Study On The Role And Mechanism Of Procyanidins On Oxidative Damage Of HEI-OC1 Cells Induced By T-BHP

Objectives 1 To explore the effect of Nrf2 signaling pathway in the oxidative damage of HEI-OC1 cells induced by t-BHP.2 To investigate the role and mechanism of procyanidins in oxidative damage induced by t-BHP in HEI-OC1 cells.Methods 1 The cochlea hair cells were treated with different concentration of t-BHP,Control,50μΜ,100μM,200μΜ,respectively.After 12 h,counting Kit-8(CCK8)was used to detected the viability of cell and intracellular level of ROS and the rate of apoptosis were detected by flow cytometry to verify whether construction of oxidative damage model is successful or not.2 Real time fluorescence quantitative PCR test method was used to test the expression levels of Nrf2,HO-1,GSTα1,GCLC,GCLM and NQO1 m RNA in HEI-OC1 cells.3 Using the Western Blot method to evaluate the protein expression levels of HO-1,Bax,Bcl-2 and cleaved-caspase-3 in HEI-OC1 cells.4 The effects of procyanidins on the survival rate of HEI-OC1 cells in normal state and the effect of procyanidins on the survival rate of HEI-OC1 cells under the oxidative damage model were observed by CCK-8 assay,to determine appropriate time point and concentration for drug treatment.5 The HEI-OC1 cells treated with procyanidins were tested by flow cytometry,the ROS level in HEI-OC1 cells was measured by DCFH-DA probe method,and the Annexin V-FITC/PI double staining method was used to detect the apoptosis of HEI-OC1 cells.6 The m RNA expression of Nrf2,HO-1,GSTα1,GCLC,GCLM and NQO1 in HEI-OC1 cells was detected by real time fluorescence quantitative PCR test on the HEI-OC1 cells treated with procyanidins.7 Western Blot method was used to detected the protein expression levels of HO-1,Bax,Bcl-2 and caspase-3 on the HEI-OC1 cells treated with procyanidins.Results 1 The results of CCK8 showed that with the increase of the concentration of t-BHP,the proliferation of cells showed a significant decrease(F=322.8,P<0.001).The results of DCFH-DA probe labeling and Annexin-V/PI double staining flow cytometry showed that as the concentration of t-BHP increased,intracellular ROS levels increased,and the early and late apoptosis rates of the cells were also a certain rising trend.2 RT-PCR results showed that the levels of HO-1,GCLC,and GST-α1 genes were up-regulated,and the gene expression levels showed a certain upward trend with the increase of exposure concentration of t-BHP.Nrf2 and GCLM gene expression levels were statistically significant at 200 μM(F=10.39,P<0.01;F=13.33,P<0.01).NQO1 gene expression levels were up-regulated,but there was a statistical difference only at 100 μM(F=4.77,P<0.05).3 Western Blot results showed that the level of cleaved-caspase-3,Bax and HO-1 protein expression increased with increasing concentration of t-BHP(P<0.05),and Bcl-2 protein expression was down-regulated.4 The cell viability was measured by CCK8.The results showed that compared with the control group,0-20 mg/L procyanidins(PC)had no significant changes in cell viability,while 40 and 80 mg/L procyanidins significantly decreased cell viability(85.66±3.0,39.74±2.04,F=311.3,P<0.001).In the oxidative damage cell model,after different concentrations of PC intervention for 6,12 and 24 hours,the cytoprotective effect of different concentrations of PC showed a certain increase trend after intervention for 12 hours,and the cytoprotective effect of 20 mg/L PC was relatively larger(F=26.23,P<0.001).5 The results of DCFH-DA probe labeling and Annexin-V/PI double staining flow cytometry showed that after procyanidins intervention,the intracellular ROS fluorescence Mean values in procyanidins group(5,10,20 mg/L)were respectively 0.82 times(P<0.05),0.70 times(P<0.001)and 0.56 times(P<0.001)those of the injury group.The apoptotic cells in procyanidins group(5,10,20 mg/L)were 0.55-fold(P<0.001),0.64-fold(P<0.001),and 0.85-fold(P<0.05),respectively,in the injured group.6 RT-PCR results showed that after intervention with procyanidins,only the expression levels of HO-1,GCLM,and GST-α1 m RNA were up-regulated,and there was a statistically significant difference at low concentrations(P<0.05).Compared with the injury group,the NQO1,GCLC,and Nrf2 m RNAs showed a decreasing trend with increasing intervention concentrations.7 Western Blot results showed that the expression of Bcl-2 and HO-1 protein increased with the increasing concentration of procyanidins(P<0.05),and the expression of cleaved-caspase-3 and Bax protein was down-regulated.Conclusions 1 t-BHP induces oxidative stress and apoptosis in HEI-OC1 cells.2 t-BHP activates the Nrf2/ARE signal pathway and promotes the expression of downstream protective genes.3 It is possible that the protective effect of procyanidins on HEI-OC1 cells works by inhibiting the pathway of mitochondrial apoptosis.