Ultrashort wave alleviates oxygen - glucose deprivation/reoxygenation injury via up - regulation of SPCA1 expression in N2a cells

Abstract

Objectives: Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca²⁺-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level.

Methods: N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting.

Results: Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001).

Conclusions: USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

Keywords: Golgi apparatus stress; apoptosis; oxygen-glucose deprivation/reperfusion; secretory pathway Ca 2+-ATPase 1; ultrashort wave.

图1

倒置相差光学显微镜下N2a细胞的形态 Figure 1 Morphology of N2a cells under the inverted phase-contrast optical microscope A: Con group. N2a cells are spindle shaped, with axonal like structures of nerve cells. The cell boundary is clear, the surface is smooth, and the adhesion to the wall is good. The dead cells are rare. B: OGD/R group. The morphology and contour of the N2a cells become blurred and shrunk. The adhesiveness is decreased. The number of living cells is significantly decreased, with more suspended dead cell clusters visible. C: USW group. The morphology of N2a cells gradually recovers, the contour is clear, and the adhesion performance improves. The number of living cells is more than that in the OGD/R group, with less suspended dead cell clusters visible. Con: Control; OGD/R: Oxygen-glucose deprivation/reperfusion; USW: Ultrashort wave. Scale bar=100 μm.

图2

3组细胞活性的比较 Figure 2 Comparison of cell activity among the 3 groups ***P<0.001. Con: Control; OGD/R: Oxygen-glucose deprivation/reperfusion; USW: Ultrashort wave.

图3

3组细胞凋亡率的比较 Figure 3 Comparison of cell apoptotic rate among the 3 groups ***P<0.001. Con: Control; OGD/R: Oxygen-glucose deprivation/reperfusion; USW: Ultrashort wave.

图4

3组细胞SPCA1蛋白质表达水平的比较 Figure 4 Comparison of level of SPCA1 protein expression among the 3 groups **P<0.01, ***P<0.001. Con: Control; OGD/R: Oxygen-glucose deprivation/reperfusion; USW: Ultrashort wave; SPCA1: Secretory pathway Ca²⁺-ATPase 1.