Gyy4137

Sulfide donors used in this study include sodium sulfide (Na₂S) (65,122-06, VWR SCIENTIFIC), GYY4137 (13,345, Cayman Chemical) and diallyl trisulfide (DATS) (10,012,577, Cayman Chemical), sodium disulfide (Na₂S2), sodium trisulfide (Na₂S3) and sodium tetrasulfide (Na₂S4) (SB02-10, SB03-10, SB04-10, Dojindo Molecular Technologies). All donors were freshly prepared in degassed distilled water and diluted with cell culture medium immediately before treatments.

Sprague–Dawley rats were exposed to cigarette smoke for 4 h/day, 6 days/week for 4 months using a dynamic smoke exposure box (diameter 700 mm, Tianjin Hope Corp., Tianjin, China). Bronchoalveolar lavage (BAL) was collected carefully and centrifuged 500 g for 5 min. Alveolar macrophages were isolated by plastic adhesion and cells (10⁵/well) were incubated in 96-well plates in the presence or absence of LPS, dexamethasone and GYY4137(morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate, Cayman chemical) for 18 hours. All animal care and experimental protocals were in compliance with the PR China Animal Management Rule and the Third Hospital, Peking University Guide for the Care and Use of Laboratory Animals.

GYY4137 [morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate] (Cayman Chemicals, USA) and NaHS (Alfa Aesar, USA) were used as sources of H₂S and were prepared according to the manufacturer’s instructions. While NaHS releases H₂S instantaneously, GYY4137 is a water soluble H₂S donor that releases H₂S slowly for weeks both in vitro and in vivo^{24 (link),27 (link)}, and more closely parallels the biological effect of endogenous H₂S. For H₂S exposure experiments, a 10 mM GYY4137 stock solution was prepared in water in a 5 ml tube and was immediately diluted to make a working stock and added to cultures to achieve the desired final concentration. Experimental control samples received time-spent 25 µM GYY4137 (referred to as spent GYY) that was previously aerated for at least 120 days. An NaHS stock solution (100 mM) was prepared by equilibrating phosphate-buffered saline (PBS), made anoxic by bubbling with argon, with NaHS in a septum-sealed glass syringe. In order to avoid any trace metals catalyzing the oxidation of H₂S, the metal chelator DTPA (diethylenetriaminepentaacetic acid) was added to the buffer to a final concentration of 50 µM prior to addition of NaHS. Photo-oxidation was prevented by wrapping the solution-containing syringe with aluminum foil. Despite these precautions, the presence of polysulfide in stock solutions cannot be completely excluded.