啶虫脒对睾丸间质细胞的毒性作用

Effects of acetamiprid toxicity on testicular interstitial cells

目的探究啶虫脒(Acetamiprid,ACE)对睾丸间质细胞TM3的毒性作用,为ACE致生殖毒性机制研究提供理论基础。方法应用DMEM-F12培养基培养TM3细胞。以不同浓度ACE处理TM3细胞24 h和48 h,采用CCK8法测定细胞活力,计算24 h IC_(50)。根据IC_(50)值选择后续TM3细胞暴露的适宜ACE浓度。ACE处理TM3细胞24 h,流式细胞术测定细胞凋亡率,qPCR测定细胞增殖指数PCNA、Ki 67 mRNA,及睾酮合成酶基因Star、Hsd3b1、Cyp11a1 mRNA的表达。结果细胞活力在4、6、8、10、12 mmol/L随ACE浓度升高而下降,且在8、10、12 mmol/L组,48 h细胞活力显著低于24 h(P<0.05)。计算24 h IC_(50)为7.20 mmol/L,故后续以0、2、4、6、8 mmol/L ACE处理TM3细胞24 h。与0 mmol/L组相比,PCNA mRNA在6、8 mmol/L组表达显著降低(P<0.01),Ki67 mRNA在4、6、8 mmol/L组表达显著降低(P<0.05)。流式细胞术结果发现,TM3细胞凋亡率随ACE浓度升高而增加。ACE处理还可以导致Star和Hsd3b1 mRNA在6、8 mmol/L表达显著升高(P<0.05),Cyp11a1 mRNA在所有剂量组均显著升高(P<0.01)。结论ACE暴露可通过抑制细胞增殖和促进细胞凋亡导致细胞活力降低,同时,ACE可通过上调Star、Hsd3b1、Cyp11a1基因表达影响睾酮合成过程,但相关机制还需要进一步研究。

Objective To investigate the effects of acetamiprid(ACE)on testicular interstitial cells TM3,in order to provide theoretical basis for the study of reproductive toxicity of ACE.Methods TM3 cells were cultured in DMEM-F12 medium and were treated with different concentrations of ACE for 24 h and 48 h.Cell viability was determined by CCK8 method,and IC_(50) at 24 h was calculated.The appropriate concentration for ACE exposure to TM3 cells was determined according to the IC_(50) value.After exposure to ACE for 24 h,the apoptosis rate of TM3 cells was detected by flow cytometry,and the mRNA levels of PCNA,Ki 67,Star,Hsd 3b1 and Cyp11a1 were detected by qPCR.Results Cell viability decreased with the increase of ACE concentration at 4,6,8,10 and 12 mmol/L.In 8,10 and 12 mmol/L groups,cell viability at 48 h was significantly lower than that at 24 h(P<0.05).The IC_(50)(24 h)was calculated to be 7.20 mmol/L,so TM3 cells were treated with 0,2,4,6 and 8 mmol/L ACE for 24 h.Compared with 0 mmol/L group,PCNA mRNA in 6 and 8 mmol/L groups and Ki67 mRNA in 4,6 and 8 mmol/L groups were significantly decreased(P<0.01,P<0.05).Results of flow cytometry showed that the apoptosis rate of TM3 cells after ACE exposure was increased in a dose-dependent manner.ACE treatment also significantly increased the mRNA levels of Star and Hsd3b1 in 6 and 8 mmol/L groups(P<0.05),and the mRNA level of Cyp11α1 mRNA in all dose groups(P<0.01).Conclusion ACE can reduce the cell viability by inhibiting cell proliferation and promoting cell apoptosis.Meanwhile,ACE may affect the testosterone synthesis by up-regulating the expression of Star,Hsd3b1 and Cyp11a1.However,the relevant mechanisms need to be further studied.