Effect of silencing beclin1 gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation

doi:10.16098/j.issn.0529-1356.2020.01.001

Abstract

Abstract:

Objective To investigate the effect of Beclin1 gene on apoptosis of HT22 of mouse hippocampus neuron treated with oxygen and glucose deprivation/reoxygenation(OGD/R). Methods HT22 cells in logarithmic growth phase were randomly divided into 4 groups: normal, model, beclin1^-/-and control. All groups were reoxygenated after 6 hours of oxygen and glucose deprivation except for normal group. The beclin1 interference sequence was designed for mouse cDNA sequence using RNAi technology, and was transfected into HT22 cells by liposome Lipo2000. The transfection efficiency was observed under fluorescence microscope and Western blotting after 48 hours of transfection. Cell viability was detected by CCK-8 method. Cell damage was detected by lactate dehydrogenase(LDH) method , Bax and Bcl-2 were detected by immunofluorescence staining and the expression of LC3, P62 and Caspase-3 were tested by Western blotting after 24 hours of reoxygenation. SPSS 19.0 statistical software was used for data analysis. Results Compared with the normal group, the cell viability and P62 expression decreased significantly (P<0.01), the LDH leakage rate, LC3Ⅱ/LC3Ⅰ，Caspase-3 expression and Bax/Bcl-2 increased significantly in model group (P<0.01). Compared with the model group, the cell viability and LC3Ⅱ/LC3Ⅰ decreased significantly (P<0.01), the LDH leakage rate, Bax/Bcl-2，P62 and Caspase-3 expression increased significantly in beclin1^-/- group (P<0.01). There was no difference between the control group and the model group. Conclusion Silencing beclin1 inhibites autophagy, which aggravates the damage of OGD/R HT22 cells and further increases apoptosis.

Key words: Beclin1, Gene silencing, Oxygen and glucose deprivation/reoxygenation, Autophagy, Western blotting

CLC Number:

ZHANG Yi JIN Xiao-fei ZHOU Xiao-hong DONG Xian-hui ZHANG Ying YU Wen-tao CHENG Yuan GAO Wei-juan. Effect of silencing beclin1 gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation[J]. Acta Anatomica Sinica, 2020, 51(1): 3-8.

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