Effect Of Different Selenium Forms On Activity And MRNA Expression Of Selenoenzymes In Primary Cultured Chicken Hepatocytes

Selenium is an essential trace element for animals and human.Its functions include improving immunity,preventing cancer,resisting free radicals,postponing aging, detoxification from some heavy metals,and preventing some local epidemic etc.In many countries,Supplementation of selenium in animal diet is a routine step for preventing Se shortage on animal husbandry.Selenoproteins,most of which are also called selenoenzymes that possess important functions in organism,are the main selenium forms in animals.Many achievements have been obtained in the acitivity and gene expression study of selenoenzymes,however,there still are many unclear problems to solve.In this paper,the serum-free culture for primary chicken hepatocytes and the real-time PCR method for measuring mRNA levels of GPx4 and IDâ… were developed and then the effect of different concentrations of Se-car,Se-Met and Na-Se on the activity of GPx1 and the mRNA expressions of GPx4 and IDâ… in primary cultured chicken hepatocytes was investigated.Experiment 1.Development of method for determination of GPx4 and IDâ… mRNA levels in chicken hepatocytes.According to the published mRNA sequences of gene ofβ-actin,GPx4 and IDâ… ,three pairs of specific primers were designed by biological software and synthesized.Total RNA was extracted from chicken liver and the cDNA was synthesised using Oligo dT primer.Using the synthesised cDNA as templates,three target fragments were amplified by polymerase chain reaction(PCR),respectively.The PCR products were electrophoresed on 2%agarose gels and extracted.The concentrations of purified PCR products were measured and diluted by 10-folds.Real-time PCRs were performed based on the 10-fold dilution series of standard DNA templates.In addition,the contents and annealing temperatures for the three pairs of specific primers were optimized, respectively.The results showed that one target band,which was consistent with the fact, was amplified for each specific primer pair.The optimal contents and annealing temperatures were 10μmol/L and 58℃,respectively.Amplification efficiencies of the primer pairs for gene ofβ-actin,GPx4 and IDâ… were 93.5%,91.4%and 98.4%, respectively.The melting curve analysis showed only one peak for each PCR product.Experiment 2.Development of isolation and serum-free culture methods for primary chicken hepatocytes.Chicken hepatocytes were obtained from white Leghorn chicken(30～40-day old) by collagenase perfusion.Total cell count and survival rate of freshly isolated hepatocytes were obtained by applying trypan blue exclusion.Hepatocytes were seeded into a 6-well microplate at a density of 2.5×10⁶ cells/2.5ml/well and cultured at 37℃with free atmosphere exchange.Morphological assessment of hepatocyte monolayers and determination of LDH activity in the culture supernatant were performed. The results showed that the cell yield was(4.1±0.2)×10⁸ hepatocytes per liver.The survival rate of freshly isolated hepatocytes was 92%±1%.Cultured in this condition,the LDH activity decreased gradually from 4h to 96h after seeding and the chicken hepatocytes could maintain good state up to 6 days.Experiment 3.Effect of different concentrations of Se-Car,Na-Se,and Se-Met on GPx1 activity in primary cultured chicken hepatocytes.Chicken hepatocytes were cultured for 24 h,then the old media were removed and the cells were washed three times and cultured in fresh media supplemented with 0(control),0.5,1,1.5,2,3,4,or 5μmol·L^-1 Se as Se-Car,Na-Se,or Se-Met for another 24 h.Activities of LDH in culture media,GPx1 activities and concentrations of GSH in hepatocyte cytosol were measured.The results showed that the LDH activities rised with the increased doses of Se,however,the activities of LDH in the culture media supplemented with Se-Met at doses of 1-3μmol/L were significantly lower than those of controls(P＜0.05).When compared to controls, concentrations of GSH decreased significantly at 1.5-2μmol·L^-1 Se-Car(P＜0.01),1.5μmol·L^-1 Na-Se(P＜0.05),and 1-2μmol·L^-1 Se-Met(P＜0.01),while GSH concentrations increased significantly at 4-5μmol·L^-1 Se-Car(P＜0.01),3-5μmol·L^-1 Na-Se(P＜0.01), and 4-5μmol·L^-1 Se-Met(P＜0.05),respectively.Activities of GPx1 reached the maximums at 2μmol·L^-1 Se-Car(2.18 folds vs.control,P＜0.001),1.5μmol·L^-1 Na-Se (2.16 folds vs.controls,P＜0.001),and 2μmol·L^-1 Se-Met(2.48 folds vs.control,P＜0.001).Trends of decrease in GPx1 activities were observed at 2-5μmol·L^-1 Se-Car,1.5-5μmol·L^-1 Na-Se,and 2-5μmol·L^-1 Se-Met,in a dose-dependent manner.Our results indicated that Se-Met was better than Se-Car and Na-Se in terms of security range and antioxidant ability.Experiment 4.Effect of different concentrations of Se-Car,Se-Met and Na-Se on the mRNA expression of IDâ… and GPx4 in primary cultured chicken hepatocytes.The treatment of chicken hepatocytes was the same as that in experiment 3.The mRNA levels were measured by real-time PCR.For Se-Car and Na-Se groups,the highest levels of mRNA for IDâ… were observed at 0.5-1.5μmol·L^-1 and 0.5μmol·L^-1,respectively,then they decresed dose-dependently.For Se-Met groups,the IDâ… mRNA levels increased at concentrations of 0.5-1.0μmol·L^-1,kept high and steady at concentrations of 0.5-1.0μmol·L^-1 and then decreased slightly.For each concentration of the three selenium sources, GPx4 mRNA levels declined very significantly(P＜0.001 vs.control).With the increase of selenium,GPx4 mRNA levels declined with no difference in Se-Met groups,whereas decreased significantly in the rest two.This suggested that compared with the rest two, Se-Met could keep a high level of mRNA for IDâ… in wider concentration range.All of the three could decrease the expression of GPx4 mRNA significantly with less decrease in Se-Met groups than in the rest two.