Study On The Antioxidant Effect In Vitro And Molecular Mechanism Of Apoptosis Of Human Bladder Cancer T24 Cells Induced By Isoliensinine

Objective: In this study,to compare the antioxidant effects of Isoliensinine,neferine and liensinine in vitro,the more monomer was selected as the drug to be tested,and it was applied to on human bladder cancer T24 cells and human hepatocarcinoma Hep G2 cells.To observe its effect on the proliferation rate and select a cancer cell with higher proliferation inhibition rate.To determine of the influence on their morphology,cycle,apoptotic rate,migration and invasion induced by drugs to be tested.To explore the potential molecular mechanism of cell of apoptosis induced by drugs to be tested,so as to provide a theoretical basis for the development of its pharmacological effects.Methods: Five methods including hydroxyl radical scavenging,DPPH,ABTS,reducing power,total antioxidation capacity were used to evaluate the antioxidant effects of Isoliensinine,neferine and liensinine in vitro.CCK-8 kit was applied to determine the influence of Isoliensinine on T24 cells and Hep G2 cells proliferation at different concentrations and at 24,48,72 h respectively.The apoptotic morphology of T24 cells were induced by Isoliensinine using Hoechst 33258 staining observed.Flow cytometry was used to determine the cycle distributions and apoptosis of T24 cells.Transwell was used to observe the influence of Isoliensinine on the migration and invasion of T24 cells.The expression of relative proteins,including Bax,Bcl-2,Caspase-3,MMP-2,MMP9,P65,P-P65,Ikka,I?Ba,b-Actin in T24 cells induced by Isoliensinine was determined by Western blot.The nuclear translocation of NF-?B active subunit P65 was detected by immunofluorescence.Results: Isoliensinine,neferine and liensinine have strong effect to scavenge free radicals,and Isoliensinine is the strongest.Isoliensinine has a significant inhibitory effect on the proliferation of T24 cells and Hep G2 cells,the inhibitory effect on T24 cells was stronger.Flow cytometry showed that Isoliensinine induced block of T24 cells in G0/G1 phase and S phase and apoptosis in a dose-dependent manner.Hoechst 33258 staining revealed that the number of T24 cells decreased,the morphology of apoptotic cells shrank and change,the color deepened.Western blot assay showed that Isoliensinine down-regulated the expression levels of P-P65,P-I?Ba,and up-regulated the expression levels of P65 and I?Ba proteins.Isoliensinine inhibited the transfer of P65 from the cytoplasm to the nucleus.Conclusion: Isoliensinine has strong free radical scavenging and anti-lipid peroxidation ability,and reduce cancerous or mutations caused by peroxidation products.Isoliensinine can also inhibit the migration and invasion of T24 cells,which may be related to down-regulation of MMP-2 and MMP-9 protein expression.In addition,Isoliensinine significantly inhibited proliferation and induced apoptosis of T24 cells,and its mechanism may be related to inhibition of NF-?B pathway activity.