Effect Of Hyperthermia On Expression Of Selenoproteins In Myogenic Differentiated C2C12 Cells And The Protective Effect Of Different Sources Of Selenium On Cells Heat Stress

In this study murine muscle C2C12 cell line was taken as research material to develop heat stressed cell models and two experiments were conduct:(1)To study the effect of heat stress on expression of selenoprotein in murine C2C12 cells,(2)To compare the protective effect of two different selenium sources on cells heat stress and explore its possible mechanism.The main contents and results are as following:Experiment 1:Effect of heat stress on myogenic differentiation of C2C12 cells and expression of selenoprotein.The objective of this study was to investigate the effect of heat stress(HS)on expression of selenoproteins in differentiated C2C12 cells.Cells were cultured until reached to 80%confluence,then cells were shifted to differential media containing 2%horse serum and exposed to a hyperthermia at 41.5°C(HS)or 37°C(control)for additional 4,6 or 8 days.The mRNA expression of 24 selenoprotein encoding genes and abundance of 5 selenoproteins were investigated.The results showed that:(1)HS suppressed myogenic differentiation and impaired the development of muscle myotubes,down-regulated(P<0.01)mRNA expression of MYOD and MYOGENIN,and decreased(P<0.01)MYOGENIN protein abundance,elevated(P<0.01)HSP70 and(P<0.01)the ratio of BCL-2 to BAX at both mRNA and protein level.(2)HS up-regulated(P<0.01-0.05)mRNA levels of 18,11 and 8selenoprotein encoding genes after 4,6 and 8 days of hyperthermia,and only down-regulated(P<0.01)DIO2 after 6 and 8 days of hyperthermia,respectively.(3)HS influenced expression of selenoproteins and up-regulated(P<0.01-0.05)GPX1,GPX4and SEPN1 after 6 days of HS.In conclusion,the damage to development of mouse skeletal muscle myotubes by HS accompanied with the up-regulation of both selenoprotein encoding genes and proteins,which suggested a potential protective effect of selenoprotein on hyperthermia associated damage in C2C12 cells.Experiment 2:The protective effect of different sources of Se on C2C12 cells heat stress and the possible mechanism.This study was designed to investigate the protective effect of different sources of Se(sodium selenite,SS and selenomethionine,SeMet)on C2C12 cell damage heat stress,meanwhile the expression of selenoproteins were compared.C2C12 cells were grown in complete medium at 37 ~oC until to 80%confluence,then cells were grown on medium contain 2%horse serumfor another 4,6 and 8 d under different condition:at 37 ~oC(Control),at 41.5 ~oC(HS),at 41.5 ~oC with 0.5μmol Se/L SS supplementation(SS),or at41.5 ~oC with 0.5μmol Se/L SeMet supplementation(SeMet).The qPCRs were performed to compare expressions of 24 selenoprotein genes,4 differentiation-related genes and 2 apoptosis-related genes.Expressions of heat shock protein 70(HSP70),Myogenin,and 5 selenoproteins were also investigated.The results showed that:(1)HS affected myogenic differentiation of C2C12 cells with dereasing gene expression of MYOD and MYOGENIN and protein of MYOGENIN,and increasing gene and protein expression of HSP70;(2)HS up-regulated(P<0.05)mRNA levels of 14,10 and 14 selenoprotein encoding genes after4,6 and 8 days of hyperthermia,and down-regulated(P<0.05)mRNA levels of 1,4 and3 selenoprotein encoding genes after 6 and 8 days of hyperthermia,respectively;(3)Compared with the HS group,SS and SeMet supplementation modestly recovered expression of AMPK,MYOGENIN and MYOD genes,meanwhile decreased gene and protein expression of HSP70;(4)Compared with the HS group,SS and SeMet supplementation recovered the expression of selenoprotein genes,the mRNA profiles of aboved mentioned selenoprotein genes in SeMet groups were more similar to that of CK cells compared with SS cells;(5)The addition of SS and SeMet were significantly increased(p<0.05)expression of SELENON at HS 4days,however the expression of SELENON in SeMet was close to CK groupafter 6 days hat stress.Conclusion:(1)HS inhibited the myogenic differentiation of C2C12 cells and reduced the expression of differentiation genes and proteins of C2C12 cells,up-regulated the gene and protein expression of HSP70.At the same time,the expression of selenoprotein genes were globally upregulated,indicating that there is a close relationship between expression of these selenoproteins and C2C12 cells heat stress relieving effect;Adding SS and SeMet alleviated C2C12 cells heat stress via regulation selenoprotein encoding genes or protein.In general,exhibits better protective effect than inorganic selenium(SS)in relieving the heat stress in the differentiated C2C12 cells.