Antitumor Mechanism Of Alkylresorcinols In Wheat Bran On HepG2 Cells

Wheat bran is a by-product of wheat flour processing.Due to its poor taste and difficult to digest,and it is rarely used as a food raw material.It is often used as animal feed or industrial papermaking and brewing raw material,and its comprehensive value has not been fully utilized.In recent years,more and more studies have shown that a variety of natural compounds in wheat bran have anti-tumor activities.Alkylresorcinols,the fat-soluble compounds in wheat bran,can inhibit the proliferation of a variety of cells.However,the specific mechanism is still non-deterministic.In our experiment,Alkylresorcinols were extracted and isolated from wheat bran,and the regulation on the growth and movement ability of HepG2cells was studied,as well as the anti-tumor mechanism from the cellular and molecular level.The main research results are as follows:?1?Extraction,separation and identification of AlkylresorcinolsThe target compounds were isolated from wheat bran by ethyl acetate extraction,and purified with thin layer chromatography,column chromatography and preparative high performance liquid chromatography.Fast Blue RR?ZnCl₂ diazo salt color reaction and UV characteristic absorption peak of Alkylresorcinols were used as the guidance of the separation process.The final isolates identified by LC-MS were homologues of Alkylresorcinols C_19:0 and C_21:0?defined as ARs?.?2?Effects of ARs on proliferation,migration and invasion of HepG2 cellsMTT assay was used to detect the proliferation inhibition effect of ARs on HepG2 cells.Transwell assay was used to evaluate the effect of ARs on the motor ability of HepG2 cells.Western blotting assay was used to detect the effect of ARs on the expression levels of proteins MMP-7 and RhoA in HepG2 cells.The results showed that ARs showed a dose-and time-dependent growth inhibition effect on HepG2 cells.Compared with the control group,the migration inhibition rates of ARs at 40,80 and 160?g/mL were 16.73%,43.43%?P<0.01?,59.56%?P<0.01?,and the invasion inhibition rates on HepG2 cells were 6.32%,37.09%?P<0.01?,68.68%?P<0.001?,respectively.ARs can significantly reduce the migration and invasion ability of HepG2 cells,and the cells movement ability is negatively correlated with the ARs concentration.With ARs treatment,the expression levels of MMP-7 and RhoA were significantly decreased?P<0.01?.ARs may affect the migration and invasion ability of HepG2 cells by inhibiting the expression of MMP-7 and RhoA proteins.?3?Autophagy of HepG2 cells induced by ARsAutophagy double-labeled adenovirus was used to detect the changes in the content of autophagosome labeled by fluorescence.Beclin-1,LC3-II,p62,p-PI3K,p-Akt and p-mTOR autophagy-related proteins expression levels were detected by Western blotting.The results showed that ARs could promote the formation of autophagosomes in HepG2 cells.The number of autophagosomes increased significantly?P<0.05?and the expression of p62 decreased?P<0.05?in cells pretreated with autophagy inhibitors under ARs,and the level of autophagy was up-regulated.In the ARs-treated HepG2cells,Beclin-1?P<0.01?,LC3-II?P<0.01?expression levels were increased,and protein p62 expression was decreased?P<0.05?.Expression levels of p-PI3K?P<0.01?,p-Akt?P<0.01?and p-mTOR?P<0.01?were decreased and dose-dependent,suggesting that ARs induced autophagy in HepG2 cells may be related to inhibition of PI3K/Akt/mTOR signaling pathway.?4?Apoptosis of HepG2 cells induced by ARsNuclear morphological changes were detected by Hoechst 33342 staining.Annexin V-FITC/PI staining was used to determine the effect of ARs on apoptosis rate by flow cytometry.The change of mitochondrial membrane potential was detected by JC-1 staining.The protein expressions of Bax/Bcl-2 and cleaved caspase3were detected by western blotting.In the ARs treatment group,the nuclear deformation rate increased?P<0.05?,the nucleus shrank,and chromatin fragmented and condensed to form apoptotic bodies.ARs induced an increased apoptosis rate in HepG2 cells?P<0.01?.The mitochondrial membrane potential of the ARs group HepG2 cells decreased?P<0.001?,and the cells entered the early apoptosis state.ARs could increase the expression of Bax/Bcl-2?P<0.001?and cleaved caspase3?P<0.001?.The above results suggest that ARs may affect the mitochondrial pathway by regulating the expression of Bax/Bcl-2 protein,thereby activating the caspase pathway to produce a cascade reaction and induce apoptosis.After the inhibition of autophagy,the mitochondrial membrane potential increased?P<0.05?,and the expression of apoptotic protein increased,but there was no significant difference about cleaved caspase3 compared with the control group.After the addition of ARs,the mitochondrial membrane potential dropped?P<0.05?,and the expression of cleaved caspase3 increased significantly?P<0.05?.ARs may promote apoptosis by upregulation of autophagy.