Effects Of Vitamin E On Oxidative Stress And Inflammatory Response In Iron Overload Liver And Cells

BackgroundIron is one of the essential trace elements for our body,involving in various physiological and biochemical processes,such as oxygen transport,DNA synthesis,electron transport.Previous studies were mainly focus on iron deficiency anemia,but iron overload will exits severe toxic effects for the past few years.Liver is an important organ in regulating iron homeostasis.There are many diseases following with hepatic iron overload,such as β-Thalassemia.Due to β-globin defect,β-Thalassemia patients have rupture of red blood cells and hemoglobin separation,resulting in anemia.Meanwhile,those patients exits high serum malonyldialdehyde(MDA),pro-inflammatory cytokines and transaminase.A large number of studies have confirmed excess iron can produce reactive oxygen species(ROS),which can interact with lipid,protein and nucleotide and then cause oxidative stress,resulting in lipid peroxides,via Fenton reaction.Recently,studies indicated that the expressions of mi R-122 and mi R-146 a have an important role in inflammatory response.Interestingly,our previous study demonstrated that iron overload can inhibit the expression of hepatocyte nuclear factor 4α(HNF4α),then down-regulated mi R-122 and mi R-146 a enhance the activities of NF-κB signaling pathway,resulting in the expression of inflammatory cytokines.Vitamin E(Vit.E)as a widely used antioxidant vitamins,it can clear excess free radicals and maintain the balance of oxidative and anti-oxidation system in our body.Studies have shown that Vit.E supplement can decrease oxidative stress induced by iron overload.However,other studies indicated that high doses of Vit.E can enhance iron overload related oxidative stress.Effects of Vit.E on hepatic oxidative stress were controversial.In clinic,vitamin E was often used to prevent oxidative damage of red blood cells in patients with thalassemia.However,whether it can prevent hepatic oxidative stress and inflammation caused by iron overload has not been reported.ObjectiveOur study via animal and cell experiments to observe effects of Vit.E on iron-overload associated hepatic oxidative stress and inflammation response.Aim to provide experimental evidence for the future whether to adopt supplement Vit.E to prevent and cure iron overload liver injury related diseases in clinic.Methods 1.Animal feeding and interventionTwenty-four four-weeks-old male C57BL/6 mice were randomly divided into four groups: control group(Ctrl),iron overload(IO)group,iron overload plus corn oil(IO +corn oil)group,iron overload plus vitamin E(IO +Vit.E)group,there are six mice in each group.The control group were intraperitoneal injection of 0.9% saline twice a week,and other three groups were intraperitoneal injection of iron dextran(20 mg/ml)twice a week,0.15 ml every times,and total iron were 12 mg.IO plus Vit.E mice fed daily Vit.E dissolved in corn oil(100 mg/kg bw),IO plus corn oil group feeding with corn oil daily,and the volume equal to Vit.E,IO + corn oil group as a solvent control.The experiment maintain two weeks.2.Detection of part serum biochemical parameters and liver ironUsed automatic biochemical analyzer to detect levels of serum iron(SI),transferrin saturation(TS),total iron binding capacity(TIBC),alanine aminotransferase(ALT),and aspartate aminotransferase(AST).Detect contents of liver iron by nitrification process.Detect hepatic iron deposition by Prussian blue staining.3.Detection of hepatic tissue oxidative stress damageDetect ROS content: Preparation of liver homogenate,centrifuged supernatant by ROS detection probe DCF in 37 oC and dark for 1 h(DCF were 5 μM).Use microplate reader to detect fluorescence intensity.Detect SOD activity,contents of MDA,GSH: First liver were homogenized,and then all groups were handled according to the kit operational instructions strictly.Detect content of 8-OHd G: Used immunofluorescence method to detect content of 8-OHd G following the operational instruction.4.Hepatic tissue HE staining and pathological inflammation scoreLiver tissue were immobilizated by 4% paraformaldehyde,after embedded in paraffin,then spread sheet for a thickness of 4 μm,and stained with hematoxylin and eosin.Finally chips were photographed under a microscope,accordance with standards of inflammatory pathological inflammation score proposed by lok.Schener in 1985.5.Cell cultivation and interventionHuman hepatoma cell line Huh7 cells were divided into three groups: control group(Ctrl),Holo-Tf