Effect Of Mono(2-ethylhexyl) Phthalate On The Pluripotency Of Mouse Embryonic Stem Cell And The Underlying Mechanism

ObjectivesPhthalic acid esters(PAEs)is a commonly used industrial plasticizer,which exists in our daily life widely.PAEs have some harmful effects on organism,for example,it has chronic toxic effects on heart,liver,lung,kidney,reproductive system,and the endocrine system.Di(2-ethylhexyl)phthalate(DEHP)is one of the the most widely used compound in PAEs,and the metabolite of DEHP is mono(2-ethylhexyl)phthalate(MEHP)is the metabolite of DEHP in organism,which has more toxic.Embryonic Stem Cell(ESC)is a kind of pluripotent cell which can proliferate infinitely,self-renew and differentiate into various cells.ESC has been used as an alternative experimental material in recent years because of its good sensitivity to the tested compounds.To investigate the effects of MEHP on mouse ESCs(mESC)and its possible mechanisms,the cytotoxicity of MEHP on the maintenance of pluripotency of mESCs and the possible mechanisms are studies in this paper,which will provide the basis for the study of influencing factors and related mechanisms of early embryonic development.Methods1.mESCs were cultured supported by mouse embryonic fibroblasts(MEF)in 5%CO2incubator at37?.mESCs should be cultured with fresh medium every other day and were passaged every 5-7days.Alkaline Phosphatase Detection Kit was used to detect the differentiation of mESCs,and the undifferentiated cells could be used in the next experiments.2.Test compounds:MEHP was dissolved in DMSO,and mixture was filtered and sterilized.Differernt c oncentrations of MEHP solution(0.1,1,10,100 and 1000?mol/L)were prepared with cell culture medium(contained 0.1%DMSO)and 0.1%MSO was prepared with as control reagent.3.Morphological observation of mESC:mESCs were cultured with different medium contained different concentrations of MEHP respectively.After treatment,the effects of MEHP on mESCs were observed under the microscope.4.Cytotoxicity:mESCs were treated with different concentration of MEHP respectively.After treatment,the cytotoxicity of MEHP on mESCs was detected through the dection of cell viability which was tested by cell counting kit-8(CCK8)assay,and the detection of cell apoptosis which was tested by flow cytometry.5.The alkaline acid activity test:the treated cells were stained with alkaline phosphatase staining to analyze the effect of MEHP on the maintenance of mESC.6.Gene detection:After treatment,total RNAs of mESCs were extracted and then were reverse transcripted into cDNA.Reverse transcription-polymerase polymerase chain reaction(Real-time PCR)was performed to detect the expression the SOX 2 and OCT 4 genes.7.Protein detection:After treatment,indirect immunofluorescence assay(IFA)and Western blot assay were performed to detect the expression of SOX 2 and OCT 4 proteins.Results1.Observate the morphology of mESC,we found that as the dose of MEHP increased,the number of mESC mass decreased,and the size of mESC mass is also reduced.In the highist dose group(1000 mol/L),the change is particularly evident.2.Compared with the control group(0.1%DMSO),the highist dose group(1000?mol/L)significantly decreased the cell viability(P<0.05),while the treatment with other doses also showed a decrease in cell viability,but the effect was not significant.3.The results of the dection of cellapoptosis showed that the high dose group(1000 ?mol/L)significantly increased the apoptosis rate of mESCs(P<0.05),while the other doses had no effect.However,the other indexes of cell apoptosis,such as viable cell ratio,early cell apoptosis rate,late cell apoptosis rate,and cell debris ratio of mESCs,had no significant difference among all the dose groups and the control group(P>0.05).4.The result of alkaline phosphatase staining showed that mESC were stained dark blue or blue purple in control group and low dose groups,while lighter coloring,even without coloring in the highist dose(1000 mol/L).5.The results of Real-time PCR showed that the expressions of SOX 2 and OCT 4 genes were significantly decreased in both 100 ?mol/L and 1000 ?mol/L MEHP groups(P<0.05),and the expression in 1000 ?mol/L group was also lower than that in 100 ?mol/L group.6.The expression of SOX 2 and OCT 4 proteins were detected by IFA and Western blot,the results all showed that,compared with the control group,the expressions of the two proteins were significantly decreased in the high dose group(1000 ?mol/L)(P<0.05),while the other doses had no effect.Conclusions1.MEHP has cytotoxicity on mESC and inhibits the growth of mESC:it can affect the morphology of mESCs,reduce the cell viability and increase the total cell apoptosis rate.2.MEHP can affect the maintenance of pluripotency of mESC,thereby reducing the synthesis of alkaline phosphatase.The possible mechanism is that MEHP can decrease the gene and protein expressions of SOX2 and OCT4,the core transcription factors of mESCs,which will affect the maintenance of pluripotency of mESCs and even affect the development of early embryos.