Study On The Anti-tumor Effect Of Ampelopsis Based On Autophagy Regulatio

Autophagy is an important biological process of eukaryotes,which plays a regulatory role in the homeostasis of cells,tissues and organisms,and is widely involved in the regulation of physiological and pathological processes including anti-tumor.On the one hand,its inhibitory effect on the development of tumor has attracted much attention,some traditional Chinese medicine including monomer have been reported to promote apoptosis and inhibit proliferation of tumor cell through regulation of autophagy to achieve the effect of anti-tumor;On the other hand,autophagy has a protective effect on normal cells(including normal cells in the early stage of formation of tumor).Autophagy can maintain the stability of intracellular environment through regulation of autophagy.To clarify the mechanism of regulation of autophagy in the anti-tumor effect of monomers of traditional Chinese medicine,combined with the current preclinical research on autophagy target LC3,p62,ATG7,etc.will provide more clear pharmacological evidence for the clinical application of anti-tumor of traditional Chinese medicine.The first chapter of this study is mainly to select research subjects that are more suitable for studying the effect of autophagy on anti-tumor.In the reported anti-tumor effects of traditional Chinese medicine monomers AMP and curcumin,verified by preliminary toxicity experiments on cell proliferation and apoptosis,the results of CCK-8 method and flow cytometry respectively showed that AMP and curcumin both inhibit the proliferation of HCT116 cells,induce apoptosis and have the effects of anti-tumor.From these two Chinese medicine monomers,AMP with wide dosage range was selected to explore the effect of autophagy regulation on the anti-tumor of Chinese medicine monomers.The second and third chapters are about the study of the effect of autophagy on the antitumor of AMP.The cell line HCT116-RFP-GFP-LC3 was conducted by successfully constructing an over-expressing autophagy marker fluorescent protein LC3 through lentiviral infection in this part to study the induction of autophagy,while investigating autophagy marker proteins and autophagy pathway-related proteins to confirm the drug’s induction of autophagy.Constructed a recombinant cell line ATG7 si RNA-HCT116 cells with autophagy target protein ATG7 knockdown and an inhibitor of autophagy Baf A1 to explores the corresponding changes in cell proliferation and apoptosis after autophagy inhibition to confirm the effect of the regulation of autophagy on the anti-tumor of AMP.The HCT116-RFP-GFP-LC3 cells treated with AMP for 24 hours and found that the autophagy fluorescenct spot of LC3 II protein increased significantly,and the expression of autophagy-related proteins LC3II/I and p62 increased and decreased,respectively,indicating that AMP can significantly induce autophagy.Furthermore,ATG7 si RNA recombinant cells combined with autophagy inhibitor Baf A1 were used to explore the regulation of autophagy on cell proliferation and apoptosis.After blocking the autophagy process by the autophagy inhibitor Baf A1,the results show that the apoptotic rate of cells affected by AMP(300 μg/m L)increased by 15.68%,and the expression of the corresponding anti-apoptotic protein Bcl2,the pro-apoptotic protein Bax and nuclear transcription factor NF-κB were significantly changed,indicating that the blockade of autophagy can promote the anti-tumor of AMP;however,the blockade of autophagy inhibitors and the knockdown of the key autophagy protein ATG7 did not affect the inhibitory effect the proliferation of AMP on HCT116 cells.There is no significant difference between the control and the treatment group,which shows that AMP does not inhibit the proliferation of HCT116 cells directly through autophagy,suggesting that AMP may inhibit cell proliferation by inhibiting other pathways such as cell cycle.