Hydrogen sulfide (H₂S) has been shown to protect against oxidative stress injury and inflammation in various hypoxia-induced insult models. However, it remains unknown whether H₂S protects human skin keratinocytes (HaCaT cells) against chemical hypoxia-induced damage. In the current study, HaCaT cells were treated with cobalt chloride (CoCl₂), a well known hypoxia mimetic agent, to establish a chemical hypoxia-induced cell injury model. Our findings showed that pretreatment of HaCaT cells with NaHS (a donor of H₂S) for 30 min before exposure to CoCl₂ for 24 h significantly attenuated CoCl₂-induced injuries and inflammatory responses, evidenced by increases in cell viability and GSH level and decreases in ROS generation and secretions of IL-1β, IL-6 and IL-8. In addition, pretreatment with NaHS markedly reduced CoCl₂-induced COX-2 overexpression and PGE₂ secretion as well as intranuclear NF-κB p65 subunit accumulation (the central step of NF-κB activation). Similar to the protective effect of H₂S, both NS-398 (a selective COX-2 inhibitor) and PDTC (a selective NF-κB inhibitor) depressed not only CoCl₂-induced cytotoxicity, but also the secretions of IL-1β, IL-6 and IL-8. Importantly, PDTC obviously attenuated overexpression of COX-2 induced by CoCl₂. Notably, NAC, a ROS scavenger, conferred a similar protective effect of H₂S against CoCl₂-induced insults and inflammatory responses. Taken together, the findings of the present study have demonstrated for the first time that H₂S protects HaCaT cells against CoCl₂-induced injuries and inflammatory responses through inhibition of ROS-activated NF-κB/COX-2 pathway.

Hydrogen sulfide (H₂S) has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl₂) is a well-known hypoxia mimetic agent. We have demonstrated that H₂S protects against CoCl₂-induced injuries in PC12 cells. However, whether the members of mitogen-activated protein kinases (MAPK), in particular, extracellular signal-regulated kinase1/2(ERK1/2) and p38MAPK are involved in the neuroprotection of H₂S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl₂ induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α), decreased cystathionine-β synthase (CBS, a synthase of H₂S) expression, and increased generation of reactive oxygen species (ROS), leading to injuries of the cells, evidenced by decrease in cell viability, dissipation of mitochondrial membrane potential (MMP) , caspase-3 activation and apoptosis, which were attenuated by pretreatment with NaHS (a donor of H₂S) or N-acetyl-L cystein (NAC), a ROS scavenger. CoCl₂ rapidly activated ERK1/2, p38MAPK and C-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 or p38MAPK or JNK with kinase inhibitors (U0126 or SB203580 or SP600125, respectively) or genetic silencing of ERK1/2 or p38MAPK by RNAi (Si-ERK1/2 or Si-p38MAPK) significantly prevented CoCl₂-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl₂-induced ROS production, but also phosphorylation of ERK1/2 and p38MAPK. Thus, we demonstrated that a concurrent activation of ERK1/2, p38MAPK and JNK participates in CoCl₂-induced injuries and that H₂S protects PC12 cells against chemical hypoxia-induced injuries by inhibition of ROS-activated ERK1/2 and p38MAPK pathways. Our results suggest that inhibitors of ERK1/2, p38MAPK and JNK or antioxidants may be useful for preventing and treating hypoxia-induced neuronal injury.

Hydrogen sulfide (H₂S) is a signaling molecule which plays regulatory roles in many physiological and/or pathological processes. Therefore, regulation of H₂S levels could have great potential therapeutic value. In this work, we report the design, synthesis, and evaluation of a class of N-mercapto (N-SH)-based H₂S donors. Thirty-three donors were synthesized and tested. Our results indicated that controllable H₂S release from these donors could be achieved upon structural modifications. Selected donors (NSHD-1, NSHD-2, and NSHD-6) were tested in cellular models of oxidative damage and showed significant cytoprotective effects. Moreover, NSHD-1 and NSHD-2 were also found to exhibit potent protective effects in a murine model of myocardial ischemia reperfusion (MI/R) injury.