Study On Azadirachtin A Induced Autophagy And Apoptosis In Spodoptera Litura Cells

Azadirachtin,which has the strongest antifeedant activity,is well-known in the world as a typical representative of botanical insecticides.Azadirachtin not only shows stable biological activity in the aspect of insect growth regulation but also takes on proper dose-effect relationship,so it is considered as one of the best biopesticide and has the potential to be a commercial botanical insecticide.The special activity feature changed the traditional concept about pesticide and pest control,and furthermore gave a direction for pesticide research.To date,there is no more information about the mechanism of action of azadirachtin,so it is difficult to explore azadirachtin and develop new pesticide molecular design basing on it.In this study,we researched the key proteins PI3 K,AKT and Tor of PI3K-AKT-Tor pathway in Spodoptera litura cells treated with azadirachtin.The expression of key proteins and molecular switch Atg5 will be further evaluated to discover the molecular mechanism of induced autophagy and apoptosis causing by azadiractin.It provided a guidance for insect growth inhibition caused by azadirachtin,and main results of this study were as follows:1.Azadirachtin cytotoxicity was evaluated in SL-1 cells firstly.The results showed that cell viability decreased with the concentration after azadirachtin treated 24 h in SL-1 cell.The cell viability rates were 87.26% and 49.84% at the concentrations of 0.625,and 80 ?g/m L,respectively.2.It was demonstrated that azadirachtin induced autophagy in SL-1 cells by inhibiting the activation of the PI3K/AKT/Tor signalling pathway using Morphological and molecular biology method.Autophagolysosomes were detected by Lyso-Tracker Red probe of azadirachtin-induced in SL-1 cells.Compared with control group,12 h after azadirachtin treated,the number of autophagolysosomes increased 13.8 fold;24h later,the number was up to 15.9 fold;72h later,the number of autophagolysosomes decreased with time expending.The m RNA expression levels of Atg8 and the marker protein Atg8 were detected by q RT-PCR or Western blotting,respectively.The results showed that the expression of Atg8 was significantly upregulated with 6.3 fold after azadirachtin treatment.The expression levels of Atg8-PE protein were significantly higher than the controls(*p<0.05),the results accorded with the starvation of PBS treated group.In addition,the expression of fusion genes GFP-Atg8 was detected,and the expression level of azadirachtin treated group was significantly higher than control in the overexpression of GFP-Atg8 assay.In conclusion,azadirachtin can induce autophagy in SL-1 cell.The expression of autophagy related pathway protein was detected using Western blotting.Compared with controls,the expression level of P13 k,p-AKT and p-Tor in azadirachtin treated group and PBS treated group showed significant inhibition(*p<0.05),and the total protein expression level of AKT and Tor did not change.These results indicated that Aza induced autophagy by the way of inhibiting protein phosphorylation of PI3K-AKT-Tor signal pathway.3.Compared with the control groups,the I type apoptosis depended Caspace 3 could be induced by azadirachtin in SL-1 cells.After 12 h of inducing,apoptosis signal could be detected and had significant difference with controls(**p<0.01),The protein expression level increased with the inducing time expending.Further testing on apoptosis pathway proteins,the expression level of anti-apoptotic cytokine Bcl-x L was inhibited in cells treaded with azadirachtin.Cyt C was released from mitochondria,apoptotic molecular Caspase-3 was activated and cut protein PARP.Azadirachtin induced apoptosis through mitochondrial pathway in SL-1 cell.4.According to the sequential expression analysis results,the fastigium of autophagy expression appeared after 24 h of induced by azadirachtin,and the fastigium of apoptosis expression appeared after 48 h of induced by azadirachtin,autophagy precedes apoptosis.Furthermore,the function of truncated molecule Atg5 in the process of apoptosis caused by autophagy induced with azadirachtin was studied.The overexpression of t Atg5 and Atg5?193-201 which contained mutant site were treated by azadirachtin.24 h later,the apoptosis indicator Cyt C protein expression level in t Atg5 overexpression group was higher than that in Atg5?193-201 group.We found that azadirachtin could induce autophagy by the way of inhibited phosphorylation of P13K/AKT/Tor signal pathway,and induced apoptosis by the activation of Caspase-3 through mitochondrial pathway in this study.In addition,autophagy preceded apoptosis,and autophagy transformation to apoptosis via t Atg5.It provided important idea for the research of azadirachtin molecular target.