| ObjectiveTo investigate the effect of Perillaldehyde(PAE)on D-galactose-induced senescence HEI-OC1 cells,and to further explore its anti-aging molecular mechanism.Methods1.Establishment of senescence model in the HEI-OC1 cells: HEI-OC1 cells were treated with 15mg/ml,30mg/ml,45mg/ml,60mg/ml D-galactose for 24 h,36h,48 h,and CCK8 assay was used to estimate the cells viability and for determining the optimum conditions of D-galatose which will be used to construct the aging model in HEI-OC1 cells(45mg/ml D-galactose for 36h).Real-time fluorescent quantitative PCR was used to detect the m RNA expression of P16 、P21 in the aging model of HEI-OC1 cells;Flow cytometry was used to detect the cell cycle,and the DEFH-DA probe was used to detect the expression level of ROS in HEI-OC1 cells to verify the aging model.2.Experimental groups:(1)Group Control: Control group,cells grew in normal medium;(2)Group D-gal group: HEI-OC1 cells were cultured with a medium containing D-galactose at 45 mg/ml;(3)D-gal+0.25mg/ml PAE group: 0.25mg/ml PAE was mixed in the medium containing 45mg/ml D-gal;(4)D-gal+0.5mg/ml PAE group: 0.5mg/ml PAE was mixed in the medium containing 45mg/ml D-gal;3.Studies on mechanism of PAE Inhibiting apoptosis of on aging HEI-OC1 cells induced by D-galactose;(1)CCK8 assay was used to observe the effect of PAE on the cell viability of HEI-OC1 aging cell;(2)The changes of reactive oxygen species(ROS)in HEI-OC1 cells were detected after treatment at different concentrations for 36h;(3)The mitochondrial membrane potential changes were detected by JC-1 staining.;(4)TUNEL staining was used to detect the apoptosis in aging HEI-OC1cells;(5)To observe the location and expression of Caspase-3,Bax and Bcl-2 by immunofluorescence in aging HEI-OC1 cells;(6)The protein expression levels of Bax,Bcl-2 and Caspase-3 by Western-blot were used to further evaluate apoptosis in aging HEI-OC1 cells.Results1.According to the results of CCK8,the optimum treatment of D-galactose(45mg/ml,for 36 hours)was used to establish a pseudo-aging model in HEI-OC1.Compared with the control group,the m RNA expression levels of P16 and P21 in the D-galactose-induced aging group were up-regulated;the proportion of cells in the G1 phase was significantly increased,and the proportion of cells in the S phase was significantly decreased(P<0.05);the data of reactive oxygen species(ROS)showed that the intracellular ROS content of the D-galactose group increased significantly.The above experimental results showed that pseudo-aging model induced by D-galactose in HEI-OC1 cells was established successfully and ready for the next experiments.2.Researches on the inhibiting effect of PAE on D-galactose induced aging HEI-OC1 cells:(1)CCK8 essay results showed that the 0.25mg/ml,0.5mg/ml PAE-treated cells groups compared with the D-galactose group,cell viability increased significantly in dose-dependent(P<0.05).(2)Reactive oxygen species(ROS)detection results showed that compared with the D-galactose group,the intracellular ROS content in the perillaldehyde 0.25mg/ml and 0.5 mg/ml groups was significantly reduced,and showed a dose-dependent manner.(3)JC-1 staining explored that the mitochondrial membrane potential of the control group was higher(showing more stronger red fluorescence),but the lower in the D-galactose induced group significantly(showing more stronger green fluorescence);compared with the D-galactose group,The mitochondrial membrane potential in the HEI-OC1 cells changed slightly induced by0.25mg/ml PAE;Conversely,the mitochondrial membrane potential in the HEI-OC1 cells was significantly increased induced by 0.5 mg/ml PAE(he red fluorescence was enhanced);TUNEL staining showed that apoptosis were dose-dependent inhibited by PAE treatment in D-galactose induced aging HEI-OC1 significantly.(4)The analysis of apoptosis-related proteins(Caspase-3,Bax,Bcl-2)by immunofluorescence staining showed that,compared with the control group,the Caspase-3 and Bax proteins in the D-galactose group increased,which were dose-dependent depressed by PAE treatment;and the expression of Bcl-2decreased in the D-galactose group,but dose-dependent up-regulated by PAE treatment.(5)Western Blot results showed that after 45mg/ml D-galactose treating for36 hours in HEI-OC1 cells,compared with the control group,the expression of Bcl-2(P<0.01)decreased,and the expressions of Bax and Caspase-3increased(P<0.01);PAE treatment clearly increased the Bcl-2,but reduced(P<0.05).ConclusionsD-galactose induced aging model in HEI-OC1 cell was established successfully;PAE attenuated cell aging in HEI-OC1 by inhibiting apoptosis. |
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