Research On Injuries Of PC12 Cell Induced By TDF And Possible Mechanisms

Objective: The study aims to observe the process of cell proliferation and apoptosis and investigate the possible mechanism of neuron injuries of Tenofovir disoproxil fumarate(TDF) by utilizing PC12 cells treated with different concentrations of TDF.Methods: The effects of TDF on proliferation and injuries of PC12 cells were examined by MTT assay and LDH assay respectively. Morphological variation of nucleus was labeled by Hoechst-33342. Apoptosis rate was measured by TUNEL and flow cytometry. The MDA and ROS in PC12 cells were tested by enzyme linked immunosorbent assay and DCFH-DA respectively. Protein expressions of Bax was detected with Western blotting analysis.Results: Cell livability of PC12 cells was 100±3.07%, 97.29±1.74%, 99.08±1.54%, 92.47±0.81%, 81.86±2.53% and 48.74±4.65%respectively when treated with TDF at the concentration of 0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 200μ mol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01) which indicates that there is a concentration dependent inhibition of TDF on PC12 cells proliferation. Cell activity of PC12 cells was 118.84±17.88, 137.87±26.0, 144.85±25.26, 189.58±24.98, 214.51±22.30, 262.86±21.71 respectively when treated with TDF at the concentration of 0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 200μmol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01) which indicates that there is a concentration dependent injuries of TDF on PC12 cells. With the increasing concentration of TDF, apoptosis rate of PC12 was seen a significantly increase which was indicated by Hoechst-33342. Apoptosis rate of PC12 cells was 1.21±0.45%、11.96±1.64、33.60±5.87%and 51.71±2.50%respectively when treated with TDF at the concentration of 0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01) which indicates that cell injuries of PC12 cells was able to be induced by TDF. ROS fluorescence intensity in PC12 cells was 25540.26±2584.27、37264.90±2098.7、68924.21±330.14 and 92540.88±4545.79 respectively when treated with TDF at the concentration of 0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01). MDA in PC12 cells was 6.64±0.54、8.88±0.78、9.88±0.70 and 16.22±0.38 respectively when treated with TDF at the concentration of 0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01). Protein expression of Bax in PC12 cells was 1.14±0.04、1.28±0.04、1.61±0.06 and 2.19±0.07 respectively when treated with TDF at the concentration of 0 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L. Comapred with the control(0μmol/L), there was a significant difference at the concentration of TDF decreasing to 100 μmol/L(P<0.05), 200 μmol/L(P<0.01), 400 μmol/L(P<0.01) which indicates that the injuries of PC12 cells induced by TDF was related ro the pathway of mitochondrial apoptosis.Conclusions: There is a dose-dependent inhibitory effect of TDF on cell proliferation which is related to cell apoptosis. And this apoptosis relates to mitochondrial apoptosis pathway.