Mechanism Of MBP Disrupting Growth Of Sertoli Cells During Prepuberty

Over the past decades,environmental chemicals are a heterogeneous group of substances that began to attract attention due to possible harmful effects.Dibutyl phthalate(DBP),one of the most dominant phthalate esters,is used as a plasticizer in PVC products.Monobutyl phthalate(MBP)is the active metabolite of DBP.The reproductive organs are particularly sensitive to DBP exposure.A large number of studies have found that DBP has reproductive and developmental toxicity,testis is the main target organ,and Sertoli cells(SCs)are the target cells of DBP.In the previous study,we found that the SCs from the testis of 9-day rats exposed to different concentrations of MBP could cause abnormal cell growth.The differential expression profiles of mRNA during the abnormal proliferation of SCs induced by low concentration MBP were further screened by mRNA high throughput chip technology.We found that there is a potential negative regulatory mechanism between E3 ubiquitin ligase Pellino 2(Peli2)and Interleukin-1 receptor-associated kinase 1(IRAK1)based on the chip result analysis and database review.Therefore,we first observed the effects of MBP on the growth and function of SCs.Then we discussed the mechanism of Peli2 in cell proliferation and the related molecular mechanisms of the effect of MBP exposure on SCs growth.Part I The effect of MBP on the growth of SCsObjectiveThe effect of different doses of MBP exposure on the growth of SCs.Methods1.Primary culture of testicular Sertoli cell from 9 days old rat.2.SCs were divided into 4 groups to detect:Control,0.1 mM,1 MM,and 10 mM MBP groups.Fluorescent photomicrographs of cells dyed with FDA/PI was used to observe the effects of MBP on live or death of cells.3.RT-PCR was used to validate the mRNA chip data.4.TM4 cells were treated with different concentrations of MBP for 24 h,48 h,and 72 h.TM4 cells viability was tested by using CCK-8.5.EdU imaging kit was used to examine the influence of MBP on DNA synthesis in TM4 cells.6.The cell cycle distribution and apoptotic rate were measured using flow cytometry assay.7.The levels of adenosine nucleoside adenosine triphosphate(ATP)and cell release of lactate dehydrogenase(LDH)were detected in TM4 cells after exposure to different concentrations of MBP 24 h.8.The levels of tight junction proteins were detected in TM4 cells after exposure to different concentrations of MBP 24 h,48 h,or 72 h.Results1.There were no significant changes in Sertoli cells morphology after exposing up to 10 mM.However,cells number were decreased significantly in 10 mM group.2.FDA/PI double staining test showed that the proportion of SCs apoptosis decreased and then increased with the increase of exposure concentration,and the activity of cells increased and then decreased.3.Chip verification results show that the expression of Peli2,Trim2,Rnf135,and Cpsf2 is consistent with the chip results,which proves the accuracy of the chip.The expression of Peli2 was more significant.Further mechanism study will take mouse testicular Sertoli cell line TM4 as the research object.4.Cell viability was significantly increased after 1,2,3,4,or 5 days MBP exposure at a concentration of 0.1 mM and 1 mM,and was decreased at 10 mM.5.We further examined the influence of MBP on DNA synthesis in TM4 cells utilizing the EdU imaging Kits.Compared to the control,0.1 mM and 1 mM MBP increased the EdU-positive cells;10 mM MBP significantly inhibited the replication of DNA.6.Flow cytometry analysis confirmed that cell apoptosis was decreased after 24 h,48 h,and 72 h MBP exposure at a concentration of 0.1 mM and 1 mM,and was increased at 10 mM.Compared to the control,0.1 mM MBP increased the DNA synthesis in TM4 cells.7.The results of ATP detection showed that the low concentration of MBP increased the ATP level of cells,and the high concentration of MBP significantly reduced the level of ATP.The results of LDH detection found that the low concentration of MBP reduced the release of LDH significantly,and the high concentration of MBP significantly increased the LDH release.8.After exposed to low concentration of MBP,the expression of tight junction proteins in TM4 cells increased,indicating that MBP could promote the maturation of SCs,and high concentration of MBP could inhibit the expression of tight junction proteins between TM4 cells,indicating that the high concentration of MBP could destroy the SCs maturation process.Conclusion1.Low concentration of MBP promotes abnormal proliferation of SCs,and high concentration of MBP leads to apoptosis.2.Low concentration of MBP promoted SCs maturation,and high concentration of MBP destroyed SCs maturation process.Part II Mechanism of MBP interfering growth of SCsObjectiveTo investigate the mechanism of low concentration MBP promoting SCs proliferation and high concentration MBP promoting SCs apoptosis.Method1.The expression of Peli2 in TM4 cells exposed to MBP was verified by RT-PCR,Western Blot and immunofluorescence.2.RT-PCR,Western Blot,immunofluorescence and co-immunoprecipitation were used to detect the changes of IRAK1 gene and protein ubiquitination level.Western Blot was used to detect the activation of MAPK/JNK signaling pathway and to study its effect on cell proliferation.3.To investigate the effects of MBP-induced the activation of extrinsic apoptosis pathways and intrinsic apoptosis pathways by Western blot in TM4 cells after exposure to various concentration of MBP.Results1.The expression of Peli2 was consistent with the array data suggesting the reliability of the array.2.After TM4 exposure to low concentration(0.1 mM)MBP,the K63 ubiquitination level of IRAK1 increased,resulting in the activation of the downstream JNK signaling pathway and thus promoting cell proliferation.After TM4 cells exposed to high concentration(10 mM)MBP,the K63 ubiquitination level phosphorylation level of IRAK1 decreased,inhibiting the activation of downstream JNK signaling pathway and inhibiting cell proliferation.3.Apoptosis related signal pathway analysis confirmed that after TM4 exposure to low concentration MBP,extrinsic apoptosis pathway was activated,and high concentration MBP inhibited the activation of extrinsic apoptosis pathways.It is also found that after TM4 exposure to low concentration of MBP,the cell intrinsic apoptosis pathways is inhibited and the ratio of apoptosis index Bax/Bcl-2 decreased;high concentration MBP activates the intrinsic apoptosis pathways and increases the ratio of apoptosis index Bax/Bcl-2.Conclusion1.MBP at low concentration promotes the abnormal proliferation of SCs by inhibiting the expression of Peli2,breaking the ubiquitination balance of IRAK1,and activating downstream MAPK/JNK signaling pathway.2.Activation of extrinsic apoptosis pathways by low concentration of MBP may be related to abnormal proliferation of SCs.3.MBP at high concentration promotes the apoptosis of SCs by activating the intrinsic apoptosis pathways.