| [Objective]This study was aim to investigate sulforaphane active Nrf2 pathway and regulate the activities of II detoxifying enzymes and autioxidant enzyme and the protective effect of vitro sulforaphane in cadmium-induced mouse's TM3 cells.And provide theoretical foundation for prevent reproduction toxicity of CdCl2.[Methods]Used CdCl2 and sulforaphane cultivate TM3 cells together,and then establish experiment model.Detect TM3 cells relative survival rate?testosterone production?LDH active?antioxidant level?ROS producation?cell apoptosis condition and measured mRNA levels of Nrf2,HO-1,NQO1,?-GCS by RT-PCR.Measured protein expression levels of Nrf2,HO-1,NQO1,?-GCS by western blot.[Results]1 Cd can damage TM3 cells by MTT method detect.TM3 cells relative survival rate decrease when Cd concentration was 10 ?mol/L(P<0.05).TM3 cells relative survival rate significantly decrease when Cd concentration was equal or greater than 20 ?mol/L(P<0.01).And measured IC50 of TM3 cells is 51.4 ?mol/L.To a certain range,sulforaphane can protect TM3 cells,however when sulforaphane concentration was equal or greater than 20 ?mol/L,there was some inhibiting effect to TM3 cells(P<0.05).CdCl2 and sulforaphane cultivate TM3 cells together,sulforaphane can remission Cd caused cytotoxicity.And when sulforaphane concentration was 2.5?mol/L,best effect.2 Measured LDH and antioxidant index discovered that compared with the control group,Cdgroup TM3 cells content of GSH.active of T-SOD and GSH-PX significantly decrease(P<0.01),active of LDH and content of MDA increase(P<0.05).SFN group TM3 cells active of LDH and content of MDA significantly decrease(P<0.01),content of GSH.active of T-SOD and GSH-PX increase(P<0.05).Compared with the Cd group,Cd+SFN groups TM3 cells content of GSH.active of T-SOD and GSH-PX tent to increase;active of LDH and content of MDA increase tend to decrease.Indicate SFN can remission Cd caused TM3 cells toxic effect.3 By ELISA measure detect testosterone production found that compared with the control group,Cd group TM3 cells testosterone production significantly decrease(P<0.01),SFN group TM3 cells testosterone production tent to increase.Compared with the Cd group,Cd+SFN2.5 groups TM3 cells testosterone production significantly increase(P<0.05),Cd+SFN5 groups TM3 cells testosterone production increase.Indicate Cd can inhibiting testosterone secret,SFN can habiting testosterone secret.4 Used DCFH-DA tested TM3 cells ROS production found that control group ROS production little,compared with the control group,other groups TM3 cells ROS production significantly increase(P<0.01),especially Cd group ROS production significantly increase.However compared with the Cd group,SFN group and Cd+SFN groups TM3 cells ROS production significantly decrease.It turned out that anything can stimulate TM3 cells product ROS and especially Cd.5 By AO/EB and FITC-Annexen-V/PI detect TM3 cells apoptosis,compared with the control group,Cd group TM3 cells apoptosis rate significantly increase(P<0.01),SFN group TM3 cells apoptosis rate significantly decrease.And compared with the Cd group,SFN group TM3 cells apoptosis rate significantly decrease(P<0.01),Cd+SFN groups TM3 cells apoptosis rate tent to decrease.Indicate SFN can remission Cd caused TM3 cells damage.6 Used RT-PR and western blot detect TM3 cells mRNA and protein expression found that compared with the control group,Cd group Nrf2?HO-1??-GCS?NQO1 mRNA and protein expression up-regulated,GSH-PX mRNA and protein expression downstream.SFN group significantly increase(P<0.01,P<0.05).SFN can significantly up-regulated mRNA and protein expression of Nrf2 andits downstream target genes HO-1,NQOO1,?-GCS(P<0.01).Compared with the Cd group,Cd+SFN groups TM3 cells Nrf2?GSH-PX?HO-1??-GCS?NQO1 mRNA and protein expression significantly up-regulated[Conclusion]Sulforaphane has a protective effect on mouse's oxidative damaged TM3 cells which caused by cadmium,and its activation maybe achieved through of Nrf2/are pathway. |
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