Molecular Mechanism Of Azadirachtin Regulated Cell Growth And Identification Of Its Target Protein

Abstrast: Azadirachtin is a bioactive component in neem. Azadirachtin is one of the best botanical insecticides. Because of its high efficiency, low toxicity and low resistance,azadirachtin has wide application and development prospect. It has been proved to have strong antifeeding and growth inhibitory activity against most insects. However, the exact target proteins of azadirachtin are not fully identified.In order to further elucidate the mechanism of Azadirachtin, we focused on interaction between target protein and Azadirachtin. 3 kinds of mammalian cells are used as test groups,referring to SL-1 cell. Cell growth and proliferation was detected. Added growth factor agonist and PI3 K inhibitors, we research the relationship between Azadirachtin and growth factor receptor, PI3 K. By genetic engineering technology, recombinant growth factor receptor was got. In vitro interaction of receptor and Azadirachtin was researched using fluorescence spectrum quenching method and plasma resonance method. On the basis, we conclude the mechanism of the inhibition of cell growth and proliferation.Main results of this study were as follows:1. Growth and proliferation of 4 tested cell lines were detected by MTT assay in in vitro cultured cells. In the concentrations of 3 μg/m L, 12 μg/m L, 48 μg/m L, and 192 μg/m L,azadirachtin obviously inhibited the growth and proliferation of the 4 cell lines in a concentration-dependent manner. According to the results, 3 μg/m L and 12 μg/m L were selected as working concentrations for further research.Rhodamine fluorescence can be used as a measure of membrane polarization in live cell assays, which relies on the fact that rhodamine 123 accumulates in membranes in a manner which is dependent on membrane polarization. Flurescence microscopy observed results indicated that after cell treatment with azadirachtin, mitochondrial membrane potential decreased, which proved the functionary mechanism to involve mitochondrial pathway in the 4 tested cell lines.2. Proteins regulation was researched by stimulation on signal pathway. Results indicated azdirachtin inhibited FGFR-2 activated by FGF-2, and the inhibition showed positive correlation with FGF-2 ability.3. An inhibitor complex of FGF2-FGFR2 was established for the in vitro experiments by fluorescence quenching and SPR to be confirmed the influence by azdirachtin. Results showed that azdirachtin not only interacted with FGFR-2, but also inhibited the combination of FGF-2 and FGFR-2.This study indicated that azdirachtin regulated mammalian cell growth and proliferation by inhibiting the combination of FGF and FGFR. In vitro complex analytical data showed that FGF2-FGFR2 may be one of the binding proteins. By inhibiting membrane receptor signal activation, azdirachtin affected downstream signal, inhibiting cell growth and proliferation, and inducing apoptosis.