| Objective:Lead can cause damage to multiple tissues and organs of animals,but its toxic effect on chicken embryo fibroblast(CEF)is still unclear.This experiment studies the toxic effect of lead nitrate on CEF cells,and on this basis,preliminary explores the protective mechanism of N-acetylcysteine(NAC)on lead-induced CEF cytotoxicity,and provides a certain theoretical basis for further exploration of drugs that effectively prevent and control lead poisoning in animals.Methods:This experiment uses primary CEF cells cultured in vitro as a model,by adding different concentrations of lead nitrate(the final concentrations are 0,2,10,50,125,250,500 μM)and different concentrations of NAC(the final concentrations are 0,0.1,0.25,0.5,1,2,5,10 mM)after incubating at 37℃ and 5%CO2 for 24,48 and 72 h,the CCK-8 method was used to detect the effects of lead nitrate and NAC on CEF cells viability.On this basis,48 h was selected as the best time for lead nitrate treatment of CEF cells,2,10,and 50 μM were the low,medium and high concentration levels of lead nitrate treatment of CEF cells,2 mM was the optimal protective concentration for NAC treatment of CEF cells,and then performed follow-up experiments.Observed the morphological changes of CEF cells by an inverted microscope;detected the level of reactive oxygen species(ROS)in CEF cells by fluorescence staining and flow cytometry;detected superoxide dismutase(SOD)and total glutathione peroxidation with a microplate reader Changes in the activity of GSH-Px,catalase(CAT),malondialdehyde(MDA)content,total antioxidant capacity(T-AOC);Hoechst 33258 method and Annexin V-FITC method was used to detect CEF apoptosis rate,qRT-PCR method was used to detect the mRNA expression of Caspase-3,Caspase-9,Bcl-2 and BI-1.Results:1.Compared with the 0 μM lead nitrate group,lead nitrate can inhibit the activity of CEF cells.The survival rate of CEF cells in each lead-exposed group was extremely significantly reduced(P<0.01),and the toxicity showed a dose-effect relationship.CEF cells in the lead-exposing group showed a decrease in cell density,and a gradual increase in dead cells and irregular deformed cells.2.The oxidative damage test results showed that compared with the 0 μM lead nitrate group,the ROS levels of the lead-exposing groups were extremely significantly increased(P<0.01),and the antioxidant enzymes SOD,GSH-Px,and CAT activities were extremely significantly decreased(P<0.01),the content of MDA increased significantly(P<0.01),T-AOC decreased significantly(P<0.05),and the oxidative damage showed a dose-effect relationship.Compared with the 50 μM lead nitrate group,the ROS level of the 50 μM lead nitrate+2 mM NAC group was significantly decreased(P<0.01),the antioxidant enzyme SOD,GSH-Px,and CAT activities were significantly increased(P<0.05),and the MDA content significantly decreased(P<0.05),T-AOC increased,but there was no significant difference(P>0.05).3.The cell apoptosis test results showed that compared with the 0 μM lead nitrate group,the apoptosis rate of CEF cells in each lead-exposed group increased significantly(P<0.05),and the expression of Caspase-3 mRNA increased significantly(P<0.01).Caspase-9 mRNA expression increased significantly(P<0.05),Bcl-2 mRNA expression decreased significantly(P<0.05),BI-1 mRNA expression decreased extremely significantly(P<0.01),and cell apoptosis showed a dose-effect relationship.Compared with the 50 μM lead nitrate group,the Caspase-3 and Caspase-9 mRNA expression in the 50 μM lead nitrate+2 mM NAC group were extremely significantly decreased(P<0.01),and the Bcl-2 mRNA expression was extremely significantly increased(P<0.01),BI-1 mRNA expression increased,but there was no difference(P>0.05).Conclusion:Lead nitrate can inhibit the activity of CEF cells,leading to a decrease in the antioxidant capacity of CEF cells,thereby inducing oxidative damage and leading to cell apoptosis.The mitochondrial pathway plays an important role in the process of apoptosis.At the same time,the damage effect of lead nitrate on CEF cells presents a dose-effect relationship,and NAC has a protective effect on lead-induced damage to CEF cells. |
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