Effect Of Different Iron Sources On The Activity And Gene Expression Of Iron-containing Key Enzymes In Primary Cultured Hepatocytes Of Broiler Embryos

Three experiments were conducted to investigate the effects of iron?Fe?source and Fe level on the Fe concentration,Fe-containing key enzyme activities and their gene expression,and ferritin gene expression in primary cultured hepatocytes of broiler embryos,so as to reveal the mechanism of the metabolic utilization of different Fe sources in broiler hepatocytes at the cellular and molecular level.Experiment 1 was conducted to establish a stable and reliable primary culture model of broiler embryo hepatocytes,and establish experimental foundation for the subsequent experiments on Fe level and Fe source.The 14-d-old Arbor Acres healthy chick embryos were selected as the cell donor in this experiment,and Leibovitz's l-15 culture medium was used as the basic medium for in vitro culture.The results indicated that isolated hepatocytes are of high purity,complete differentiation,sound function,and good viability.The results of hepatocyte morphology,LDH activity and MTT cell viability were basically consistent,and the optimal incubation time range for optimal viability of primary culture of hepatocytes was from day 3 to 6.Therefore,a stable and reliable model of primary cultured hepatocytes of broiler embryo has been successfully established,which can be used in the subsequent experiments.Experiment 2 was conducted to investigate the effects of Fe levels and incubation time on Fe concentration,Fe-containing key enzyme activity and their gene expression,and ferritin gene expression in primary cultured hepatocytes of broiler embryos,so as to determine the optimal added Fe level and incubation time in hepatocyte primary culture and establish the foundation for the subsequent experiment on Fe source.A completely randomized design involving a 5×3 factorial arrangement of treatment was used in the current experiment.The added Fe levels were 0,0.25,0.5,0.75 and 1.00mmol/L^-1,and the incubation time were 12,24 and 48 h,respectively.The cells were randomly assigned to 15 treatments with 6 replicates per treatment.The results showed that:?1?On 24,48 or 72 h after incubation,as added Fe level increased,the Fe concentration in hepatocytes increased linearly or quardratically?P<0.05?,catalase?CAT?activity increased quardratically?P<0.05?,cytochrome c oxidase?COX?changed linearly?P<0.05?,and succinate dehydrogenase?SDH?decreased linearly or quardratically?P<0.05?.Hepatocytes from the 0.25 or 0.50 mmol/L Fe group had higher Fe concentration,and CAT,SDH and COX activities than those from other groups on 24 h after incubation?P<0.05?.?2?As added Fe level increased,SDH and FTH1 mRNA in hepatocytes increased linearly?P<0.05?,and CAT and COX7A2L mRNA increased linearly or quardratically?P<0.05?.On 24,48 or 72h after incubation,compared with the Fe-unsupplemented treatment,supplemental 0.50,0.75 or 1.0mmol/L Fe increased COX1 mRNA levels?P<0.05?.?3?The CAT,SDH and COX1 protein expression levels in hepatocytes increased linearly with supplemental Fe level increased?P<0.05?.In addition,the SDH protein expression levels in hepatocytes were higher on 24 and 72h than on 48 h?P<0.05?.On 24,48 or 72h after incubation,supplemental Fe increased FTH1 protein level?P<0.05?.In general,with regard to increase the Fe concentration,activitis and gene expression of the above enzymes,and FTH1gene expression in epatocytes,the optimal added Fe level was 0.25 or 0.50 mmol/L,and the optimal incubation time was 24 h.Experiment 3 was conducted to study the effect of Fe source on the Fe concentration,Fe-containing key enzyme activities and their gene expression,and ferritin gene expression in primary cultured hepatocytes of broiler embryos,and to further explore the difference in the metabolic utilization of different forms of Fe in hepatocytes of broiler embryos and its mechanism.A completely randomized design involving a 4×2+1 factorial arrangement of treatment was used in the current experiment.The 4 supplemental Fe sources were iron sulfate?FeSO₄?7H₂O?;Fe-Met with weak chelation strength?Fe-Met W;Q_f=1.37?;an iron proteinate with moderate chelation strength?Fe-Prot M;Q_f=43.6?;or an iron proteinate with extremely strong chelation strength?Fe-Prot ES;Q_f=8,590?.The 2 added Fe levels were 0,0.25,0.50 mmol/L,and incubation time were 24 h.The cells were randomly assigned to 9 treatments with 6 replicates per treatment.The results were as follows:?1?Compared with the control group,supplemental Fe significantly increased the Fe content in hepatocytes?P<0.05?.The Fe concentrations in hepatocytes on the FeSO₄?7H₂O,Fe-Met W and Fe-Prot M group were higher than those on the Fe-Prot ES group?P<0.05?,but no differences were detected among the FeSO₄?7H₂O,Fe-Met W and Fe-Prot M groups?P>0.05?.Compared with the 0.25 mmol/L Fe group,supplemental 0.50 mmol/L Fe increased Fe concentration in hepatocytes?P<0.05?.?2?Compared with the control group,supplemental Fe had no effect on the SDH,CAT and COX activities in hepatocytes?P>0.05?.And supplemental Fe source,Fe level and their interaction did not affect the SDH,CAT and COX activities?P>0.05?.?3?Compared with the control group,supplemental Fe significantly increased the CAT,SDH and FTH1 mRNA levels in hepatocytes?P<0.05?.The hepatocytes supplemented with0.50 mmol/L Fe had lower CAT mRNA levels but higher FTH1 mRNA levels than those supplemented with 0.25 mmol/L Fe?P<0.05?.The hepatocytes supplemented with Fe-Prot M and Fe-Met W had higher SDH mRNA levels than those supplemented with Fe-Prot ES or FeSO₄?7H₂O?P<0.05?.No differences were found in SDH mRNA levels among Fe-Prot M and Fe-Met W groups or Fe-Prot ES and FeSO₄?7H₂O groups?P>0.05?.?4?Compared with the control,supplemental Fe decreased CAT and SDH protein levels?P<0.05?,and increased COX1 and FTH1 levels?P<0.05?.Additionally,the hepatocytes supplemented with 0.25 mmol/L Fe had higher FTH1 protein levels than those supplemented with 0.50 mmol/L Fe?P<0.05?.However,supplemental Fe source,Fe level and their interaction did not affect the CAT,SDH and COX1 protein levels in hepatocytes?P>0.05?.In conclusion,added Fe could directly regulate the gene expression of CAT,COX1 and FTH1 in hepatocytes of broiler embryos at the level of transcription and translation.The organic Fe source with moderate or weak chelation strength and Fe sulfate had better effect in increasing the Fe concentration in hepatocytes than the organic Fe source with extremely strong chelation strength,and the organic Fe source with moderate or weak chelation strength was more effective in enhancing the SDH mRNA levels in hepatocytes than the organic Fe source with extremely strong chelation strength and Fe sulfate.These results suggest that organic Fe source with extremely strong chelation strength,which is difficult to release,might be poorly utilized in broiler hepatocytes compared with organic Fe source with moderate or weak chelation strength.The aforementioned new outcomes can provide scientific experimental basis for elucidating the metabolic utilization of different Fe sources in hepatocytes of broilers and its mechanism.