group(Holo-transferrin)and Holo-Tf + Vit.E group.Holo-Tf group were added Holo-Tf(final concentration were 30 μM),Holo-Tf + Vit.E group added Holo-Tf(final concentration were 30 μM)and Vit.E(final concentration were 50 μM).simultaneously,the control group were added an equal volume of sterile distilled water.All group interfered for 24 h.Detection of cell SOD activity,and contents of ·OH,MDA and GSH: After intervention,all groups cell were handled according to the operational instructions strictly.6.Real-time quantitative fluorescent PCRUsing Trizol to lysis cells or tissue,then added chloroform according to the ratio of extraction.After Centrifugation,the upper aqueous phase was precipitated with isopropanol precipitation.Washed precipitation by 75% ethanol precipitate twice,then given a white flocculent precipitate is RNA.Add appropriate amount of RNase Free dd H2 O to dissolve RNA.Then detect concentrations of RNA.Used reverse transcription kit RNA transcribed RNA into c DNA.Add forward primers and reverse primers of target gene,SYBR Green,c DNA,DEPC water to real-time quantitative polymerase chain reaction.Animal internal reference was 18 S,and cell internal reference was β-actin,RQ values were calculated based on the Ct value,and then statistics data.7.Western blotLiver tissue or cells were homogenized,lysed(volume ratio of lysis buffer,protease inhibitors,phosphatase inhibitors and PMSF were 1 ml:1 μl:10 μl:5 μl),and then centrifuged.The supernatant protein draw liquid supernatant and loading buffer by 4: 1 ratio for protein denaturation.The supernatant protein using the BCA protein quantification kit to detect concentrations of protein,and incubated for 30 min in 37 oC,measured at 562 nm using absorbance microplate reader.To calculate the concentration of protein according to the standard curve,then obtain the final sample volume of protein.Protein by PAGE-SDS-polyacrylamide gel electrophoresis,transferred to membrane,blocked,incubated with primary antibody,membranes were washed.Then membranes were incubated with secondary antibody,wash the membrane,visualizate protein bands,and analysis bands gray.The final step is to statistics expression of protein.8.Statistics and analysisAll data were determined by x ±s,using SPSS 21.0 statistical test for analysis.Four ! 10! groups using single factor analysis of variance(one-way ANVOA).With P <0.05,P <0.01,P <0.001 indicates the difference was statistically significant.Results 1.Effects of Vit.E on iron status in iron overload mice and Huh7 cells 1.1 General conditions of miceIO mice compared to control,body weight and dietary intake have no significant difference.IO+ Vit.E and IO+ corn oil mice showed no significant difference in body weight and dietary intake.The body weight and dietary intake in IO+ Vit.E and IO+ corn oil group were lower than control mice.1.2 Iron overload status of mice 1.2.1 Levels of serum iron-related parameters and liver iron in iron overload miceCompared with control mice,levels of SI,TS and liver iron were significantly increased in IO,IO+ corn oil and IO+ Vit.E mice.While after giving Vit.E to iron overload mice simultaneouly,levels of serum iron content,transferrin saturation and liver iron have no significant difference in IO,IO+ corn oil and IO+ Vit.E mice.Compared with the control group,it had seen significant iron deposition in IO,IO+ corn oil and IO+ Vit.E mice by liver Prussian blue reaction.1.2.2 The expression of hepatic iron metabolism regulators in iron overload miceCompared with control mice,m RNA and protein expression of FTL1 and HAMP were significantly increased,while m RNA and protein expression of TFRC was decreased in IO mice.After interfered with Vit.E,m RNA and protein expression of FTL1,HAMP and TFRC had no significant difference in IO,IO+ corn oil,IO+ Vit.E mice.1.3 The expression of iron metabolism regulators in iron overload Huh7 cellsCompared with control,m RNA and protein expression of FTL1 and HAMP,two kinds of iron metabolism regulators,were significantly increased,while m RNA and protein expression of TFRC was decreased in Huh7 cells which were cultivated with culture medium added with 30 μM Holo-Tf for 24 hour.Compared with 30 μM Holo-Tf added only culture medium group,iron metabolism regulators FTL1,HAMP and TFRC’s m RNA and protein expression had no significant difference in Huh7,which were cultivated in cell supernatant added with 30 μM Holo-Tf and 50 μM Vit.E.2 Effects of Vit.E on oxidative stress in iron overload liver cells 2.1 Effects of Vit.E on hepatic oxidative