Photoactivated Activity Of 2-Thiophenylfuranocoumarin On Ovarian Cell Sf9 Of Spodoptera Frugperda

Furancoumarins,the secondary metabolites of plants,may protect against the invasion of pathogenic microorganisms and herbivorous insects through light-dependent or light-independent mechanisms,so furancoumarins are considered as natural insecticides and fungicides.Many furancoumarins have photosensitive properties,which are mostly used in photodynamic therapy of skin diseases and cancer cells,but their photosensitive toxicity to insects is not significant,so there are few studies.In the early stage of our laboratory,a series of 2-substituted-furanocoumarin compounds were synthesized by modifying the chemical structure of furancoumarins.In order to evaluate the bioactivity of these compounds,the photoactivated toxicity of 2-thiophenylfuranocoumarins（LX76）on Sf9 cells was studied by flow cytometry,confocal microscope,RT-q PCR,Western bloting and other cell biology and molecular biology methods from the perspectives of cell proliferation,oxidative stress,cell cycle and cell apoptosis.The mechanism of cytotoxicity induced by LX76 in Sf9 cells was discussed.The main results are as follows:1.MTT colorimetric method was used to determine the toxicity of LX76 on the proliferation of Sf9 cells.The results showed that photoactivated LX76 showed good inhibitory activity on the proliferation of Sf9 cells through concentration dependent and time dependent.The inhibitory concentration(IC₅₀)of photoactivated LX76 on Sf9 cells for 48 h was 2.424μg/m L（95%confidence interval 2.209～2.654）.Through the morphological observation of the cells,it was found that the treated cells swelled and enlarged,the morphological structure of the cell membrane was blurred,and the cell division was inhibited.Under the condition that photoactivated LX76 produced certain toxicity to Sf9 cells and maintained a certain cell survival rate,2.5μg/m L LX76 was used as the concentration of subsequent experiments to study the photoactivated toxicity of LX76 to Sf9 cells for different time.2.The effect of LX76 on the cell cycle of Sf9 cells was detected by flow cytometry.The results showed that the number of cells in G0/G1 phase and S phase decreased significantly after Sf9 cells were treated with LX76 for 48 h（p<0.05）,while the number of cells in G2/M phase increased from（29.87±6.30）%to（53.53±2.77）%,indicated that the cell cycle of Sf9 cells blocked by LX76 remained in G2/M phase.3.The oxidative stress of Sf9 cells induced by LX76 was detected.The results showed that the intracellular ROS level of Sf9 cells treated with LX76 for 3 h,6 h,9 h and12 h was 1.67,1.43,1.21 and 1.11 times higher than that of 0 h,respectively,and the intracellular ROS increased rapidly to the peak at 3 h of LX76 treatment.At the same time,the activities of SOD,CAT and POD enzymes related to ROS scavenging were detected, and it was found that the enzyme activities decreased significantly after 6 h treatment（p<0.05）,which indicated the excessive accumulation of ROS in the cells and causing a certain degree of oxidative damage.4.Apoptosis was detected by Annexin V-FITC/PI double staining and TUNEL staining.The results showed that the ratio of normal cells decreased and the ratio of apoptotic cells increased after treatment with LX76 for different time.After 48 h of treatment,the percentage of apoptotic cells increased from（5.96±0.48）%to（58.30±1.40）%.There was no significant change in the percentage of dead cells in Sf9 cells treated with LX76 for 12 h（p<0.05）,but the cell death rate increased to（17.73±4.25）%after 24 h treatment.In addition,TUNEL assay detected the DNA breakage caused by LX76 induced apoptosis in Sf9 cells.5.Cell ultrastructure observation and mitochondrial membrane potential change detection.The results showed that the mitochondrial membrane potential of Sf9 cells decreased after 3 hours of treatment,and the mitochondrial membrane potential decreased obviously with the prolongation of treatment time.After 12 h treatment,part of the mitochondria showed swelling and the crest structure began to blur,and after 36 h treatment,most of the mitochondrial crest disappeared and the mitochondria showed vacuolization,so it was speculated that LX76 may induce mitochondrial structural damage and dysfunction in Sf9 cells.6.The detection of Caspase family of key proteases in apoptosis.The results showed that the activities of caspase-9 and caspase-3 in Sf9 cells treated with LX76 increased at first and then decreased.The activities of caspase-9 and caspase-3 in Sf9 cells treated with LX76 for 12 h were the highest,and the expression of Caspas-3 in cells treated with LX76for 12 h was detected by immunofluorescence.7.The expression of related apoptosis genes in Sf9 cells was detected by RT-q PCR.The results showed that the m RNA expression levels of Sf-Caspase-1,Sf-Caspase-5,Sf-Apaf1,Sf-Cytochrome C and Sf-p53 were significantly increased in Sf9 cells treated with LX76 for 12 h（p<0.05）.The m RNA expression of Sf-Cathepsin B and Sf-Cathepsin L genes related to cathepsin expression was significantly up-regulated after 12 h treatment,and the m RNA expression level of Sf-Cathepsin D began to decrease significantly after 6 h treatment.8.The expression of related apoptosis proteins in Sf9 cells was detected by Western bloting,the results showed that the expression of Phospho-Akt（Ser473）decreased and the expression of Cyt C increased in Sf9 cells treated with LX76 for 6 h（p<0.05）,and the expression of Phospho-Bad（Ser112）and Bcl-XL,the key proteins in mitochondrial apoptosis pathway,was significantly inhibited,while the expression of Cleaved-PARP and p53 was significantly increased after 12 h treatment.LX76 is a kind of photosensitive substance and has obvious photoactivation toxicity to Sf9 cells.Photoactivated LX76 induces the production of ROS in Sf9 cells,caused oxidative damage to cells to a certain extent,made the cell cycle stay in G2/M phase,and inhibits the phosphorylation of Akt,which is the key nutritional factor for Sf9 cell survival,so as to inhibit cell division and proliferation.It further inhibits the expression of anti-apoptosis factor Bcl-XL in the Bcl-2 family on the outer membrane of mitochondria,dephosphorylates phosphorylated Bad rapidly,and promoted the change of mitochondrial membrane potential.Induced the release of Cyt C into the cytoplasm,activated Caspase-9 and 3,cut the key protein PARP,in the process of apoptosis to promote the expression of p53 protein,caused cell DNA breakage,and finally induced Sf9 cell apoptosis by activated Caspase-dependent mitochondrial pathway.