| PurposeDi-2-ethylhexyl phthalate(DEHP),an important part of the plasticizer phthalate(PAE),is a colorless,odorless,low volatility and water-insoluble oily liquid.With the development of modern industry,DEHP is widely applied among industries like plastic bags,cosmetics and medical supplies.Plastic products are easy to release DEHP and cause environmental pollution to soil,air and water.DEHP enters the body through the respiratory tract,digestive tract and contact and affects the body’s functions.Studies have shown that DEHP has great damage to the liver,brain,prostate and testes.The testis is the main target organ for the reproductive toxicity of DEHP.Androgens are mainly derived from the stromal cells in the testes.The Leydig cells will decrease the secretion of androgen testosterone under the damage of DEHP,which will trigger a series of clinical symptoms.Therapies to low levels of androgen involve exogenous hormones supplement,testicular transplantation and Leydig cells transplantation.However,these ways are not able to substitute physical pattern of testosterone secretion and exist problems of lacking seed cells,rejection and ethical issues.Currently,a wide range of studies are looking for a normal testosterone secretion mode.Through researching bone mesenchymal stem cell(BMSC),adipose tissue-derived mesenchymal cell(AMSC),umbilical cord blood-derived mesenchymal stem cell(UMSC)and embryonic stem cells(ESCs),more seed cell source of Leydig cells can be found.This study aimed to evaluate the impact of DEHP exposure on C57 mice reproductive toxicity.More specifically,the goal of this study was to research the mechanism on inducing differentiation of BMSCs into Leydig-like cells in vitro and the impact of MHEP to Leydig TM3 apoptosis.We adopted cocultivation of BMSC to study the mechanism of Leydig-like cells repairing TM3.We observed testicular function change of C57 mice infected by DEHP afer transplanting Leydig-like cells derived from BMSC.Our study explored the toxicity mechanism and repair mechanism of cells and tissues under the action of DEHP from the study of signal pathways,m RNA and protein levels.The therapeutic effect of transplanting Leydig-like cells derived from BMSC on the functional impairment of leydig cells in DEHP-infected mice was also indicated.As a result,our study provided new options and scientific basis for cell transplantation to treat low androgen.Materials and Methods This study is mainly divided into the following 5 parts:1.12 C57 rats(4-week old)were randomly divided into 2 groups: 6 rats in control group(corn oil)and 6 rats in DEHP group(900mg/kg.d DEHP mixed with corn oil).Both control group and DEHP group were given gavage at 3 pm(6 days per week and add up to 4 weeks).The body weight were recorded each week,and the gavage does was adjusted based on the weight.After gavage,3 rats in each group were randomly selected and weighed,and venous blood from the eyeball was taken to determine the serum testosterone level.Two sides of testicular epididymis were cut and weighed respectively,and the epididymis coefficient was calculated.One testis was stored in paraformaldehyde for performing HE staining and immunohistochemical staining to observe the changes in the testicular tissue structure of rats.The other side of testis was stored at-80℃ for storage.After homogenization,the levels of P450 scc,3β-HSD and CYP17 in testis were determined by ELISA.2.Combined with Percoll liquid separation method and whole bone marrow adherence method,mouse BMSCs were extracted and purified,and then cultured,passaged,frozen and recovered.Then the extracted cells were cultured,subcultured,cryopreserved and recovered,and the morphology and cell surface markers CD44 and CD45 were identified by immunofluorescence.In vitro,BMSC was induced with cytokines(HMG 0.75U/ml+HCG 40U/ml +PDGF 10ng/ml+IL-1α 0.0005ng/ml)for 28 days.The Leydig cell-specific marker Hsd3β1 was identified by cell immunofluorescence,and the culture process was measured at the same time The level of testosterone in the culture supernatant at 7 days,14 days,21 days,and 28 days,and the expression of Hsd3β6,Hsd3β1,St AR,Cyp11A1,Hsd17β3 and