Study On The Role And Molecular Mechanism Of MSTN In Offspring Mice Skeletal Muscle Inhibitation By DEHP Maternal Exposure

Background and purposePlasticizers,also called phthalic acid ester compounds(phthalatic esters,PAEs),are a kind of environmental endocrine disruptors.They have potential harmful effects on the reproduction,liver,kidney,embryo,and so on.Currently,researchers are mainly focusing on studies of the effects of plasticizers on the metabolism and reproduction,while reports on skeletal muscle development are rare.In our previous experiments,we found that the weight of the skeletal muscle of mice obviously decreased after maternal exposure to DEHP,and the mRNA expression of MSTN was up-regulated after maternal exposure to DEHP.The result shows that MSTN may play a role in muscle dysplasia after DEHP exposure.In this study,we use the Cre-lox P technology to establish a MSTN-knockout mice model,and study the role and molecular mechanism of MSTN in offspring mice skeletal muscle inhibitation by plasticizer DEHP maternal exposure.Method1.The Cre-loxp system was used to establish the MSTN-knockout mice model,and then the genotype of the offspring was identified by PCR assay.2.The weight of leg skeletal muscles and the body weight of MSTN-knockout mice were compared with the wild-type mice(at 0,7,14,21d).The sizes of leg skeletal muscle fibers of wild type and MSTN-knockout mice were tested by Immunofluorescence assay.The m RNA expression level of Atrogin-1 and MuRF-1 was detected by RT-qPCR.3.Construct the pregnant mice model of wild-type mice(WT)and MSTN-knockout mice(KO).Pregnant mice were randomly divided into four groups: WT-Con group,WT-DEHP group,KO-Con group,and KO-DEHP group.Mice were given gavage with corn oil from gestation to weaning(at 21d).DEHP group mice were administered by gavage with DEHP(250mg/kg/d)from gestation to weaning(at 21d).The weight of leg skeletal muscles(at 21d)and the body weight of DEHP mice were compared with the control mice(at 0,7,14,21d).The m RNA expression level of synthesis and degradation gene was detected by RT-qPCR.4.In order to explore the mechanism of DEHP on skeletal muscle development,C2C12 myoblasts were exposed to various concentrations(0,62.5,125,250,500,1000 ?M)of MEHP(MEHP is the activated metabolite of DEHP)over different time periods(48,72,96 h),and viability of cells were assessed using CCK-8 assay.After treatment of skeletal muscle C2C12 cells with MEHP,the mRNA expression levels of related synthesis and degradation genes were tested by RT-q PCR.The influence of MSTN promoter after treatment with MEHP was detected by luciferase assay.After MEHP treatment of C2C12 cells with interfered C/EBP?,the mRNA expression levels of related synthesis and degradation genes were tested by RT-q PCR.Result1.Compared with the wild-type mice,the body weight of MSTN-knockout mice were significantly heavier at 7d and 21d(P<0.05),and the weight and the size of leg skeletal muscle fibers were also bigger(P<0.01).Besides,the mRNA expression level of Atrogin-1 and Mu RF-1 significantly decreased after knocking out MSTN in mice(P<0.01).2.Compared with the control mice,the body weight of the offspring of WT-DEHP mice were lower(at 0,7,14,21d),and the weight of leg skeletal muscle was also lower(at 21d).Compared with the control mice,the body weight of the offspring of KO-DEHP mice were not lower(at 0,7,14,21d),and the weight of leg skeletal muscle was also not lower(at 21d).3.Compared with the control mice,the m RNA expression level of Atrogin-1 and Mu RF-1 increased,and that of Myo D and Myogenin decreased in the offspring of WT-DEHP mice.Compared with the control mice,the m RNA expression level of Atrogin-1 and MuRF-1 did not increase,but that of Myo D and Myogenin were up-regulated in the offspring of KO-DEHP mice.Compared with the control mice,the protein expression levels of MyoD and p-Akt were downregulated in the offspring of WT-DEHP mice.Compared with the control mice,the protein expression levels of Myo D and p-Akt did not decrease in the offspring of KO-DEHP mice.These consequences indicate that the inhibition effect of DEHP on skeletal muscle development through maternal exposure was mediated by myostatin.4.C2C12 myoblasts were exposed to various concentrations of MEHP.With the increasing of the concentration and the exposure time of MEHP,the viability of C2C12 cells significantly decreased.It suggests that MEHP has inhibition effects on proliferation of C2C12,which also shows a time-dose relation.Compared with the control,the fluorescence levels of MEHP(125 and 500 ?M)increased(P<0.05).It indicates that MEHP activated MSTN promoter,and the result was consistent with that from our animal experiments.Compared with the control,the mRNA expression levels of MSTN,Atrogin-1,Mu RF-1 and increased(P<0.05),while the mRNA expression levels of MyoD and Myogenin decreased(P<0.05).In addition,compared with the control,the mRNA expression level of C/EBP? increased.5.The expression of C/EBP? genes in C2C12 cells was interfered using siRNA technology and then treated with MEHP.The result shows that interfering C/EBP? inhibited the effects of MSTN not only on up-regulation of mRNA expression level of Atrogin-1 and Mu RF-1,but also on down-regulation of m RNA expression level of MyoD and Myogenin.These results reveal that C/EBP? plays an important role in up-regulating expression of MSTN in C2C12 through MEHP.Conclusion1.A MSTN-Knockout mice model is established by the Cre-loxp system technology.Knocking out MSTN gene results in increasing of the body and skeletal muscle weight,and the skeletal muscle fibers in newborn mice.This result might be closely related to the down-regulation of expression of Atrogin-1 and MuRF-1.2.MSTN plays a role in offspring mice skeletal muscle inhibitation by DEHP maternal exposure.After MSTN-Knockout,the skeletal muscle inhibitation decrease,with up-regulation of the mRNA levels of MyoD and Myogenin and down-regulation of the m RNA levels of Mu RF-1 and Atrogin-1.After MSTN-Knockout,the muscle inhibitation decrease,with up-regulation of the protein expression levels of MyoD and p-Akt.3.MEHP increases the expression of MSTN by activating the promotor of MSTN via transcription factor C/EBP?,resulted skeletal muscle cell wasting.