Figure 5
Hyperglycemia reduces 3-MST activity without affecting 3-MST protein expression. (A) bEnd3 cells were grown in full DMEM media containing 5.5. mmol/L glucose (low-glucose) or 40 mmol/L glucose (high glucose) for 14 d. Cell lysates were then analyzed by Western blotting. No differences were found in the expression levels of 3-MST and CSE in bEnd3 cells following 14 d of hyperglycemia, while a tendency for increased levels of CBS was noted. The blots show a representative experiment of n = 3 independent determinations conducted on different experimental days. (B) The H2S probe AzMC was employed to measure 3-MST activity (estimated as the ability to convert the substrate 3-MP [10–100 µmol/L] to H2S) in live bEnd3 cells. Cells grown in high glucose conditions, unlike those grown in normal glucose, showed a marked decrease in the intracellular fluorescent signal stimulated by 3-MP. The figures show a representative experiment of n = 3 independent determinations conducted on different experimental days. Colocalization studies with the mitochondrial localization marker Mitotracker show that 3-MP-induced H2S production shows partial mitochondrial localization, consistent with the known presence of 3-MST both in the mitochondria and in the cytoplasm. (C–E) Functional impairment of the 3-MP/3-MST/H2S pathway was further evidenced by the loss of the in vitro proliferative, wound-healing-stimulatory and scratch-wound-closing-stimulatory effect of 3-MP in hyperglycemic bEnd3 cells (experimental conditions as in Figure 2). Note that 3-MP no longer stimulates these responses, while NaHS continues to exert a significant effect (*p < 0.05 versus vehicle; n = 4/group). (E) The presence of LA restored the stimulatory effect of 3-MST on bEnd3 cell migration in high glucose conditions (*p < 0.05 versus vehicle; n = 4/group), but did not affect the effect of NaHS. (F) In STZ-diabetic rats subjected to burn-induced wound healing model, 3-MP did not accelerate wound closure, whereas the combination of LA and 3-MP had a significant effect. Similar to its effect in nondiabetic rats, NaHS remained effective in stimulating burn wound healing in diabetic animals. Burn wound healing in STZ-diabetic animals was studied and analyzed similar to the approach used in nondiabetic rats in Figure 4. (*p < 0.05 versus vehicle; n = 8/group). In all bar graphs, mean ± SEM values are shown.