Study On The Molecular Mechanism Of Isothiocyanate Extraction From Broccoli And Induction Of Autophagy In Mouse Melanoma B16 Cells

The glucosinolates(GSL)in broccoli seeds can be hydrolyzed by myrosinase or intestinal bacteria to isothiocyanate(ITCs)with multiple biological activities,It has been shown that the 4-methyl-butyl isothiocyanate(Erucin)and Sulforaphane(SF)in ITCs have anticancered effects,and SF has higher anticancer activity than Erucin.In order to improve the content of active substances of ITCs in broccoli seeds and to study the molecular mechanism of autophagy induced by sulforaphane in mouse melanoma B16 cells.In this paper,broccoli and mouse melanoma B16 cells were used as subjects to study the effect of extraction methods on ITCs content in broccoli seeds,the effect of different stir-fry methods on ITCs content in broccoli and the activity of quinone oxidoreductase(NQO1)in B16 cells,and to preliminarily explore the molecular mechanism of sf-induced autophagy in B16 cells.The main achievements of this paper are as follows:(1)Taking broccoli seeds as the research object,the effects of enzymatic hydrolysis before extraction(EBE),enzymatic hydrolysis and extraction simultaneously(EES)on the extraction of ITCs content were studied.The results showed that there was no significant difference in the total ITCs between the EBE group and the EES group(P>0.05),but the Erucin content in the EES group was significantly increased(P<0.05).Similar results were obtained with unpurified myrosinase and radish glucosinolates.However,when pure myrosinase and sulforaphane were used as the research subjects,the two extraction methods had no significant influence on the content of ITCs and Erucin in the extraction(P>0.05).(2)Stir-fry is a common cooking method of broccoli in China.In this paper,mouse melanoma B16 cells were taken as the object,the ITCs content of the extracts of raw broccoli(RB),direct stir-fried broccoli(DS),hydrolyzed and stir-fried broccoli(HS)were compared,and then the proliferation inhibitory effect and NQO1 induced activity of broccoli extract of RB,DS and HS groups on mouse melanoma B16 cells were studied.The results showed that there was no significant difference in the proliferation inhibitory effect of broccoli extracts on B16 cells.The extracts from the RB and HS groups significantly induced the ITC content and the activity of NQO1 in B16 cells,which was significantly different from that of DS group(P<0.05).(3)Immunofluorescence cytochemical staining was used to detect the effect of SF on the formation of autophagosomes in B16 cells,it was found that SF at 5 ?M and 10 ?M had no significant effect on the formation of autophagosomes,while SF at 20 ?M and 30 ?M could significantly promote the formation of autophagosomes in B16 cells.(4)The expression of Atg5 and LC3 proteins in B16 cells and the relationship between sf-induced autophagy in B16 cells and PI3K/ERK/AMPK signaling pathway were investigated,The results showed that SF could up-regulate the expression of Atg5 and LC3 proteins,SF can induce autophagy in B16 cells through the PI3K/ERK signaling pathway,and PI3K/ERK/AMPK signaling pathway inhibitor can promote the effect of SF on inducing B16 cell apoptosis.However,when SF concentration was low,inhibition of AMPK signaling pathway had no significant effect on apoptosis of B16 cells.