| Five experiments were conducted to study the anti-heat stress effect and possible mechanisms of manganese(Mn)by using the model of primary chick embryo Myocardial Cells.The first experiment was conducted to establish the primary chick embryo myocardial cells model and evaluate the chick embryo Myocardial cells continued viability to prepare for subsequent experiment.As for the primary chick embryo myocardial cells model,the myocardial tissue was digested with 0.12% collagenase II for 8 min at 37℃.The digested mixtures were centrifuged at 800 r/min for 5 min to recover cells.Collect cells and these cells were then re-suspended and removed into a culture flask to two-time difference velocity adherent culture to remove out fibroblasts.These cells were then cultured at 40°C in a humidified incubator with 5% CO2 and 95% air.After cultured for 24 h,it can be observed that myocardial cells have a marked autonomic pulsation.After 72 h in the culture medium,the proportion of cells showing spontaneous contraction was over 95%,and then these cells were used for the following trials.As for the Myocardial cells continued viability,the AST activities in the supernatants of myocardial cells were significantly(P < 0.05)influenced by incubation time and increased with incubation time and reached the maximum on the ninth day.The CK and LDH activities displayed the similar tendencies and decreased significantly(P < 0.05)on the sixth day and then increased gradually.The activities of AST,CK and LDH indicate that the cell vitality could last for 6 days and then decreased with incubation time.The second experiment was conducted to investigate the effect of heat stress on chick embryo Myocardial 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.Under the heat stress,the CK 、 LDH and AST enzymatic activities in the suspension of myocardial cells were increased(P < 0.05),suggesting that the integrity of myocardial cells were altered.High temperature up-regulated(P < 0.05)the HSF1 and HSF2 m RNA expressions and the gene expression of HSP70 and HSP90.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 2 h and 4 h,respectively.The protein expression of HSP70 and HSP90 increased with the culture time.We also found that HSP70 and HSP90 m RNA were more sensitive than their protein expression.These results indicate that the gene expression of HSP70 and HSP90 in the primary chick embryo 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 2 h and 4 h were the best time points,which provides a foundation for the experiment 4.In order to estimate the optimal Mn level and effective incubation time,we investigate the effect of manganese(Mn)on the expression of the manganese superoxide dismutase(Mn SOD)gene in cultured primary myocardial cells.In this trial,we used a completely randomized design involving a 5 Mn levels in the culture medium(no Mn supplemenation,0.5,1,2 and 4 m M of Mn as the inorganic Mn Cl2)× 3 incubation time points(12,24 and 48 h)factorial arrangement of treatments(n=6).As for the Mn SOD activity,an interaction(P < 0.05)between Mn level and incubation time was observed.At 12 h,there were no differences(P > 0.05)among Mn levels.At 24 h,however,compared with the control with no Mn addition and 0.5 m M Mn level,the addition of 1.0 m M Mn significantly increased(P < 0.05)Mn SOD activity with no difference(P > 0.05)between the control and 0.5 m M Mn level,and the similar results were observed at 48 h.For the Mn SOD m RNA expression level,both Mn level(P < 0.05)and incubation time(P < 0.05)had significant effects on Mn SOD expression.Compared with the control,the additions of 0.5 and 1.0 m M Mn levels significantly increased(P < 0.05)the Mn SOD m RNA expression levels with no difference(P > 0.05)between 0.5 and 1.0 m M Mn levels.The Mn SOD m RNA expression was significantly greater(P < 0.05)at 48 h than at 12 h or 24 h.The Mn SOD protein level was not affected(P > 0.05)by Mn level,incubation time and their interaction.The results may also suggest that the Mn-induced Mn SOD gene expressions in the primary chick embryonic myocardial cells could be modulated at transcriptional and post-translational levels,or through modified stability of synthesized m RNA and protein.the results showed that supplementation of 1 m M Mn and culturing 48 hours were the most suitable in the cultivation of chick embryo primary myocardial cells.Experiment 4 was conducted to investigate the effect of temperature and manganese(Mn)treatment on antioxidant status and the expression of heat shock protein/ factors in the cultivation of chick embryo primary myocardial cells.A completely randomized design involving 2 incubation temperatures×3 Mn 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 of 44 ℃(HT).The three Mn treatments were no Mn supplementation(C,medium),1 m M of Mn as the inorganic Mn CL2(i Mn)or 1 m M of Mn as Mn proteinate(o Mn).The 2 heat stress time points were at 2 or 4 h.There were total 12 treatments with 6 replicates.High temperature increased(P < 0.05)Mn SOD activity,the Mn SOD,HSP70,HSP90,HSF1,HSF2 and HSF3 m RNA expression,and the HSP70 protein expression.Compared with control,addition Mn increased(P < 0.05)the Mn SOD activity,the myocardial cells treated with o Mn had higher(P < 0.05)Mn SOD activities than that treated with i Mn.Mn source,temperature and their interaction influenced the gene expression of Mn SOD(P < 0.05).Under the NT,adding Mn increased(P < 0.05)the gene expression of Mn SOD.Under the HT,compared to control group,only o Mn increased(P < 0.05)the Mn SOD m RNA expression.However,both i Mn and o Mn increased(P < 0.05)the Mn SOD protein expression,and o Mn showed more effective than i Mn(P < 0.05).Mn source,temperature and their interaction influenced the gene expression of HSP70 and the m RNA expression of HSP90(P < 0.05).Under the NT,Mn didn’t effect the gene expression of HSP70 and the m RNA expression of HSP90(P > 0.05).Under the HT,compared to control group,adding Mn decreased(P < 0.05)the HSP70 and HSP90 m RNA expression,and o Mn showed more effective than i Mn(P < 0.05).However,i Mn and o Mn were equivalent in decreasing the HSP70 protein expression(P < 0.05).Compared with control,addition Mn increased(P < 0.05)the protein expression of HSP90.The results showed that the Mn,especially o Mn proteinate with a moderate chelation strength could improve the antioxidant capacity and inhibit the oxidative stress induced by heat stress.In experiment 5,we further investigated the effect of temperature and Mn treatment on the epigenetic of chick embryo myocardial cells based on the experiment 4.addition either i Mn or o Mn had lower(P < 0.05)DNA methylation of Mn SOD and HSP70 promoter,which was related to the high m RNA expression of Mn SOD.High temperature increased(P < 0.05)the expression of gga-mi R-34c-5p and gga-mi R-29c-3p,and decreased(P < 0.05)the expression of gga-mi R-10a-5p,gga-mi R-10b-5p and gga-mi R-125b-5p.both i Mn and o Mn increased(P < 0.05)the expression of gga-mi R-142-5p,gga-mi R-34b-5p,gga-mi R-34c-5p,gga-mi R-29c-3p,gga-mi R-10a-5p,gga-mi R-10b-5p and gga-mi R-125b-5p.The results indicated that Mn can alleviate the oxidative damage caused by high temperature stress by alterations of of DNA methylation of the specific gene promoter and the corresponding mi RNA expression.In summary,we investigate the anti-heat stress effect and mechanisms of manganese in the cultivation of chick embryo primary myocardial cells by using the above five experiments in vitro.The results indicated that Mn,especially o Mn proteinate with a moderate chelation strength could improve the antioxidant capacity and inhibit the oxidative stress induced by heat stress by alterations of of DNA methylation of the specific gene promoter and the corresponding mi RNA expression. |
PDF Full Text Request