Anti-heat Stress Effect And Mechanisms Of Zinc In The Cultivation Of Chick Embryo Primary Hepatocytes

Four experiments were conducted to investigate the anti-heat stress effect and possible mechanism of Zinc(Zn)by using the model of primary chick embryo liver cells.The frist experiment was conducted to establish the chicken embryo hepatocytes and evaluate the chick embryo hepatocytes cells continued vitality to prepare for subsequent experiment..Embryonic liver cells were acquired from the 14-dayold chick embryos and plated into 6-well culture-plates.the medium Trypan blue staining showed that the cell survival rate was above 95%,glycogen staining results showed that isolated liver cells had higher purity can reach more than 90%;After 72 h in the culture medium,the proportion of cells showing spontaneous contraction was over95%,and the model was established successfully,and the cell vitality could last for 3days and then decreased with incubation time.Experiment two aimed to estimate the optimal Zn level and effective incubation time.A completely randomized design involving a 5 Zn leveis in the culture medium(no Mn supplemenation,50,100,2 00 and 400 u M of Zn as the inorganic Zn SO4)×3 incubation time points(12,24 and 48 h)was used in this trial.And there were 15 treatments with 6 replicates.At the12 h,only addition 50 u M Zn significantly increased(P < 0.05)Cu Zn SOD activity;compared with the control group,addition Zn were significantly up-regulated(P<0.0001)MT m RNA expression.and there was no difference between the Zn levels(P>0.05).the results showed that supplementation of 50 u M Zn and culturing 12 hours were the most suitable in the cultivation of chick embryo primary liver cells.Experiment three was conducted to investigate the effect of heat stress on chick embryo liver cells in vitro to estimate the optimal heat stress time.A completely randomized design involving 2 incubation temperatures(normal(40℃)temperature or high(44℃)temperature.)×5 time treatments(1,2,4,6 and 8 h)was used in this trial.And there were 10 treatments with 6 replicates.Compared with the normal group,the high temperature group significantly increased liver cells culture medium LDH activity at 4,6 and 8 h(P<0.0001);Under high temperature,the expression of HSP 70 and HSP90 m RNA increased firstly and then decreased,and the expression of HSP 70 and HSP 90 m RNA reached the peak at 4 h and 6 h,respectively.These results indicate that the m RNA expression of HSP70 and HSP90 in the primary chickembryo myocardial cells is sensitive to heat stress.And they are play an important role in opposing and alleviating the heat stress.From the above results,we conclude that the myocardial cells in high temperature at 4 h and 6 h were the best time points,which provides a foundation for the experiment 4.Experiment 4 was conducted to investigate the anti-Heat Stress Effect and Mechanisms of Zinc in the cultivation of chick embryo primary hepatocytes.A completely randomized design involving 2 incubation temperatures×3 Zn treatments×2 heat stress time points was used in this experiment.The two incubation temperatures were a normal temperature of 40 ℃(NT)and a high temperature of44 ℃(HT).The three Zn treatments were no Mn supplementation(C,medium),50 u M of Zn as the inorganic Zn SO4 or 50 u M of Zn as Zn-Lys.The 2 heat stress time points were at 4 or 6 h.There were total 12 treatments with 6 replicates.The results showed that the addition of Zn SO4 could increase the activity of Cu Zn SOD(P=0.0048)in hepatocytes at normal temperature.At high temperature,the activity of Cu Zn SOD enzyme in liver cells of Zn-Lys group was significantly higher than that of the non zinc group and Zn SO4 group(P=0.049).The MDA content of hepatocytes was significantly higher than that of normal temperature(P=0.006).Compared with non zinc group,adding zinc could significantly increase the expression of hepatocyte MT m RNA(P<0.035);compared with the normal group,the high temperature significantly increased the expression of hepatocyte Cu Zn SOD m RNA;compared with the normal group,the high temperature significantly increased the expression of hepatocyte HSP70 m RNA(P<0.0001).Under the high temperature,compared with non Zn group,adding Zn SO4 group significantly reduced the HSP70 expression of m RNA(P<0.05);under normal temperature condition,the expression of Zn-Lys was added HSP90 m RNA was significantly lower than that without zinc and Zn SO4 group(P<0.005).Under the high temperature,the treatment of 4h,the expression of HSP90 m RNA in Zn SO4 group was significantly lower than the non plus zinc group and ZnLys group(P<0.0003);compared with the normal group,the high temperature significantly increased the expression of HSP70 protein in liver cells(P<0.0001).When 6h was treated with high temperature,the expression of HSP70 protein was significantly decreased in Zn SO4 group and Zn-Lys group(P<0.0001).In summary,Zn could improve the antioxidant capacity of chick embryo liver cells.Zinc improved chick embryo liver cell Cu Zn SOD enzyme activity and MTm RNA expression,especially organic Zn increased the Cu Zn SOD enzyme activity under the high temperature.oxidative injury of broiler chick embryo liver cells caused by heat stress(high temperature increased the expression of HSP70 gene and HSP90 m RNA in chick embryo liver cells);and adding Zn could alleviate the negative effect of the heat stress in chick embryo liver cells.