Mechanisms Of Mitochondrial Pathway In Apoptosis Of Human Embryonic Lung Fibroblasts Induced By Beryllium Sulfate

Objective The research observed the influences of the early apoptosis rate and the mitochondrial membrane potential caused by different doses of beryllium sulfate(BeSO4)on the human embryonic lung fibroblast(MRC-5),and the changes of apoptosis inducing factor(AIF),the apoptotic protease activating factor 1(Apaf-1)m RNA expressions and protein expressions.To study the functions of the mitochondrial permeability transition pore(mPTP)in apoptosis of MRC-5 cultured in vitro induced by BeSO4,and the interventions caused by mPTP opening inhibitors Cyclosporine A(CsA)and promotive opening Atractyloside(ATR)were observed.Method Established the experimental model of MRC-5 in vitro and CCK-8 method was used to determine the CsA and ATR doses of intervention.There were blank control group,BeSO4 infected group(dose respectively 1,10,100?mol/L),intervention group(10?mol/L BeSO4+10?mol/L CsA and 10?mol/L Be SO4+10?mol/L ATR),intervention of control group(10?mol/L CsA and 10?mol/L ATR)in our experiment.Then infected and intervened respectively after 24 h,48h,72 h and collected the cells for testing,using the flow cytometry to detect early apoptosis rate.Fluorescent probe JC-1 was marked and to detect the changes of mitochondrial membrane potential,qRT-PCR was used to detect AIF,Apaf-1 mRNA expressions,Western Blot was used to detect the protein expressions of AIF and Apaf-1 in each of dose groups.Results1.After infected 24 h,48h,72 h,we determined the CsA and ATR intervention were all10?mol/L by using CCK-8 method.2.The low,medium and high dose groups of BeSO4,infected 24 h,48h,72 h,MRC-5 early apoptosis rate decreased in different degree.Infected 72 h,the low,medium and high dose groups of Be SO4 were lower than blank control group at the same time(P<0.05).CsA intervention group was lower than blank control group at the same time(P<0.05).ATR intervention group compared with blank control group(P<0.05),and compared with the medium dose group of Be SO4,the early apoptosis rate increased without statistical significance(P>0.05).3.The low,medium and high dose groups of BeSO4,respectively infected 48 h,72h,mitochondrial membrane potential increased in different degree.Infected 48 h,the high dose group of BeSO4 was higher than blank control group and the medium dose group of BeSO4(P<0.05);infected 72 h,the medium dose group of BeSO4 was higher than blank control group at the same time(P<0.05);in 72 h point,CsA and ATR intervention group compared with the medium dose group of BeSO4,the mitochondrial membrane potential were lower(P<0.05),The difference between CsA,ATR control group and the Be SO4 medium dose group at the same time were significant(P<0.05).4.The low,medium and high dose group of BeSO4,respectively infected 24 h,48h,72 h,three dose groups of AIF m RNA expressions compared with blank control group reduced at the same time(P<0.05);within 24 h,48h,72 h point,CsA intervention group compared with blank control group decreased at the same time(P<0.05),but compared with the medium dose group of BeSO4 at the same time had no significance(P>0.05);within 24 h,72h point,the ATR intervention group compared with the medium dose group of BeSO4 increased at the same time(P<0.05).5.The low,medium and high dose group of BeSO4 respectively infected 24 h,48h,72 h,the expressions of Apaf-1 mRNA of three dose groups compared with blank control group reduced at the same time(P<0.05);within 24 h,72h point,CsA intervention group compared with blank control group reduced at the same time(P<0.05),compared with the medium dosegroup of Be SO4 reduced at the same time(P<0.05);within 24 h,48h,72 h point,the difference between ATR intervention group and blank control group were statistically significant(P<0.05),within 24 h,48h point,the ATR intervention group compared with the medium dose group of Be SO4 were higher at the same time(P>0.05).6.Infected 48 h,72h,the AIF protein expressions of the medium and high dose group of BeSO4 compared with blank control group reduced(P<0.05);within 72 h point,CsA intervention group compared with blank control group decreased at the same time(P<0.05),and compared with the medium dose group of BeSO4 decreased at the same time(P>0.05),the ATR intervention group were higher compared with the medium dose group of BeSO4(P<0.05).7.Infected 24 h,48h,the Apaf-1 protein expressions of the medium and high dose group of BeSO4 compared with blank control group reduced(P<0.05);within 24 h point,CsA intervention group compared with blank control group decreased at the same time(P<0.05),the ATR intervention group were higher compared with the medium dose group of BeSO4(P<0.05).Conclusion Be SO4 inhibits human embryonic lung fibroblast apoptosis;mPTP played a role in MRC-5 early apoptosis caused by BeSO4.Through CsA and ATR intervention proved the role of mPTP.