Study On The Role Of MAPK Signaling Pathways In Carbendazim-induced Damage In Mouse Sertoli Cells

【Objective】 To explore the possible mechanism and the role of MAPK signaling pathways in carbendazim-induced damage in mouse Sertoli cells,in order to provide theory basis about the prevention and treatment of carbendazim.【Methods】 We selected TM4 cells to establish damage model.TM4 cells were treated with different concentrations of carbendazim(1μM,2μM,5μM,10μM,20μM)and cells treated with 0.1%DMSO served as solvent control group.After treatment for 24 h,48h and 72 h,MTT assay was used to test the proliferation of TM4 cells.We used different concentrations of carbendazim(0-5μM)to treat TM4 cells for 48 h,the cell cycle distribution,the fraction of apoptotic cells and the reactive oxygen species level were analyzed by flow cytometry,the expression of Bax m RNA and Bcl-2 m RNA were detected by q RT-PCR.TM4 cells were pretreated with 2μM U0126(the inhibitor of ERK),5μM SP600125(the inhibitor of JNK)and 10μM SB203580(the inhibitor of P38 MAPK)for 1h respectively.TM4 cells were divided into four groups: solvent control group,inhibitor group,5μM carbendazim group and inhibitor+5μM carbendazim group,MTT assay was used to test the proliferation of TM4 cells,flow cytometry utilized to test the cell cycle distribution and the fraction of apoptotic cells,Western blot analysis was used to detect the expression level of ERK,JNK,P38 MAPK and their phosphorylated proteins.1.General toxicity of carbendazim in mouse Sertoli cells Carbendazim could inhibit the proliferation of TM4 cells.The survival rate of(5-20μM)carbendazim groups for 24 h were significantly lower than that of solvent control group(P<0.01).The survival rate of(2-20μM)carbendazim groups for 48 h and 72 h were significantly lower than that of solvent control group(P<0.05,P<0.01).【Results】Carbendazim could induce G2/M arrest in TM4 cells.After treated with carbendazim for 48 h,the proportion of G2/M phase cells in 2μM and 5μM carbendazim groups increased gradually compared with the solvent control group(P<0.01),and the proportion of G0/G1 phase cells decreased significantly(P<0.05).Carbendazim could induce apoptosis in TM4 cells.After treated with carbendazim for 48 h,the apoptotic rate of 2μM and 5μM carbendazim groups were significantly increased compared with the solvent control group(P<0.05,P<0.01).Carbendazim could increase the level of ROS in TM4 cells.Compared with the solvent control group,the level of ROS in(1-5μM)carbendazim groups significantly increased(P<0.01).Carbendazim could increase the expression of Bax m RNA and decrease the expression of Bcl-2 m RNA in TM4 cells.The expression of Bax m RNA was significantly increased after 48 h treated with carbendazim(P<0.05,P<0.01),and the expression of Bcl-2 m RNA was significantly lower than the solvent control group(P<0.01).2.The role of MAPK signaling Pathways in carbendazim-induced damage in TM4 cells.Carbendazim could increase the expression of p-ERK,p-JNK,p-P38 protein in TM4 cells.Compared with the solvent control group,the expression of p-ERK,p-JNK,p-P38 protein were significantly increased in a dose-dependent manner(P<0.01).U0126 could inhibit the expression of p-ERK protein in TM4 cells.SP600125 could inhibit the expression of p-JNK protein in TM4 cells.SB203580 could inhibit the expression of p-P38 MAPK protein.The expression of p-ERK protein in 2μM U0126+5μM carbendazim group decreased gradually compared with the solvent control group(P<0.01),the expression of p-JRK protein in 5μM SP600125+5μM carbendazim group decreased gradually compared with the solvent control group(P<0.05),The expression of p-P38 protein in 10μM SB203580+5μM carbendazim group decreased gradually compared with the solvent control group(P<0.01).Both SP600125 and SB203580 inhibited the inhibitory effect of carbendazim on the proliferation in TM4 cells.The survival rate of(5μM SP600125,10μM SB203580)+5μM carbendazim groups were significantly higher than that of 5μM carbendazim group(P<0.05,P<0.01).Both SP600125 and SB203580 inhibited G2/M arrest induced by carbendazim.Compared with 5μM carbendazim group,the percentage of G2/M phase cells in(5μM SP600125,10μM SB203580)+5μM carbendazim groups were decreased(P<0.01).Both SP600125 and SB203580 inhibited the apoptosis of TM4 cells induced by carbendazim.Compared with 5μM carbendazim group,(5μM SP600125,10μM SB203580)+5μM carbendazim groups decreased significantly(P<0.01).【Conclusions】 1.Carbendazim could inhibit the proliferation of TM4 cells,induce apoptosis and cell cycle arrest.The injury was accompanied by increases in the production of reactive oxygen species and in the level of pro-apoptotic protein Bax m RNA.2.Carbendazim could activate ERK,JNK and P38 MAPK signaling pathways in TM4 cells.3.JNK and P38 MAPK signaling pathways could participate in carbendazim-induced the inhibition of proliferation,G2/M arrest and apoptosis in TM4 cells.