Mechanism Of Chronic Liver Damage In Mice Induced By Combined Chromium-nickel Exposure Via TLR4/mTOR Pathway

In order to further clarify the effects of combined chromium-nickel exposure on liver damage and the toxicological mechanisms of TLR4/mTOR pathway-mediated inflammation and autophagy in mice,160 C57BL/6J female mice were randomly divided into four groups:the Con group（ultrapure water）,the Cr group（K₂Cr₂O₇ 141 mg/L）,the Ni group（Ni Cl₂ 450mg/L）,and the Cr+Ni group（K₂Cr₂O₇141 mg/L+Ni Cl₂ 450 mg/L）.Liver samples were collected at weeks 4,8 and 16 of the experiment.Effects of combined chromium-nickel exposure on liver damage in mice were determined by analyzing the results of liver weight,liver index,pathological changes,ELISA assay and ICP-MS assay.With same time,q RT-PCR,Western Blot and immunohistochemistry were used to detect the changes of TLR4/mTOR pathway-related factor expression.The results showed that:（1）The liver weight and liver index of mice induced by chromium,nickel and their combined exposure increased with increasing time.The hepatocytes of each exposed group were different degrees of degeneration and swollen,with obvious focal necrosis and inflammatory cell infiltration.At the same time,the hepatocyte mitochondria were damaged to varying degrees,with blurred cristae,and dilated endoplasmic reticulum.（2）The levels of ALT and AST in liver tissues of each exposed group were increased significantly with increasing time（P<0.05）,while the expression levels of MDA,T-AOC,and T-SOD were increased significantly with increasing time（P<0.05）.（3）The contents of various micronutrients such as chromium,nickel,copper,selenium,zinc and manganese in the liver tissue of each exposed group were increased significantly with increasing time（P<0.05）,while the contents of each micronutrient in the Cr+Ni group were decreased significantly compared with the single exposed group（P<0.05）.In addition,the factorial analysis of micronutrients showed an interaction between chromium and nickel with chromium and nickel contents showing antagonistic effects.（4）Compared with the Con group,TLR4,TRAM,TRIF,TBK-1,IRF-3,My D88,IRAK-4,TRAF6,TAK-1,IKKβ,NF-κB,IL-1β,IL-6,TNFα,ULK1,Beclin 1 and LC3m RNA expression levels were significantly up-regulated in each exposed group（P<0.05）,while IL-10,mTOR and p62 m RNA expression levels were significantly down-regulated（P<0.05）.In addition,the factorial analysis showed an interaction between chromium and nickel with TLR4,My D88,NF-κB,mTOR,LC3 and p62 m RNA expression levels showing antagonistic effects.（5）Compared with the Con group,TLR4,My D88,TRAF6,NF-κB p50,IL-6,TNFα,ULK1 and LC3II/LC3Ⅰprotein expression levels were significantly up-regulated in each exposed group（P<0.05）,while mTOR protein expression levels were significantly down-regulated（P<0.05）.At the same time,immunohistochemical results showed that the positive expression of TLR4 protein was the most significant in the Cr group,and the positive expression of LC3B protein was the most significant in the Ni group.In conclusion,combined chromium-nickel exposure could induce liver inflammation and autophagy in mice through the TLR4/mTOR pathway,leading to chronic liver damage.The resulting liver toxicity was time-dependent and there was an antagonistic effect between chromium and nickel.Our experiment provided an important theoretical basis for studying the mechanism of combined chromium-nickel exposure-induced liver damage in mice.