Study On The Toxic Effect Of Alcohol Extraction Of Polygonum Multiflorum Thunb On L02 Cells And The Mechanism Of ROS-Induced Apoptosis

Objective:Taking normal human liver cell（L02 cells）as an in vitro model,we studied the toxic effects of Polygonum multiflorum alcohol extract（PME）and its one of main components Rhein on L02 cells and the mechanism of ROS inducing apoptosis via mitochondria and death receptor pathway,and to provide a basis for the rational and safe administration of Polygonum multiflorum in clinic.Methods:L02 cell model was established.MTT assay was used to detect cell viability of PME and rhein at different concentrations and different time.The morphological changes of cells were observed by optical microscope.Nuclear morphology was observed by Hoechst 33342 staining.The release rate of lactate dehydrogenase（LDH）,the activity of superoxide dismutase（SOD）and the content of malondialdehyde（MDA）in the cells were detected using the corresponding kits.The changes of apoptosis rate,mitochondrial membrane potential（MMP）and reactive oxygen species（ROS）were detected by flow cytometry.The protein levels of Bcl-2,Bax,Caspase 8,9,and 3 proteins in the PME-administered group were detected by Western blot.Meanwhile,TNF receptor-associated factor 1（TRAF-1）,tumor necrosis factor receptor 1（TNFR1）,tumor necrosis factor receptor-associated death protein（TRADD）,Caspase 8,9,3,Cleaved Caspase 3and Bax expression levels in the rhein-administered group were detected by Western blot.Results:Treated with the concentration of PME（5、10、20 mg/m L）and Rhein（25、50、100μmol/L）for 24 and 48 h,the survival rate of L02 cells were decreased in concentration and time depended manner.Two time points of half inhibitory concentration(IC₅₀)were 12.29,5.84 mg/m L and 108.10,66.12μmol/L,respectively.Different concentrations of PME and rhein treated L02 cells for 24 h,respectively.The number of cells decreased,the cells shrank,rounded,and partially fell off.The rate of apoptosis increased,and the nucleus stained stained with Hoechst 33342 were shrinking,chromatin agglutinated and apoptotic bodies appeared.Compared with the normal control group,the release rate of LDH was significantly increased（P<0.01）,the intracellular ROS level was significantly increased（P<0.01）,and the SOD activity was significantly decreased（P<0.01）in PME and rhein-administered groups.There was no significant change in MDA content in PME group,but MDA content in Rhein group was significantly increased（P<0.01）;the low MMP rate in the PME group was decreased significantly（P<0.05,P<0.01）,and the rhein group was also decreased significantly（P<0.01）.With increasing concentration of PME,Caspase 3 and Caspase 9 were significantly decreased,while the expression of pro-apoptotic protein Bax was increased and the expression of anti-apoptotic protein Bcl-2 was decreased.Compared with the normal control group,in rhein-administered the expression of death-associated pathway-associated protein TNF-αwas increased（P<0.01）.Further studies found that the expression levels of TRAF-1 and TRADD showed an upward trend（P<0.05,P<0.01）.The zymogen cleavage of Caspase 8 was activated（P<0.05,P<0.01）,the expression of Cleaved Caspase 3 was increased（P<0.01）,and the expression of pro-apoptosis protein Bax was significantly increased（P<0.01）,and the levels of Caspase 9 and 3 were down-regulated（P<0.01）in the mitochondria-associated pathway.Conclusion:The study illustrated that PME and rhein have toxic effects on L02 cells,which may change the morphology of liver tissue and destroy the structure of hepatocytes to a certain extent,promote ROS levels,induce oxidative stress,activate the mitochondrial pathway,then activate apoptosis-related proteins to cause cells damage.It is suggested that ROS-mediated mitochondrial pathway was involved in PME-induced apoptosis,and Rhein-induced liver injury may be a combination of death receptor and mitochondrial pathway.