Fig. 4

The protection of STAT3 against the cytotoxicity of isoflurane was related to its antioxidative effect. (A) Western blot analysis and immunofluorescence staining of STAT3 in U251 cells treated with STAT3-ASO or STA-21 (n = 3). STAT3-ASO efficiently reduced STAT3 expression (P < 0.001). STA-21 treatment for 6 h significantly inhibited the nuclear translocation of STAT3 without affecting its total protein levels. Scale bar, 30 μm. ASO, antisense oligonucleotide. The blots were cropped according to the ladders and then incubated with different antibodies. The contrast and brightness of the blot images were adjusted uniformly. (B) The levels of intracellular ROS were detected using carboxy-H2DCFDA in U251 cells and presented as a percentage of that in controls (upper, n = 6). STAT3 disruption aggravated (P < 0.001), but STAT3 overexpression mitigated (P < 0.001) ROS accumulation induced by isoflurane. Representative images of carboxy-H2DCFDA staining in U251 cells are shown in the bottom panel. Scale bar, 30 μm. (C) The percentage of Annexin V positive cells was analyzed with flow cytometry (n = 6). The apoptosis induced by isoflurane was obviously augmented in cells with STAT3 knockdown (P < 0.001) or STA-21 pretreatment (P = 0.001), but was mitigated in cells with STAT3 overexpression (P = 0.002). (D) TUNEL staining (green) confirmed the protective effects of STAT3 overexpression. Magnification is 200 ×. Arrows indicate TUNEL-positive U251 cells (3 wells per group, 6 images per well). (E) Western blot analysis revealed that isoflurane downregulated the protein level of MnSOD in a STAT3-dependent manner (n = 4). STAT3 overexpression restored the decline in MnSOD and cleavage of caspase-3 after isoflurane exposure in U251 cells (P < 0.001). ## P < 0.01 versus control. * P < 0.05, ** P < 0.01 versus isoflurane only