Effects Of PGC-1? Against Lead-induced Oxidative Stress And Energy Metabolism Dysfunction In Testis Sertoli Cells

Objective:The reproductive system is particularly sensitive to lead toxicity.In recent years,the reproductive toxicity of heavy metal lead has been paid attention to.However,the mechanism of toxicity of lead on the reproductive system is not clear.The TM4 cell line and the TM4 overexpression PGC-1? cell line(PGC-1?(+)TM4 cell line)and the low expression of PGC-1?(TM4)cell line(referred to as PGC-1?(-)TM4 cell line)to study the effect of PGC-1? on lead-induced testicular cell Stress and energy metabolism.Methods:After treated by lead acetate for 24 h,the expression of PGC-1? and SIRT3 mRNA in the TM4 cells?PGC-1?(+)TM4 cells and PGC-1?(+)TM4 cells was measured by RT-PCR.The cells were stained with DHE fluorescent probe,and the intracellular ROS content was detected by flow cytometry,fluorescence microscopy and fluorescence microscopy.The contents of ATP and LD in the cells were measured by ATP content assay kit and LD content determination kit,The contents of LDH,SDH,Na+-K+-ATPase and Ca2+-Mg2+-ATPase were measured by LDH activity assay kit,SDH activity assay kit,Na+-K+-ATPase activity assay kit and Ca2+-Mg2+-ATPase activity assay kit.Results:(1)The expression of PGC-1? and SIRT3 mRNA in TM4 cells and PGC-1?(+)TM4 cells increased first and then decreased with the increase of exposure concentration.The levels of PGC-1 a and SIRT3 mRNA in the cells decreased gradually,and there was significant difference with the control group(P<0.05).The expression of PGC-la and SIRT3 mRNA in TM4 cells was significantly lower than that in PGC-1?(+)TM4 cells and PGC-1?(-)TM4 cells after treated with the same concentration of lead acetate.(2)The results of flow cytometry showed that the levels of ROS in TM4 cells,PGC-1?(-)TM4 cells and PGC-1?(+)TM4 cells increased gradually with the increase of lead acetate concentration,and ROS in the PGC-1?(-)TM4 cells>TM4 cells>PGC-1?(+)TM4 cells(P<0.05),but When treated with the same concentration of lead acetate,the concentration of ROS in PGC-1?(+)TM4 cells was reduced by 33.7%?45.2%compared with that of TM4 cells,and the ROS level in TM4 cells was 65%?85%lower than that of PGC-1?(-)TM4 cells.The results of fluorescence microscopy showed that the fluorescence intensity of the three groups increased gradually with the increase of lead acetate concentration,which indicated that the intracellular ROS level was increased.And the ROS level in the PbAc exposure group was higher than that in the control group.The fluorescence intensity was lower than that of PGC-1?(-)TM4 cells,but higher than PGC-1?(+)TM4 cells.(3)Compared with the control group,the content of ATP in the PGC-1? TM4 cells,TM4 cells and PGC-1?(+)TM4 cells decreased,the difference was statistically significant(P<0.05);And with the increase of lead acetate concentration,ATP content in TM4 cells,PGC-1?(-)TM4 cells and PGC-1?(+)TM4 cells showed a downward trend.When treated with the same concentration of lead acetate,the content of ATP in the three kinds of cells was PGC-1?(+)TM4 cells>TM4 cells>PGC-1?(-)TM4 cells,the difference was statistically significant(P<0.05).(4)The results of LD content showed that the intracellular LD level of PGC-1?(-)TM4 cells,TM4 cells and PGC-1?(+)TM4 cells decreased gradually with the increase of lead acetate concentration,compared with the control group,the LD level in PGC-1?(-)TM4 cells decreased by about 40%?70%(P<0.05),the LD content was decreased by about 45%?75%(P<0.05)in TM4 cells,the intracellular LD level of PGC-1?(+)TM4 cells decreased by about 30%?45%(P<0.05).The LD levels of the three cells were significantly different from those of the same concentration of lead acetate,and the LD level of TM4 cells was 15%?34%higher than that of PGC-1?(-)TM4 cells,and PGC-1?(+)TM4 cells had an LD level of about 20%to 50%higher than that of TM4 cells.The LD content in PGC-1?(-)TM4,TM4 and PGC-1?(+)TM4 cells decreased from 71.22,100,127.60 nmol/mg pro to 16.41 when the concentration of lead acetate was increased from 0?M to 160?M,30.88,63.00 nmol/mg pro.(5)The activity of LDH,SDH,Na+-K+-ATPase and Ca2+-Mg2+-ATPase in the cells were decreased by exposure to lead acetate.In addition,when the concentration of lead acetate was increased to 160?M,the intracellular LDH activity of PGC-1?(-)TM4 cells,TM4 cells and PGC-1?(+)TM4 cells decreased from 89.9%,100%,117.7%to 37.7%,41.9%?62.7%respectively.The intracellular SDH activity of PGC-1?(-)TM4 cells,TM4 cells and PGC-1?(+)TM4 cells was reduced by 6 times,5 times and 5 times,respectively.The SDH activity of PGC-1?(+)TM4 cells increased by 54.7%?161.8%compared with that of TM4 cells,while the SDH activity of TM4 cells increased by 84.4%?150.3%compared with PGC-1?(-)TM4.The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were decreased in the three kinds of cells after exposure to lead acetate,and the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in TM4 cells was higher than that of PGC-1?(-)TM4 cells when treated with the same concentration of lead acetate,and lower than that of PGC-1?(+)TM4 cells(P<0.05).Conclusion:Lead acetate exposure can lead to intracellular oxidative damage and energy metabolism disorders.PGC-1? has a protective effect on lead-induced oxidative stress and cellular energy metabolism in mice induced by lead.