| The momordicins are a series of cucurbitane triterpenoid compounds,and have samiliar structures.The momordicins are important anti-insect secondary metabolites,isolated from extracts of Momordica charantia leaves.Momordicin I and momordicin II are a great number of compounds in extracts of momordica charantia leaves,while charatin B is a new compound.The purpose of this article is to clarify the anti-insect action mechanism of momordicin I and provide scientific basis for selecting biopesticide with new anti-insect mechanism.The larval Ostrinina furnacalis hemocytes（Ofh）and Trichoplusia ni ovary（Hi-five）cells were used to study the toxic effect of momordicin I and charatin B on larval Ostrinina furnacalis hemocytes（Ofh）at the cellular level,and study the effects of momordicin I on expression of Hi-five cells proteins.1.The cytotoxicity of Momordicin and charantin B to ofh cellsThe inhibition effects of momordicin I,momordicin II,charantin B and azadirachtin A on Ofh cells were compared by using CCK-8 assay.The effects of momordicin I and charantin B on Ofh cell morphology structure were observed by using inverted phase contrast microscopy and fluorescence microscopy,and the induced necrosis effects on Ofh cells were studied by using trypan blue staining and flow cytometry（FCM）technologies.Results shows Momordicin I,momordicin II and charantin B had apparent inhibition effects on Ofh cell proliferation,with the IC50 values at 36 h after treatment of 7.566,24.340 and 8.514μg/mL,respectively.The cytotoxicity of momordicin I and charantin B were significantly higher than that of momordicin II and azadirachtin A.Using inverted phase contrast microscopy,we found that after exposure to momordicin I and charantin B, Ofh cell shapes changed to circular and swelling increased,and the cellular membrane bubbled,suggesting that both momordicin I and charantin B could destruct cell morphology structure.Further research indicated that the mortality rates of cells in the groups treated with 8μg/m L momordicin I and charantin B were significantly higher than that of the control group at 12 to 48 h after treatment,and in a time-dependent manner,indicating that the cytotoxicity of momordicin I is significantly higher than that of charantin B.Fluorescence microscopy observation revealed that nuclear morphology was irregular,the cell membrane dissolved and the nucleus appeared to be severely damaged after AO/EB staining,and cells exhibited a non-uniform orange-yellow fluorescence after treatment with 8μg/m L momordicin I and charantin B,showing a typical characteristic of necrosis.Flow cytometry analysis showed that Ofh cells treated with momordicin I and charantin B were dramatically induced to undergo necrosis,and the total cell necrosis rates were 74.92%±2.02%and 49.77%±1.69%,respectively,after 36 h treatment with 8μg/mL momordicin I and charantin B.Conclusion:Momordicin I and charantin B have significant inhibition effects on cell proliferation and can induce necrosis,which may account for the antifeedant effect of momordicins on O.furnacalis and the inhibition of growth and fecundity of O.furnacalis treated by momordicins.2.The effects of momordicin I on expression of Hi-five ovary cells proteins.The inhibition effects of momordicin I and charantin B on Trichoplusia ni ovary Hi-five cells were tested by using MTT assay.Results shows momordicin I and charantin B had apparent inhibition effects on Hi-five cells,and the inhibition effects of momordicin I significantly higher than charantin B.The IC50 values was 19.239μg/mL at 12h treatment and 12.000μg/mL at 36h treatment after Hi-5 cells were treated with momordicin I.According to the IC50 values at 36h treatment,the Hi-5 cells were treated with 12μg/mL,with the application of Two dimensional gel electrophoresis in Proteomics,to further study the action mechanism.The total protein expression profiles of treatment ground which the Hi-five cells were treated with 12μg/mL,compared with control group,that the protein spots of treatment group were obviously less than control group,meanwhile,there were1563 protein spots in control group,and 1070 protein spots in treatment group,which was less 493 protein spots than that in control group,and there were 17 protein spots that the gap of protein content was more than 1.2 times.And 2 protein spots have disappeared,and 15 protein spots have declined,and there were 16 differential expressed proteins have been successfully identified by using MALDI-TOF/TOF-MS,including Isocitrate dehydrogenase,Ribonucleoside-diphosphatereductasesmallchain,POX-A,Phosphoglyceromutase,NADH ubiquinone oxidoreductase 30 kDa,Glycerol-3-phosphate dehydrogenase-1,Stathmin,GMP reductase 1-like,Ecto-ATPase,Alpha-tubulin,Glutamate dehydrogenase,Elongation factor 1 alpha,PolyA-binding protein,Transactivator,Vasa. |
