cell count suggested that luteolin ameliorated anemia induced by I-Hg.?2?The results of biochemical parameters analysis suggested that luteolin reduced the increase of aspertate aminotransferase and alanine aminotransferase activities induced by I-Hg.?3?The observation of pathological sections suggested that luteolin alleviated acute liver injury induced by I-Hg.?4?The results of GSH and MDA determinations suggested that luteolin attenuated oxidative stress level increased by I-Hg.?5?The results of TUNEL analysis suggested that luteolin attenuated hepatocyte apoptosis induced by I-Hg.?6?The data of western blot analysis suggested that luteolin activated nuclear factor?erythroid-derived 2?-like 2?Nrf2?signaling pathway and increased metabolism-related protein levels,including Nrf2,heme oxygenase 1,NAD?P?H:quinone oxidoreductase 1,Krüppel-like factor 9,silent mating type information regulation 2 homolog 1?Sirt1?,and mammalian target of rapamycin.The data also suggested that luteolin decreased protein levels of inflammation-and pro-apoptotic proteins increased by I-Hg,including nuclear factor kappa-light-chain-enhancer of activated B cells,tumor necrosis factor alpha?TNF-??,phosphorylated p38,p53,and Bcl-2-associated X protein,and increased protein level of B-cell lymphoma 2 decreased by I-Hg,an inhibitor of apoptosis protein.?7?The data of qPCR showed that luteolin and HgCl₂ did not affect miR-146a expression,which is one of the upstream genes of Nrf2,indicating that luteolin regulated Nrf2 signaling pathway repressed by I-Hg via a miR-146a independent way.?8?The results of vitro experiments suggested that luteolin prevented the decrease of hepatocyte viability and the aggravation of ROS production induced by I-Hg.In conclusion,luteolin effectively alleviated anemia and acute liver injury induced by I-Hg via regulating Sirt1/Nrf2/TNF-?signaling pathway.Dietary intake of luteolin may become a novel and safe method to protect the health of poultry and livestocks against I-Hg exposure and the security of animal food.