Cyp17A1 in the testosterone synthesis pathway at the m RNA level.The Cyp11A1 and Hsd17β3 proteins were determined by western blot,and the differentiation of BMSCs into Leydig-like cells was identified from the function,immune markers,m RNA and protein levels.3.In vitro,the mouse testicular interstitial TM3 cells interfered by MEHP were adopted to detect the changes in testosterone secretion function,the protein molecule of Caspase-8,Caspase-3,Caspase-9,Bax and Bcl-2 apoptotic proteins in the apoptosis signaling pathway,and the content of MDA and GSH as well as the activity of SOD and GSH-PX in the TM3 cell.The Leydig-like cells derived from BMSCs and TM3 damaged cells were co-cultivated to detect the secretion of testosterone,the changes of Caspase-8,Caspase-3,Caspase-9 and Bax apoptotic proteins after co-cultivation,and the content of MDA,GSH and SOD,GSH-PX activity.4.42 DEHP infected rats were randomly divided into three groups with 14 rats in each group: control group(normal saline),Leydig-like cell transplantation group(Leydig-like cell derived from differentiated transplantation BMSC),BMSC transplantation group(P3 generation of BMSC transplantation).In 7 days,14 days and 21 days after transplantation,eyeball venous blood was taken to detect serum testosterone.Bilateral testis and epididymis were cut to calculate testicular coefficient and epididymis coefficient.One side of the testis was saved in paraformaldehyde for performing HE staining and immunohistochemical staining to observe the rat testicular tissue structure change.The other side of testis reserved-80 ℃ cold storage.After homogenization,the levels of P450 SCC,3β-HSD and CYP17 in testicular tissues were determined by ELISA.The changes of apoptotic proteins of Caspase-8,Caspase-3,Caspase-9 and Bax in each group were detected.The contents of MDA and GSH and the activities of SOD and GSH-Px were determined.ResuLts:1.The testicular coefficient and epididymal coefficient of DEHP group were0.41±0.002 vs 0.40±0.004 and 0.06±0.002 vs 0.06±0.002.These results were not statistically significate(P>0.05).P450 scc,3β-HSD,CYP17 in testis and serum testosterone in DEHP group were significantly different from those in control group(P<0.01),the contents were 62.7±4.63 vs 213.4±9.844(ng/m L),339.5±17.87 vs418.6±11.09(pg/m L),545.9±15.25 vs 743.6±20.03(pg/m L),0.508±0.087 vs1.819±0.049(ng/m L),respectively.Through HE,immunohistochemical staining and Tunel staining,HE staining of testicular tissues found that there were vacuoles in the testicular tissues of the DEHP-exposed group,the seminiferous tubules swelled,and the number of spermatogonia layers was reduced and disordered.The number of Leydig cells increased and aggregated,and there were apoptotic cells.The apoptotic proteins Caspase-8,Caspase-3,Caspase-9 and Bax were increased,while Bcl-2 was decreased.In testis of DEHP group and control group,MDA,GSH-Px,SOD and GSH were 8.053±0.339 vs 1.158±0.075(nmol/mg),6.116±0.178 vs 19.301±0.46(U/mgprot),59.132±1.538 vs 125.168±3.675(U/mgprot)and 31.954±1.988 vs80.659±1.894(μg/mgprot).There was statistically significant difference between two groups(P<0.01).2.Combining the Percoll liquid separation method and the whole bone marrow culture adherence method to successfully extract and purify BMSC,and establish a stable BMSC culture system.In the P3 generation of BMSC cells,the positive rate of CD44 was 92.27%,and the expression rate of CD45 was 1.14%.BMSCs were induced by cytokines to express Leydig cell marker Hsd3β1.The culture supernatant testosterone ELISA test was performed on 7 days,14 days,21 days,24 days,and 28 days.The testosterone in the supernatant of the experimental group was significantly increased.It reached its peak in 21 days after induction and gradually stabilized after24 days,which was statistically significant compared with the control group supernatant testosterone(P<0.05).Cellular immunofluorescence was performed on Day 7,Day 14,Day 21,Day 28.It was found that the experimental group all expressed Leydig cell marker Hsd3β1,and the expression rate was 91.2% on Day 21.Hsd3β6,Hsd3β1,St AR,Cyp11A1,Hsd17β3,and Cyp17A1 m RNA levels indicated that their