| Fluoride pollution became more and more serious in recent years.Accumulation of excess fluoride pose a negative effect on the environment,endangering human health,affecting organism growth and development,and leading to biological chain damaged,thereby affecting ecological environment balance.Numerous studies focused on the molecular mechanism of fluoride toxicity,however,the toxic effects and mechanisms of fluoride on insect cells remain unclear.In present study,Sf9 cells were used to study the effects of sodium fluoride?NaF?on cell morphology,cell viability,cell proliferation,apoptosis and necrosis,protein expression and cell metabolism,striving to elucidate the toxicity mechanism of NaF to insect cells.These results provide valuable information for the cytotoxicity caused by NaF pollution,and also provide a theoretical basis for the treatment of fluorine pollution.The main findings are as follows:1.Effect of NaF on Sf9 cells morphology and proliferation.?1?Morphological changed of Sf9 cells after NaF exposure:Sf9 cells were treated with different concentrations of NaF(0,1×10-5,1×10-4,1×10-3,1×10-22 M,and 1×10-11 M)for 0 h,48 h or 72 h,and then inverted microscope was used to observe changes in cell morphology.Results showed that cells treated with NaF performed gradual larger and clustered,cell membrane blurred,cell fragmentation and contents flowed out.Cells density was very low when treated with higher concentration of NaF,indicating that NaF was toxic to Sf9 cells in a dose-and time-dependent manner.?2?Sf9 cell viability reduced and cell proliferation inhibited after NaF exposure:Sf9 cells were treated with different concentrations of NaF(0,1×10-5,1×10-4,1×10-3,2×10-3,2.5×10-3,5×10-3,7.5×10-3,1×10-22 M)for 72 h,and their viabilities were detected by CCK-8 assay.Compared with control cells,treated cells demonstrated reduced viability with increased concentration of NaF.The half inhibitory concentration of NaF(IC50)on Sf9 cells at 72 h is 5.919x10-33 M.The results of cell proliferation experiments showed that high concentrations of NaF(0,2×10-3,5×10-3,1×10-22 M)inhibited the proliferation of Sf9 cells in a time-and dose-dependent manner.2.Effect of NaF on apoptosis and necrosis of Sf9 cellsCells were treated with different concentrations of NaF(0,1×10-5,1×10-4,1×10-3,1×10-22 M)for 72 h.The morphological apoptosis results demonstrated that normal cells showed green fluorescence gradually decreased,while apoptotic cells stained with orange or red fluorescence increased significantly with NaF concentration increased.The rate of apoptosis and necrosis was detected after cells were treated with different concentrations of NaF for 72 h or IC500 of NaF for 0 h,24 h,48 h or 72 h.The data showed that,with increased NaF concentration and prolonged treated time,the number of early apoptotic,late apoptotic and necrotic cells increased significantly,while the normal cell obvious decreased.DNA ladder experiment showed that DNA fragmentation occurs in both high and long-term treated groups.The above results showed that NaF may induce apoptosis and gene damage in Sf9cells in a time-dose dependent manner.3.Effect of NaF on protein expression of Sf9 cellsTwo-dimensional electrophoresis and mass spectrometry techniques were used to separate and identify proteomes from both control and NaF-treated(at IC50)Sf9 cell for 72 h.A total of 17 differentially expressed proteins was successfully obtained,including 7 up-regulated proteins and 10 down-regulated proteins.GO analysis suggested that these proteins are involved in metabolic processes,catalytic,cellular processes,and binding.Results of RT-qPCR showed that the expression of most of these differential expressed proteins is consistent with their transcription levels.4.Effect of NaF on the metabolism of Sf9 cellsCells were treated with different concentrations of NaF(0,1×10-5,1×10-4,1×10-3,1×10-22 M)for 72 h or IC500 of NaF for 0 h,24 h,48 h or 72 h.Cells mitochondrial membrane potential and intracellular ROS production were measured.Results showed that,the ratio of red-green fluorescence gradually decreased with increased NaF concentration or treated time,and ROS production increased in a time-dose dependent manner.The above results indicated that NaF may cause decreased cell mitochondrial membrane potential and increased the ROS level,then resulting in enhanced mitochondrial damage and abnormal metabolism of Sf9 cells. |
PDF Full Text Request