Fig. 1

A, SH?SY5Y cells underwent oxygen/glucose deprivation for 4 hours. After 24 hours of reoxygenation, the amount of reactive oxygen species was detected by H2DCFDA via FACS analysis. Shock wave?treated cells showed reduced oxidative stress. Data are means±SEM. ^# P<0.05. n=4. B, FACS analysis of Annexin V/PI staining to determine apoptotic and necrotic cell death upon oxygen/glucose deprivation. Shock wave?treated cells showed greater survival in this setting. Data are means±SEM. *P<0.05; ****P<0.0001. n=16 (Control), n=15 (OGD), n=12 (OGD + SWT). C, Immunofluorescence staining revealed an increased number of NRF2 positive SH?SY5Y cells after treatment with shock waves or Toll?like receptor 3 agonist Poly(I:C). Addition of a Toll?like receptor 3 inhibitor abolished shock wave?mediated NRF2 expression. Scale bar: 100 ?m. Data are means±SEM. *P<0.05; **P<0.01. n=5. D, Immunoblot analysis of NRF2 protein expression upon treatment with shock waves and Poly(I:C). Inhibition of TLR3 prevented NRF2?expression upon SWT. E and F, Quantitative polymerase chain reaction analysis revealed increased gene expression levels of NRF2 and its downstream target HO?1 (heme oxygenase?1) upon SWT and treatment with TLR3 agonist Poly(I:C). Data are means±SEM. *P<0.05; **P<0.01. n=6. G, SH?SY5Y cells undergoing oxygen/glucose deprivation showed increased protein expression of antioxidative NRF2 when pretreated with shock waves or Poly(I:C). Statistical comparison by ranks: Kruskal?Wallis test (A). Statistical comparisons between multiple groups: 1?way ANOVA with Tukey post hoc analysis (B, C, E, F). OGD indicates oxygen/glucose deprivation; and SWT, shock wave therapy.