expression levels increased with the induction time.Western blot analysis of Hsd3β1,St AR,Cyp11A1 and Hsd17β3 protein levels increased with the induction time,which was statistically significant compared with the control group.3.MEHP intervention in vitro decreased testosterone secretion in testicular interstitial TM3 cells of mice.The levels of testosterone secretion in MEHP group and control group were 0.93±0.05,1.45±0.07 at 48 h,0.31±0.02,1.44±0.07 mat 72h(P<0.01).The apoptotic proteins Caspase-8,Caspase-3,Caspase-9 and Bax were increased and Bcl-2 was decreased in MEHP group.In MEHP group,MDA content increased,GSH content decreased,while GSH-PX and SOD activities were inhibited.MDA content in MEHP group and control group at 48 h was 0.943±0.026,0.541±0.013,and it was 1.314±0.08,0.523±0.023 at 72h(P<0.01).The content of GSH in MEHP group and control group at 48 h was 49.22±0.63,58.95±0.005,and the content at 72 h was 40.32±3.14,57.06±0.04(P<0.01).The content of GSH-PX in MEHP group and control group at 48 h was 0.135±0.003,0.166±0.006,and the content at 72 h was 0.109±0.005,0.166±0.003(P<0.01).The content of SOD in MEHP group and control group at 48 h was 2.41±0.003,2.75±0.005,and the content at 72 h was 2.28±0.004,2.78±0.004,which was statistically significant(P<0.01).After co-culture,the content of testosterone in co-culture group increased,and the content of testosterone in co-culture group and MEHP group at 48 h was 0.7±0.01,0.31±0,and 72 h was 1.04±0.07,0.31±0.01(P<0.01).The detection of apoptotic protein in the co-culture group indicated that the protein expressions of Caspase-8,Caspase-3,Caspase-9 and Bax were decreased,while the protein expression of Bcl-2was increased.After co-culture,MDA content decreased,GSH content increased,GSH-Px and SOD activity increased: the content of MDA in TM3 cells of co-culture group and MEHP group was 0.811±0.018 vs 1.307±0.068 at 48 h,and 0.521±0.025 vs 1.314±0.08 at 72h(P<0.01).The content of GSH in the co-culture group and MEHP group at 48 h was 48.366±0.566 vs 38.902±0.224,and the content at 72 h was60.33±0.621 vs 40.318±3.143,which was statistically significant(P<0.01).The content of GSH-PX in the co-culture group and MEHP group was 0.166±0.006 vs0.107±0.006 at 48 h and 0.208±0.011 vs 0.109±0.005 at 72 h,which was statistically significant(P<0.01).The content of SOD in co-culture group and MEHP group was2.748±0.005 vs 2.112±0.084 at 48 h,and 3.027±0.06 vs 2.151±0.068 at 72h(P<0.01).4.The mice infected by DEHP were divided into Leydig-like cell transplantation group,BMSC cell transplantation group and control group.The serum testosterone at 7d after transplantation was 1.735±0.08 vs 0.532±0.039 vs 0.379±0.03.The serum testosterone of 14 d was 2.284 ±0.056 vs 0.617±0.016 vs 0.341±0.057,and at 21 d was 2.502±0.248 vs 0.677±0.012 vs 0.424±0.034,there was statistically significant difference between groups(P<0.01).The P450 scc in testicular tissue by ELISA was234.2±14.46 vs 156.4±2.778 vs 158.8±4.486 at 7d,187±3.255 vs 158.8±6.462 vs151.5±7.713 at 14 d,and 193.9±9.59 vs 156.3±4.576 vs 148.3±7.246 at 21 d.There was statistically significant difference between these groups(P<0.01).3β-HSD was402±26.79 vs 321.3±4.587 vs 315±13.45 at 7d,438.9±11.47 vs 316.9±8.539 vs322.4±12.33 at 14 d,444.1±9.611 vs 317.7±6.365 vs 306.3±5.266 at 21 d,and there was difference between groups(P<0.01).CYP17 level was 753.2± 19.32 vs 536.5±12.61 vs 518.4±5.705 at 7d,866± 43.46 vs 543.2 ±1.908 vs 536.1±15.86 at 14 d,780.1± 13.96 vs 531.2 ±19.3 vs 524.1±10.4 at 21 d.There was significant difference between these groups(P<0.01).The apoptotic protein expressions of Caspase-8,Caspase-3,Caspase-9 and Bax in Leydig-like and BMSC transplantation groups were lower than those in the control group,while Bcl-2 protein was increased.The MDA content of Leydig-like cell transplantation group,BMSC cell transplantation group and control group was 3.544± 0.148 vs 6.26±0.317 vs 7.848 ±0.487 at 7d,2.085± 0.062 vs 4.933±0.622 vs 8.383 ±0.491 at 14 d,and 1.101± 0.099 vs2.598±0.448 vs 8.326 ±0.546 at 21 d.There was significant difference between these groups(P<0.01).The content of GSH was 64.852±3.734 vs 42.19±2.484 