SO-002

Endogenous sulfur dioxide (SO₂) and hydrogen sulfide (H₂S) abnormality are newly identified gasotransmitters regulating hippocampal injury. Objective: This research aimed to investigate the effect of endogenous H₂S on the SO₂ generation in the BV2 microglial and explore its significance in the microglial inflammation in vitro and in vivo. Hydroxylamine (HA), an inhibitor of cystathionine β-synthase (CBS), was employed to induce hippocampal H₂S/CBS system deficiency in rats. Rats treated with HA exhibited reduced levels of H₂S in the hippocampus, elevated SO₂ levels, and increased hippocampal inflammatory response, and treatment with l-aspartate-β-hydroxamate (HDX), an inhibitor of endogenous SO₂, further exacerbated hippocampal inflammatory response due to H₂S deficiency. In in vivo experiment, endogenous H₂S deficiency induced by CBS knockdown increases SO₂ levels, elevates the expression levels of M1 polarization markers iNOS and IL-1β in BV2 microglial, and increases the protein expression levels of TLR4, MyD88, p-p65, TNF-α, and IL-6. Inhibition of SO₂ with HDX further exacerbates the levels of M1 polarization markers iNOS and IL-1β, as well as the protein expression levels of TLR4, MyD88, NF-κB p-p65, TNF-α, and IL-6 in BV2 cells. In contrast, SO₂ donors alleviate microglial M1 polarization and inflammatory responses induced by H₂S/CBS deficiency. Furthermore, it was found that overexpression of TLR4 reverses the inhibitory effect of SO₂ on the expression levels of iNOS, IL-1β, MyD88, p-p65, TNF-α, and IL-6 in microglia. Moreover, H₂S did not alter the expression of aspartate aminotransferase (AAT) 1, a key enzyme involved in the synthesis of SO₂, while reducing the activity of AAT in BV2 cells, thus contributing to inhibiting the production of endogenous SO₂. In terms of mechanism, H₂S caused persulfidation of the AAT1 protein in BV2 cells and purified AAT1 protein. The presence of DTT or C192S mutations in BV2 cells prevented H₂S-induced persulfidation of AAT1 and resulted in the restoration of AAT1 enzyme activity. In summary, SO₂ inhibits microglial M1 polarization by suppressing the TLR4/MyD88/p-p65 signaling pathway, thereby alleviating microglia inflammation induced by H₂S deficiency. H₂S inhibits the activity of AAT by persulfidating AAT1 at the Cys192 site, thereby reducing the production of SO₂ in BV2 microglia. These results suggest that endogenous SO₂ serves as a compensatory defense system against microglia inflammation in cases where the H₂S/CBS pathway is disrupted.

A nitroreductase (NTR)-responsive sulfur dioxide (SO₂)-releasing polyurethane-based polyprodrug system is developed to execute enzyme-responsive gas therapy under hypoxic conditions. The self-assembled characteristics of the amphiphilic polyurethanes are thoroughly investigated, and their enzyme-triggered degradation is elucidated by size exclusion chromatography (SEC), hydrodynamic diameter (D_h) measurement, and microscopic study. Additionally, NTR and SO₂-responsive small molecular and polymeric fluorescent probes are synthesized, and the respective responsiveness is studied by ¹H NMR and fluorescence spectroscopy. Hypoxia-activated anticancer drug tirapazamine (TPZ) is encapsulated into the polymeric nanoaggregates, and 63% drug release is observed at pH 6.0 in the presence of NTR. Anticancer activity of the polyprodrug (SO₂ as a cytotoxic agent) and TPZ-loaded polymeric nanoaggregate (SO₂ and TPZ as cytotoxic agents) is demonstrated with cobalt chloride (CoCl₂, hypoxia mimetic mediator). Overall, the present work reveals the impact of the NTR-responsive degradable polyprodrug as an anticancer therapeutic and gives a new perspective on enzyme-responsive gas therapy.