| The botanical pesticides are important supplements for chemical pesticides in the integrated pest management.Harmine,a ?-carboline alkaloid,is a widely spread bioactive substance in the world.It has been widely used in medicine,as well as in plant protection.Harmine was suggested to control a variety of insects effectively through antifeeding and development regulation.However,the mechanisms of its insecticidal activity and development regulation are still unclear.In this paper,we studied the inhibitory effect of harmine on the growth of Drosophila melanogaster,and on the proliferation of Spodoptera frugiperda Sf9 cells.Harmine were proved to induce autophagy in insect cell and the related mechanisms were explored.Main results were as follows:(1)The inhibitory effect of harmine on the development of Drosophila showed a dosedependent manner.With the increase of harmine from 0 to 200 mg/L in culture medium,the pupal weight,length and height,emergence rate,male-female ratio were significantly inhibited.Moreover,harmine have a comprehensive inhibitory effect on the genes' expression of developmental signaling pathways,including Wnt,Notch,TGF-?,Hippo,Hedgehog pathway,etc.But the contributions of each pathway were various in female survivals and stresses management.In addition,the PI3K/Akt pathway was also significantly inhibited,which suggested that harmine might participate in Drosophila developmental inhibition through autophagy.(2)The five ?-carboline alkaloids inhibited the proliferation of Sf9 cells,and the inhibition effect showed a time-and dose-dependent manner from 0 to 0.2 m M.The inhibitory effect was ranked as: harmine > harmol > harmaline > harmalol > tryptoline.Among them,the unsaturated ?-carboline alkaloids,harmine and harmol,showed stronger biological activity to affect cell morphology,making cell vacuolization and swelling,and so on.As the dose increasing and time prolonging,apoptosis characteristics appeared.In addition,harmine and harmol showed strong autophagy induction ability,which could significantly enhance the fluorescence intensity of autophagy fluorescent dye monodansylcadaverine(MDC)and lysosomal dye Lyso Tracker Red.The expression of autophagy-related genes and proteins,an example Atg8,were also promoted.(3)After treated with different concentrations of harmine for 24 h,the fluorescence intensity of MDC and Lyso Tracker Red staining increased since 0.05 m M,as well as the expression of autophagy-related genes and proteins.Apoptotic bodies would appeare at 0.2 m M.Therefore,0.05 m M was used as the dosage to study the m RNA transcriptional and protein expression level of harmine-induced Sf9 autophagy.The results showed that,2463 genes were up-regulated and 689 genes were down-regulated in the transcriptome.In proteome,36 proteins were up-regulated and 77 proteins were down-regulated.These differential genes were mainly enriched in the endoplasmic reticulum,extracellular matrix and metabolic pathways,which involved in transmembrane transport,immune stress,metabolic regulation,autophagy and apoptosis,etc.All these suggested the potential action model of harmine on Sf9 cells.(4)PI3K/Akt pathway is an important pathway for autophagy.Three specific inhibitors,pictilisib,MK-2206 2HCl and rapamycin,and two pathway activators,1,3-dicaffeoylquinic acid and MHY1485,were used to study the role of PI3K/Akt pathway in harmine-induced autophagy of Sf9 cells.The results showed that inhibitors could significantly promote harmine-induced autophagy,including cell morphology and fluorescent staining,while the two activators could alleviate the cell proliferation inhibition and fluorescent staining.Thus,PI3K/Akt pathway was indeed involved in harmine-induced autophagy.(5)Detection of mitochondria-specific dyes and mitochondrial membrane potential indicators showed that mitochondria had a weaker effect on harmine-induced autophagy,as well as the result of ATPase activity detection.This article focused on lysosomal-related pathways and Rab7/RILP plays an important role in the recognition and fusion of autophagosome and lysosome.Bioinformatics analysis found that Rab7 was conserved in insects and mammals,while RILP was quite different,which suggesting different modes between them.RNAi and overexpression technologies were used to confirm the role of Rab7 and RILP in harmine-induced autophagy.In addition,the interaction between Rab7-RILP in insect was investigated by yeast two-hybrid system and GST-pull down assay.A GC-rich region at the C terminal of RILP needed to be removed to interact with Rab7,and the N-terminal of RILP was also involved in the interaction.Point mutations in some amino acids could affect the interaction,but there were significant differences between mammals and insects.In this paper,insect cell autophagy induced by the new insecticidal active ingredient harmine was studied and the growth of Drosophila was also inhibited.The results would provide a theoretical basis for further study of the mechanism of harmine,as well as the chemical transformation and application of related components.Furthermoe,it also contributes to enrich the mechanism of insect development inhibition and pathway transduction of insect in autophagy process. |
PDF Full Text Request