| Objective:To investigate the protective effect of Perillae Folium with aqueous extract?PFAE?on some key factors of Adriamycin?ADR?-induced oxidative injury in human renal tubular epithelial cells?HK-2?,including the survival rate,oxidative injury indexes and cell apoptosis,and to define the underlying mechanism.Method:A model of ADR-induced HK-2 cells oxidative injury was established in vitro.The effects of different solvent extracts of Perilla leaf on cell activity were screened,the best solvent extract PFAE was selected for subsequent experiments.Cell viability was detected by cell counting kit-8?CCK-8?after intervention with positive reference N-acetylcysteine?NAC?or PFAE(5,15,45 g·L-1)at different concentrations.According to the morphological changes under microscopy,the optimum concentration of PFAE was screened out for the follow-up experiments.Then,the experiments were divided into six groups:blank group,ADR(0.05 g-L-1)group,PFAE(15 g·L-1)group,ADR+PFAE(0.05+15 g·L-1)group,NAC(0.81 g·L-1)group,and ADR+NAC(0.05+0.81 g·L-1)group.After that,malondialdehyde?MDA??superoxide dismutase?SOD?,total antioxidant capacity?TAC?were measured in the cell homogenate after 24h administration.The level of Reactive Oxygen Species?ROS?was detected by2',7-Dichloroflurescin diacetate?DCFH-DA?fluorescence probe.Flow cytometry and TdT-mediated dUTP Nick-End Labeling?TUNEL?were used to monitor the cell apoptosis.Western Blot?WB?was used to observed the expressions of mitochondrial apoptosis-associated proteins,like B lymphocyte tumor-2 gene?Bcl-2?,Bcl-2 related X protein?Bax?,cysteine aspartate protease 9?Caspase-9?,cysteine aspartate protease 3?Caspase-3?and poly ADP-ribose polymerase?PARP?,as well as their shear bodies.In addition,the phosphorylation protein expressions of p38 mitogen-activated protein kinase?p38 MAPK?,extracellular signal-regulated kinase?ERK?,c-Jun amino-terminal kinase?JNK?in mitogen-activated protein kinase?MAPK?signaling transduction pathway were detected by Western blot.Result:(DCompared with the blank group,the cell activity of the ADR group was significantly reduced?P<0.01?.Compared with the ADR group,5,15 g-L-1 PFAE and NAC could promote cell proliferation?P<0.01?,the protective effect was the most obvious when the concentration was 15 g·L-1 with PFAE.?Compared with the blank group,the levels of total antioxidant capacity?TAC?and SOD in the ADR group were significantly decreased?P<0.01?,but the levels of MDA and ROS were significantly increased?P<0.01?.Compared with the ADR group,the levels of TAC and SOD both in the ADR+PFAE group and ADR+NAC group were significantly increased?P<0.01?,and the levels of MDA and ROS were significantly decreased?P<0.01?.?Compared with the blank group,cell apoptosis rate was increased in the ADR group?P<0.01?,the ratio of apoptosis-related proteins expressions Bax/Bcl-2,cleaved Caspase-9/Caspase-9,cleaved Caspase-3/Caspase-3,cleaved PARP/PARP were increased significantly?P<0.01?,and phosphorylated protein expressions of p38 MAPK,ERK and JNK in the MAPKs pathway increased significantly?P<0.01,P<0.01,P<0.05?.Compared with the ADR group,after the intervention with PFAE or NAC,the apoptosis rate was reduced,as well as the relative ratio of apoptotic proteins and the phosphorylations of p38 MAPK and ERK proteins?P<0.01?,but it had no effect on the expression of the phosphorylated JNK proteins. |
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