vs29.186±1.102 at 7d,74.833±3.758 vs 52.07±2.673 vs 29.546±1.712 at 14 d,and84.837±3.801 vs 63.725±2.294 vs 29.518±1.163 at 21 d.There was significant difference between groups(P<0.01).SOD was 98.367±0.716 vs 72.166±1.083 vs53.654±3.464 at 7d,116.872±5.088 vs 95.885±3.487 vs 53.174±4.39 at 14 d,131.279±2.978 vs 104.109±10.393 vs 53.321±3.832 at 21 d.There was significant difference between these groups(P<0.01).GSH-PX activity was 15.707±0.575 vs11.015± 0.156 vs 5.608 ±0.442 at 7d,18.795±0.626 vs15.344±0.222 vs 5.154±0.111 at 14d,and 26.248±0.525 vs 19.366±0.388 vs 5.109±0.11 at 21 d,there was significant difference between groups(P<0.01).Histologically,the morphology and arrangement of spermatogenic cells were restored.Hsd3β1 was stably expressed in the transplanted cells which transplanted into the testicular stroma and around the spermatogenic tubules.TUNEL staining indicated that the apoptotic cells in the testicular stroma decreased.Conclusion:Through our analysis,this study can draw the following conclusions:1.DEHP can induce testicular injury in adolescent C57 mice,including decreased testicular coefficient and serum testosterone,decreased concentrations of key enzymes of testosterone synthesis such as P450 Scc,CYP17 and 3β-HSD,increased apoptotic proteins Caspase-8,Caspase-3,Caspase-9 and Bax,and decreased Bcl-2.The content of MDA increased and the content of GSH decreased.The activities of GSH-PX and SOD were inhibited,and the testicular interstitial cells had apoptosis.It was speculated that DEHP had toxic effects on the reproductive system of mice by inhibiting the activities of key enzymes for the synthesis of testosterone,reducing the secretion of testosterone in Leydig cells,and promoting the apoptosis of Leydig cells.2.BMSC can be separated and cultured by a combination of density centrifugation and whole bone marrow culture adherence method.A stable BMSCs in vitro culture system can be established by culture,passage,cryopreservation and resuscitation.BMSCs could be induced to differentiate into Leydig-like cells in vitro induced by cytokines(HMG0.75U/ml+HCG40U/ml+PDGF 10ng/ml+IL-1α0.0005 ng /ml)and expressed Hsd3β1.In the induction,the expression of key enzymes of testosterone synthesis was increased at m RNA and protein levels,and the cells had the function of secreting testosterone.3.The intervention in vitro of MEHP caused the decrease of testosterone secretion,the increase of apoptotic proteins Caspase-8,Caspase-3,Caspase-9 and Bax,and the decrease of Bcl-2 in testicular TM3 cells of mice.The content of MDA was increased and GSH was decreased.The activities of GSH-PX and SOD were inhibited,which suggested that cell apoptosis affected the function of TM3 cells.It was speculated that oxidative stress was involved in the process of cell apoptosis.Leydig-like cells derived from BMSCs were co-cultured with damaged TM3 cells,and the secretion of testosterone of TM3 cells was restored,and the proteins of Caspase-8,Caspase-3,Caspase-9 and Bax were decreased,while the protein of Bcl-2 was increased.The content of MDA content was decreased and GSH content was increased.The activities of GSH-PX and SOD were increased,suggesting that co-culture can reduce cell apoptosis and oxidative stress to restore part of the function of damaged TM3 cells.4.Leydig-like cells derived from BMSC and cells survived after BMSC transplantation,and no rejection was seen.Serum testosterone secretion was significantly increased after transplantation,and the concentrations of key enzymes in testosterone synthesis,such as P450 Scc,CYP17 and 3β-HSD,were increased,and the morphology of spermatogenic cells recovered.Apoptosis proteins Caspase-8,Caspase-3,Caspase-9 and Bax decreased,while Bcl-2 protein increased.The content of MDA was decreased and the content of GSH was increased.The activity of GSH-PX and SOD activity were increased.TUNEL staining indicated that the apoptosis of mesenchymal cells decreased.It was confirmed that both Leydig-like cells from BMSCs and BMSCs transplantation could reduce the reproductive toxicity of DEHP and restore the function of testis in mice. |
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