Study On The Effect And Mechanism Of Polyphenols From Kiwi By-Product On Programmed Cell Death In HepG2 Cells Based On Metabonomics

Polyphenols from Kiwi by-product(PKWP)have anti-tumor activity,but the specific mechanisms have not been fully elucidated.Cell metabolism is very important to maintain cell viability and function.Tumor cell metabolism is abnormal compared with normal cells,and intervention of tumor cell metabolism is the focus of anti-tumor research.Programmed cell death(PCD)such as apoptosis,pyroptosis and autophagy is an important way to affect cell survival,and suitable intervention in the PCD of tumor cells could effectively control the proliferation of tumor cells.Therefore,in this study,PKWP were used as the research object,and human hepatoma cell HepG2 was used as the model.The effects of PKWP on PCD(cell apoptosis,pyroptosis,autophagy),as well as interactions among the three PCD were analyzed based on the metabolomics.The results are as follows:(1)The effects of PKWP on cell metabolism and programmed cell death were studied.The results showed that after 24 h of treatment with PKWP,it could effectively inhibit cell(HepG2 and HL-7702)growth in a concentration-dependent manner.However,lower concentration of PKWP(12.5,25.0,50.0 μg/mL)did not affect HL-7702 cell growth.So HepG2 was treated with suitable concentration of PKWP for further study.The results showed that PKWP promoted the productions of ROS and MDA,reduced the activities of CAT and T-SOD.PKWP reduced the supply of nutrients in HepG2 cells,and affected amino acid synthesis,TCA cycle,glucose metabolism and other pathways,reducing cell life activity.PKWP increased the activities of Caspase3 and Caspase8,and promoted the expressions of Fas/FasL and cleaved PARP-1,inducing cell apoptosis.PKWP activated NALP3 inflammasome,increased the activity of Caspasel,and promoted the contents of TNF-α and LDH,leading to pyroptosis.PKWP also down-regulated the expressions of LC3 and Beclinl,inhibiting autophagy.These results showed that PKWP promoted cell oxidative stress,intervened cell metabolism,promoting apoptosis and pyroptosis and inhibiting autophagy.(2)The effects of PKWP-induced apoptosis on pyroptosis and autophagy were studied.The results showed that after intervention of apoptosis with inhibitor ZVAD-fmk,PKWP-induced oxidative stress was alleviated.Metabolic pathways such as aminoacyl-tRNA biosynthesis,glyoxylate and dicarboxylate metabolism,and glutathione metabolism had undergone significant changes.Inhibition of PKWP-induced apoptosis could also reduce NALP3 expression,Caspase1 activity,and the contents of TNF-α and LDH,inhibiting inflammatory pyroptosis.These results suggested that PKWP-induced HepG2 apoptosis could promote pyroptosis and inhibit autophagy.(3)The effects of PKWP-induced pyroptosis on apoptosis and autophagy were studied.The results showed that after intervention of pyroptosis with inhibitor YVAD-cmk,PKWP-induced oxidative stress was eased.Metabolic pathways such as glycine,serine and threonine metabolism,glutathione metabolism,and alanine,aspartate and glutamate metabolism had undergone significant changes.But PKWP-induced pyroptosis did not affect Caspase3 activity and the expressions of pro-Caspase3,LC3 and Beclinl.These results suggested that PKWP-induced pyroptosis did not affect cell apoptosis and autophagy.(4)The effects of PKWP-induced autophagy on apoptosis and pyroptosis were studied.The results showed that after intervention of autophagy with activator Rapamycin,PKWP-induced oxidative stress was weakened.Metabolic pathways such as TCA cycle,Glycolysis,and Glutathione metabolism had undergone significant changes.Activating autophagy could reduce NALP3 expression and Caspasel activity,as well as prohibit TNF-α and LDH contents,inhibiting cell pyroptosis.These results showed that PKWP-inhibited HepG2 autophagy could promote pyroptosis,but did not affect apoptosis.In summary,PKWP promoted cell apoptosis and pyroptosis,and inhibited autophagy by inducing cellular oxidative stress and disturbing cell metabolism,subsequently leading to cell death.And there are different degrees of interaction between the three programmed death modes.This study could provide a theoretical basis for the analysis of the anti-tumor mechanism of PKWP.