FIGURE 2.

Sevoflurane induced changes of Parthanatos-related proteins and mitochondrial membrane potential in neuronal cells. (A,B) Western blotting and quantitative analysis of cytoplasmic PAR polymer after 4 and 8% sevoflurane exposure for 12 h in SH-SY5Y cells, HT22 cells, and hippocampal neurons. Compared with control group, ^∗∗p < 0.01; compared with 4% sevoflurane, 8% sevoflurane significantly increased the ratio of PAR polymer to β-actin. (C–H) Western blotting and quantitative analysis of PAPR-1 and AIF both in cytoplasm and nucleus in SH-SY5Y cells, HT22 cells, and hippocampal neurons after 4 and 8% sevoflurane exposure for 6, 12, and 24 h. Compared with the control group, ^∗p < 0.05, ^∗∗p < 0.01. Significant differences in the ratio of PARP-1 and AIF to β-actin or histone were detected when sevoflurane was exposed for 6 h vs. 12 h (p < 0.01), and for 12 h vs. 24 h (p < 0.01). (I) Confocal microscopy observed that AIF accumulated in the nucleus of SH-SY5Y cells and HT22 cells after 8% sevoflurane exposure for 12 h, which were obviously alleviated by PARP-1 inhibitor 3AB at 500 μmol/L or antioxidant NAC at 5 mmol/L. (J) Fluorescence microscopy with JC-1 staining revealed that a concentration-dependent weakened red fluorescence and enhanced green fluorescence were markedly observed in SH-SY5Y cells and HT22 cells treated with 4 and 8% sevoflurane for 12 h, which were counteracted in the presence of PARP-1 inhibitor 3AB or antioxidant NAC. (K) Flow cytometry analysis showed that concentration-dependent decline of mitochondrial membrane potential after 4 and 8% sevoflurane for 12 h was attenuated in the presence of PARP-1 inhibitor 3AB or antioxidant NAC in SH-SY5Y cells and HT22 cells. Data are represented as mean ± SD from five independent experiments.