stress in iron overload mice 2.1.1 Effects of Vit.E on hepatic contents of ROS,MDA,GSH and activities of SOD in iron overload miceCompared with control group,contents of ROS and MDA were increased,however,contents of GSH and the activity of SOD were decreased in IO,IO+corn oil mice.Compared with IO mice,contents of ROS and MDA were decreased in IO+Vit.E mice,however,changing trend of the activity of SOD and contents of GSH were opposite.2.1.2 Effects of Vit.E on hepatic contents of 8-OHd G in iron overload miceCompared with control group,fluorescence intensity of hepatic 8-OHd G were increased significantly in IO,IO+corn oil mice,while fluorescence intensity of 8-OHd G in IO+corn oil mice were weak comparing with IO mice.2.2 Effects of Vit.E on oxidative stress in iron overload Huh7 cells 2.2.1 Effects of Vit.E on contents of ·OH in iron overload Huh7 cellsCompared with control,contents of ·OH were increased significantly in Huh7 cells which were cultivated with culture medium added with 30 μM Holo-Tf for 24 hour.Compared with Holo-Tf added only group,contents of ·OH were decreased in Huh7 cells,which were cultivated in cell supernatant added with 30 μM Holo-Tf and 50 μM Vit.E.And there were dose-effect relationship,however,contents OH in 50 μM and 100 μM Vit.E had no significant difference.2.2.2 Effects of Vit.E on contents of hepatic ROS,MDA,GSH and activities of SOD in iron overload miceCompared with control,contents of ROS and MDA were increased,contents of GSH and the activity of SOD were decreased in Huh7 cells which were cultivated with culture medium added with 30 μM Holo-Tf for 24 hour.Compared with Holo-Tf added only group,contents of ROS and MDA were decreased,however,contents of GSH and the activity of SOD were increased in Huh7 cells,which were cultivated in cell supernatant added with 30 μM Holo-Tf and 50 μM Vit.E.3.Effects of Vit.E on inflammatory response in iron overload liver and Huh7 cells 3.1 Effects of Vit.E on hepatic inflammatory response in iron overload mice 3.1.1 Effects of Vit.E on expression of hepatic inflammatory cytokines in iron overload miceCompared with control mice,m RNA and protein expression of p65,TNFα and IL-6 were significantly increased in IO mice.After interfered with Vit.E simultaneouly,m RNA and protein expression of p65,TNFα,IL-6 had no significant difference in IO,IO+ corn oil and IO+ Vit.E mice.3.1.2 Effects of Vit.E on hepatic inflammation score in iron overload miceCompared with control mice,hepatic inflammation score were increased in IO,IO+ corn oil and IO+ Vit.E mice.There was no significant difference in IO,IO + corn oil and IO+ Vit.E mice.3.1.3 Effects of Vit.E on levels of serum AST and ALT in iron overload miceCompared with control mice,levels of serum AST and ALT were increased in IO,IO + corn oil and IO + Vit.E mice.There was no significant difference in IO,IO + corn oil and IO + Vit.E mice.3.2 Effects of Vit.E on expression of inflammatory cytokines in iron overload Huh7 cellsCompared with control,m RNA and protein expression of p65,TNFα,IL-6 were significantly increased in Huh7 cells which were cultivated with culture medium added with 30 μM Holo-Tf for 24 hour.Compared with Holo-Tf added only group,m RNA and protein expression of p65,TNFα,IL-6 had no significant difference in Huh7 cells,which were cultivated in cell supernatant added with 30 μM Holo-Tf and 50 μM Vit.E.3.3 Effects of Vit.E on expression of HNF4α mi R-122 mi R-146 a CCL2 TRAF6 in iron overload Huh7 cellsCompared with control group,m RNA expression of HNF4α mi R-122 mi R-146 a were significantly decreased,CCL2 TRAF6 were significantly increased in Huh7 cells which were cultivated with culture medium added with 30 μM Holo-Tf for 24 hour.Compared with Holo-Tf added only group,m RNA expression of HNF4α mi R-122 mi R-146 a CCL2 and TRAF6 had no significant difference in Huh7 cells,which were cultivated in cell supernatant added with 30 μM Holo-Tf and 50 μM Vit.E.ConclusionComprehensive results of this study,it indicated that iron overload can cause oxidative stress damage and inflammation in liver and cells;Vit.E supplements can alleviate hepatic oxidative stress induced by iron overload,but it did not alleviate hepatic inflammatory response induced by iron overload.Vit.E supplement had no significant to decreased expression of HNF4α,mi R-122,mi R-146 a,and increased activity of NF-κB signaling pathway in iron overload hepatocytes.Therefore,prevention and treatment of Vit.E supplementary on iron overload liver damage-related diseases needs further study.