The Effect Of The Increase Of Hepatic Iron Induced By Microcystins-LR On Hepatocyte Apoptosis

ObjectivesThis study aims to investigate the effect of microcystins-LR?MC-LR?on iron homeostasis and its regulation mechanisms.The role of liver-specific microRNA-122?miR-122?in the dysregulation of iron homeostasis and hepatic damage,providing new molecular mechanisms and therapeutic targets for the disorder of iron homeostasis and hepatotoxicity induced by MC-LR.MethodsEight-week-old C57BL/6 mice and Hfe^-/-mice were selected.C57BL/6 mice were randomly divided into 6 groups,three groups?10?g/kg body weight?BW?saline,12.5?g/kg BW MC-LR and 25?g/kg BW MC-LR?of male and female,respectively.Hfe^-/-mice were randomly divided into four groups,two groups?10?g/kg body weight?BW?saline and 25?g/kg BW MC-LR?of male and female,respectively.Eight week-old C57BL/6 mice were selected and divided into five groups randomly?10?g/kg BW saline,12.5?g/kg BW MC-LR and 25?g/kg BW MC-LR Negative control agomir group and 25?g/kg BW MC-LR+miR-122 agomir group?.Negative control agomir group and 25?g/kg BW MC-LR+miR-122 agomir group were intraperitoneally injected with negative control agomir and miR-122agomir every 2 days for four times on the basis of daily injection of saline and 25?g/kg BW MC-LR respectively.Blood routine indexes were measured by blood routine detector.Iron/TIBC Reagent Set kit?Pointe Scientific Inc.,USA.?was used to detect serum iron and unsaturated iron binding capacity?UIBC?.Prussian blue staining was used to observe hepatic iron of C57BL/6 mice and Hfe^-/-mice.The pathologic damage of hepatic tissue was observed by H&E staining.Double staining with uranium lead was used to observe the organelles damage.TUNEL staining was used to observe the hepatocyte apoptosis of mice.The expression of iron homeostasis-related genes and apoptosis-related genes were determined by Real time-PCR.The expression of iron homeostasis-related proteins and apoptosis-related proteins in the liver,and the expression of FPN1 in the small intestine were detected by Western blotting.The content of Cyt-c in hepatocyte cytoplasm was detected by immunohistochemistry.ResultsThe detection results of iron homeostasis related indexes shwed that RBC,HGB and HCT were up-regulated in C57BL/6 and Hfe^-/-mice,but MCT,MCH and MCHC were down-regulated in C57BL/6 and Hfe^-/-mice after MC-LR teatment.Hepatic iron and spleen iron were elevated in C57BL/6 mice,but they were declined in Hfe^-/-mice.The result of Prussian Blue is consistent with the trend of hepatic iron content.However,renal iron was decreased in C57BL/6 mice,but elevated in Hfe^-/-mice.The phenomenon of hepatic iron overload was ameliorated after transfected with miR-122agomir.The results of H&E staining showed that no significant pathologic damage was observed in hepatic tissue of the control group in C57BL/6 mice,but the pathologic damage of hepatic tissue was increased with the increasing exposure dose of MC-LR.Slight pathologic damage was observed in hepatic tissue of the control group in Hfe^-/-mice,but the pathologic damage was more severe in the high exposure dose group of Hfe^-/-mice than that in C57BL/6 mice.The results of tissue transmission electron microscope showed that different degree damage of organelles were observed in the heart,liver and spleen tisuue in the control group of Hfe^-/-mice.Furthermore,these damage of organelles in the high exposure dose group was much worse than that in the control group.The hepatocyte apoptosis was increased.However,the hepatocyte apoptosis and the damage of hepatic tissue were not improved by miR-122 agomir.The results of genes showed that the expression of miR-122 was decreased in the liver of C57BL/6 mice after exposure to MC-LR,but the expression of miR-122 was significantly increased after tranfected with miR-122 agomir.Compared with the control group,the expression of hepcidin,Bmp6,and Il-6 were down-regulated in C57BL/6 and Hfe^-/-mice.The expressions of hepcidin,HJV,Hfe,Bmp6 and Il-6 were decreased in the liver,and the expressions of Tmprss6?the negative regulators of hepcidin?and Hif-1?were elevated.However,the expressions of hepcidin,HJV,Hfe,Bmp6 and IL-6 were improved after transfected with miR-122 agomir.At the same time,the expressions of Tmprss6 and Hif-1?were inhibited.Compared with the control group,the expression of Bax was significantly increased,but Bcl-w,Bcl-2,caspase-3,Caspase-8 and caspase-9 were down-regulated.However,the expressions of Bax and Bcl-w were further decreased,but the expression of Bcl-2 was elevated,and other genes were not changed after transfection of miR-122 agomir.The results of proteins showed that compared with the control group,FPN1 was up-regulated in the small intestine of C57BL/6 and Hfe^-/-mice.The expressions of p-Smad1 and p-Stat3 were decreased in C57BL/6 mice,but no significant changes in Hfe^-/-mice were observed.The expressions of Smad1/5/8,p-Stat3 and Trf-1 were decreased in liver.However,the expressions of FPN1,p-Smad1/5/8,p-Stat3 and Trf-1 were increased after transfection of miR-122 agomir.The pro-apoptotic proteins Bax,caspase-3 and caspase-8 were also up-regulated,but the expressions of Bcl-2,Bcl-w and cleaved caspase-3 were decreased.Caspase-3 and cleaved-caspase-9 were down-regulated in the low dose group,but the expression of caspase-3 increased in the high dose group.No significant change in cleaved-caspase-9 was observed in the high dose group.The expressions of Bcl-w,Bcl-2,caspase-3 and caspase-8 were decreased,but cleaved-caspase-9 was elevated after transfection of miR-122 agomir.The content of Cyt-c in the cytoplasm was increased.Furthermore,the expression of Cyt-c is further increased after transfection of miR-122 agomir.Conclusions1.MC-LR can induce the accumulation of hepatic iron,which may be contribute to the down-regulation of Hepcidin.The down-regulation of Hepcidin induced by MC-LR may be related to IL-6/STAT3 and BMP/SMAD signaling pathways,and the up-regulation of Tmprss6 and Hif-1? in liver.2.MC-LR can inhibit the expression of liver specific miR-122 and over-expression of miR-122 can ameliorate the accumulation of hepatic iron induced by MC-LR,which may be related to IL-6/STAT3 and BMP/SMAD signaling pathways,and the inhibition of Tmprss6 and Hif-1? in liver.3.MiR-122 can't ameliorate the hepatocyte apoptosis and the pathologic damage of hepatic tissue induced by MC-LR,which may be contribute to the inhibition of miR-122 on bcl-w.