ABSTRACT
Aim To investigate the role of S-sulfhydration of RhoA kinase 2 in the neuroprotection of hydrogen sulfide (H2S) against hypoxic injury. Methods Rat hippocampal neurons were primarily cultured and treated with exogenous H2S donor NaHS (50, 100, 200 (xmol • L"1 ) and S-sulfhydration inhibitor DTT (50 (xmol • L"1 during 4 hours of hypoxia and 12 hours of reoxygenation. Cell viability, the lactate dehydrogenase (LDH) activity and neuron-specific enolase (NSE) activity released from injured neuron to culture supernatant, and the proportion of apoptotic cells were measured to assess the neuroprotection of H2S, and the role of S-sulfhydration in the neuroprotection of H2S was preliminarily explored. In addition, the S-sulf- hydrated proteins in neurons were isolated and purified by modified biotin-switch assay. And then, the RhoA kinase 2 (ROCK2) expression and activity, and S-sul- fhydrated ROCK2 were detected to further confirm the role H2S on the S-sulfhydrated ROCK2 by Western blot and assay kits, respectively. Results The decrease of cell viability, and the increase of LDH and NSE released from injured neuron to culture supernatant and cell apoptosis after hypoxia/ reoxygenation ( H/R) were significantly inhibited by 100 and 200 |imol • L"1 NaHS. Compared with the effect of 200 jimol • L"1 NaHS, the neuroprotection of 200 (xmol • L"1 NaHS could be inhibited by co-application with DTT. Furthermore, 100 and 200 (junol • L"1 NaHS could reduce the expression of R0CK2 protein and restrain ROCK2 activity via promoting the S-sulfhydryl modification of ROCK2 protein in hippocampal neurons. Conclusions H2S exerts protective effect on H/R injury of rat hippocampal neurons via down-regulation of ROCK2 expression and inhibition of R0CK2 activity by S-sulfhydration modification.