The Role And Mechanism Of ARTS In DEHP-induced Apoptosis Of Mouse Leydig Cells

Objective:As a member of phthalates（PAEs）,di-（2-ethylhexyl）phthalate（DEHP）has been widely used as plasticizer in China.DEHP can enter the human body through skin contact,oral ingestion,and inhalation,and causes damage to various systems.Our previous study shows that DEHP can induce apoptosis in mouse testicular tissue and Leydig cells,but the exact mechanism is still unclear.This study focuses on the role of ARTS in DEHP-induced apoptosis of mouse Leydig cells and its potential mechanism.Methods:1.To clarify the impact of DEHP on apoptosis in mouse testicular tissue,adult male Kunming mice were continuously administrated with different doses of DEHP（0,100,200,400 mg/kg/day）for 21 d.The expression of Cleaved Caspase-8,Cleaved Caspase-3,Bcl-2 and Bax,was then evaluated by Western blot and immunohistochemical（IHC）assay.2.TM3 cells were treated with different concentrations of DEHP（0,100,300,500μM）for 48 h,cell viability and apoptosis were determined by CCK-8 assay,Western blot and Annexin V-FITC/PI double staining,respectively.3.The effect of DEHP on the expression of ARTS in the testis tissue was detected by Western blot and IHC assay,respectively;while the impact of DEHP on the m RNA and protein levels of ARTS in TM3 cells was determined by Western blot and Real-time PCR,respectively.4.Western blot and Annexin V-FITC/PI double staining were utilized to detect the influence of ARTS overexpression on apoptosis of TM3 cells.TM3 cells was transfected with si-ARTS in the presence or absence of DEHP,apoptosis was evaluated by the above methods.5.The effect of DEHP on the expression of p53 in testicular tissues was detected by Western blot and IHC assay,respectively.Real-time PCR and Western blot were used to determine the expression of p53 in the DEHP-treated TM3 cells.6.p53 overexpression plasmid was constructed and transfected into TM3 cells,and the protein and m RNA levels of ARTS were then detected by Western blot and Real-time PCR,respectively.TM3 cells were transfected with si-p53 in the presence or absence of DEHP,and the protein and m RNA levels of ARTS were then detected by Western blot and Real-time PCR,respectively.7.To elucidate the regulatory relationship between p53 and ARTS,a computational approach was employed to predict putative binding sites of p53 in the promoter region of ARTS gene.The p GL4.2-ARTS prom plasmid was constructed,and dual-luciferase reporter and Ch IP assays were then performed to identify whether p53can promote gene transcription of ARTS and the specific binding sites of p53 in the promoter region of ARTS gene.8.TM3 cells were transfected with p53 overexpression plasmid or si-p53 and then treated with DEHP,apoptois was evaluated by Annexin V-FITC/PI double staining and Western blot,respectively.9.TM3 cells were treated with 0,100,200,400μM H₂O₂for 12 h,Western blot was utilized to detect the expression of apoptosis-related proteins.Cell viability and apoptosis level were determined by CCK-8 assay,Western blot,and Annexin V-FITC/PI double staining,respectively,after TM3 cells were treated with DEHP in the presence or absence of N-acetyl-L-cysteine（NAC）.Results:1.There was a significant increase in the protein levels of Cleaved Caspase-8,Bax and Cleaved Caspase-3 in the testicular tissues,while a marked decrease in the expression of Bcl-2.IHC assay revealed that the expression of Caspase-3 was specifically increased in the Leydig cells of the DEHP-treated testes,indicating that DEHP could induce apoptosis of Leydig cells in the mouse testicular tissue.CCK-8assay showed that 300 and 500μM DEHP significantly inhibited viability of TM3 cells（P<0.05）,Western blot analysis demonstrated that DEHP could induce apoptosis of TM3 cells.The Annexin V-FITC/PI double-staining assay showed that DEHP significantly increased the apoptosis rate of TM3 cells,suggesting that DEHP could induce apoptosis of TM3 cells.2.Western blot analysis showed that there was a significant increase in the ARTS protein level in the DEHP-treated mouse testicular tissues,and IHC assay revealed that DEHP could specifically increase the protein level of ARTS in the Leydig cells of the testes,indicating that DEHP could specifically increase the expression of ARTS in the Leydig cells of the mouse testes.Meanwhile,DEHP could upregulate the m RNA and protein levels of ARTS in TM3 cells.3.The expression of Cleaved Caspase-8,Bax and Cleaved Caspase-3 was significantly elevated,while Bcl-2 expression was significantly decreased in the ARTS-overexpressed TM3 cells.Furthermore,the Annexin V-FITC/PI double staining assay showed that there was a significant increase in the apoptotic rate of the ARTS-overexpressed cells.ARTS knockdown resulted in a decreased protein levels of Cleaved Caspase-8,Bax and Cleaved Caspase-3,and a increased protein level of Bcl-2.Additionally,ARTS knockdown significantly reduced DEHP-induced apoptosis in TM3 cells,indicating that ARTS was involved in DEHP-induced apoptosis of TM3cells.4.Western blot analysis revealed there was a marked increase in p53 protein level in the DEHP-treated mouse testicular tissue.IHC assay showed that p53 expression was mainly increased in the Leydig cells of mouse testicular tissue,suggesting that DEHP could specifically elevate p53 expression in the Leydig cells of mouse testicular tissue.DEHP was also shown to increase the m RNA and protein levels of p53 in the TM3 cells.5.p53 overexpression resulted in a substantial increase in the m RNA and protein levels of ARTS,while p53 knockdown led to a notable decrease in the expression of ARTS.Furthermore,p53 knockdown significantly inhibited the up-regulation of ARTS caused by DEHP,indicating that p53 was capable of promoting the expression of ARTS in TM3 cells.6.The results of Dual-luciferase Reporter Assay and Ch IP indicated that p53 could promote ARTS gene transcription by binding to the RE4 site in the promoter region of ARTS gene.7.p53 overexpression significantly increased the apoptotic rate of TM3 cells,while p53 knockdown could not only reduce the apoptosis levels,but also alleviate the induction of apoptosis by DEHP in TM3 cells.8.TM3 cells were treated with different concentrations of H₂O₂ for 12 h,there was a significant increase in apoptotic rate,accompanied with the up-regulation of ARTS and p53.Furthermore,inhibition of oxidative stress with NAC significantly reduced DEHP-induced apoptosis and up-regulation of ARTS and p53 proteins.These findings suggested that DEHP-induced oxidative stress activated the p53-ARTS cascade to promote apoptosis of mouse Leydig cells.Conclusions:1.DEHP can induce apoptosis in the Leydig cells of mouse testicular tissue and TM3 cells.2.DEHP promotes the expression of ARTS in the Leydig cells of mouse testicular tissue and TM3 cells.ARTS can induce apoptosis in TM3 cells.3.DEHP can promote the expression of p53 in the Leydig cells of mouse testicular tissue and TM3 cells.p53 could promote ARTS gene transcription by binding to the promoter region of ARTS gene.4.DEHP-induced oxidative stress activates the p53-ARTS cascade to promote apoptosis of mouse Leydig cells.