| Deltamethrin(DM)has been widely used in the fields of agriculture,public health and veterinary medicine.Due to its large usage and wide application range,it led to global DM resistance.Therefore,it is of great significance to further study the resistance mechanism of DM.Studies have reported that DM can cause oxidative stress in insect cells.Nrf2-keap1 signaling pathway is an important antioxidant pathway in cells and plays a key role in preventing the damage of exogenous substances.Nrf2 gene regulates the antioxidant reaction and heterogenous metabolism by regulating the expression of a variety of antioxidant and phase II detoxification genes,so as to protect cells from damage caused by stimulation including endogenous substances,reactive oxygen species(ROSs),radiation and environmental toxins.We inferred that the transcription factor Nrf2 may play a role in DM stress and tolerance.In order to explore the relationship between Nrf2 pathway and insect tolerance to DM,we took Kc cells of drosophila melanogaster as model cells to analyze the effect of Nrf2 on the stress response to DM and the DM tolerance level Kc cells.The main results are as follows:1.The content of ROS in Kc cells increased rapidly after DM treatment,and the amount of ROS in stimulated cells was related to DM dosage and stimulation time.With the increase of DM dosage,the amount of ROS also increased,reaching a peak when the concentration of DM reached a certain value;at the same dose,the longer the DM induced,the more ROS is generated,reaching a peak when the stimulation time reached a certain value.2.The stimulation of DM could significantly up-regulate the expression of Nrf2 gene in cells and cause Nrf2 protein nuclear aggregation.qPCR assay showed that the transcription level of Nrf2 gene in cells reached the maximum with 20 ppm DM induced 24 h,and WB assay showed that the nuclear aggregation of Nrf2 protein in cells was the most obvious when 20 ppm DM induced 36 h.3.Flow cytometry results showed that under DM stimulation,the survival rate of Nrf2 gene overexpression(N+)treatment group was significantly higher than that of control group,Keap1 gene overexpression(K+)and Nrf2 gene interference(N-)treatment groups,while the survival rate of K+ treatment group was lower than that of N+ treatment group but higher than that of N-treatment group.4.According to the ability of Nrf2 to induce detoxification enzymes and antioxidant enzymes,four phase I detoxification enzymes — cytochrome P450 3A4(CYP 3A4),cytochrome P450-6a8(CYP 6a8),aldehyde dehydrogenase(ALDH)and alcohol dehydrogenase(ADH),and four phase II detoxification enzymes —Glutathione S transferase(GST),Glutathione peroxidase(GPx),Catalase(CAT),Superoxide dismutase(SOD)were selected experimentally.The expression levels of these enzymes in each treatment group were quantitatively analyzed by qPCR.Test data showed that although most of the selected enzymes were positively regulated by Nrf2,they were affected to different degrees.The above results showed that the stimulation of DM could activate Nrf2 gene in Kc cells,which was reflected by the increase of Nrf2 mRNA content,the increase of Nrf2 protein in the nucleus and the increase of detoxification enzyme gene expression.These results showed that the transcription factor Nrf2 regulated the expression of metabolism-related genes through Nrf2-Keap1 pathway,and played a role in the development of deltamethrin tolerance and resistance.Therefore,Nrf2 can be considered as a potential therapeutic target of oxidative stress and a potential molecular target for drug resistance control. |
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