ACS14 significantly attenuates elevation of intracellular MG levels caused by MG and high glucose in cultured cells.

<p>Cultured rat aortic vascular smooth muscle cells (VSMCs, A10 cell line) were incubated with methylglyoxal (MG, 30 µM) or high glucose (25 mM) alone or co-incubated with either ACS14 (100 µM), or aspirin (100 µM) or sodium hydrogen sulfide (NaHS, 90 µM) for 3 h or 24 h. MG levels in the cells were measured after derivatizing MG with ortho-phenylenediamine to form 2-methylquinoxaline, which was detected with HPLC. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs.</i> respective control, ^†<i>P</i><0.05 <i>vs.</i> respective MG group or high glucose group.</p

ACS14, aspirin, and sodium hydrogen sulfide, all attenuate the increase in oxidative stress caused by MG and high glucose in cultured cells.

<p>Cultured rat aortic vascular smooth muscle cells (VSMCs, A10 cell line) were incubated with methylglyoxal (MG, 30 µM) (A, C), or high glucose (25 mM) (B), alone or co-incubated with either ACS14 (100 µM), or aspirin (100 µM) or sodium hydrogen sulfide (NaHS, 90 µM) for 24 h. Oxidative stress (mainly peroxynitrite formation) was measured as oxidized dichlorofluorescein (DCF) (A, B). Western blotting was performed to measure NOX4 protein expression (C). *<i>P</i><0.05 <i>vs.</i> respective control, ^†<i>P</i><0.05 and ^††<i>P</i><0.01 <i>vs.</i> respective MG group or high glucose group.</p

Chemical structure of H₂S releasing aspirin, ACS14 [2-acetyloxybenzoic acid 4-(3-thioxo-3<i>H</i>-1,2-dithiol-5-yl)phenyl ester].

<p>Chemical structure of H₂S releasing aspirin, ACS14 [2-acetyloxybenzoic acid 4-(3-thioxo-3<i>H</i>-1,2-dithiol-5-yl)phenyl ester].</p

MG and ACS14 significantly reduce cell viability of cultured vascular smooth muscle cells.

<p>Cultured rat aortic vascular smooth muscle cells (VSMCs, A10 cell line) were incubated with methylglyoxal (MG, 30 µM) or ACS14 (30, 100 or 300 µM), alone or combined, for 3 h. Cell viability was determined with CellTiter 96 AQueous One Solution Cell Proliferation Assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097315#s2" target="_blank">methods</a>. ***<i>P</i><0.001 <i>vs.</i> respective control, ^†††<i>P</i><0.001 <i>vs.</i> MG alone group.</p

ACS14, but not aspirin, causes a significant attenuation of increase in nitrite+nitrate levels caused by MG or high glucose in cultured cells.

<p>Cultured rat aortic vascular smooth muscle cells (VSMCs, A10 cell line) were incubated with methylglyoxal (MG, 30 µM) (A), or high glucose (25 mM) (B, C), alone or co-incubated with either ACS14 (100 µM), or aspirin (100 µM) or sodium hydrogen sulfide (NaHS, 90 µM) for 24 h. Nitrite+nitrate levels in the supernatant were measured with the Griess assay kit (A, B). Expression of iNOS protein was determined with Western blotting (C). *<i>P</i><0.05 <i>vs.</i> respective control, ^†<i>P</i><0.05 and ^††<i>P</i><0.01 <i>vs.</i> respective MG group or high glucose group.</p

Effect of ACS14 on Asp-induced gastric mucosal injury in rats.

<p>ACS14 (1, 5 or 10 mg/kg) was given (i.p.) 30 min before intragastric administration of Asp (200 mg/kg). Representative photographs (A) and group data (B) showing ACS14 significantly attenuated Asp-induced gastric mucosal injury. Data are presented as means ± SE. n = 8. *P<0.05 compared with control; #P<0.05 compared with Asp; †P<0.05 compared with Asp+ACS14 1 mg/kg.</p

Effect of ACS14 on protein expressions of p22^phox, p47^phox, p67^phox and gp91^phox protein expressions of gastric tissue in Asp-treated rats.

<p>Data are presented as means ± SE. n = 4. *P<0.05 compared with control; #P<0.05 compared with Asp; †P<0.05 compared with Asp+ACS14 1 mg/kg.</p

Effect of ACS14 on levels of MDA (A) and GSH (B) and protein expressions of SOD1 (C) and XOD (D) of gastric tissues in Asp-treated rats.

<p>Data are presented as means±SE. n = 4–8. *P<0.05 compared with control; #P<0.05 compared with Asp; †P<0.05 compared with Asp+ACS14 1 mg/kg.</p

Effect of Asp at 200 mg/kg and ACS14 (430mg/kg) on the morphology of gastric mucosa in rats.

<p>ACS14 or Asp was administered to rats 3 h by intragastric administration. Representative photographs (A) and group data (B) showing that Asp, but not ACS14, induced significant gastric mucosal injury. Data are presented as means ± SE. n = 6. * P<0.05 compared with control; # P<0.05 compared with Asp.</p

Effect of ACS84 on oxidative stress induced by 6-OHDA in SH-SY5Y cells.

<p>(A) Dose dependent effect of ACS84 on ROS generation in the 6-OHDA-treated (50 µM) SH-SY5Y cells. Cells were pretreated with ACS84 at different concentrations for 4 h. DCFDAH₂ (10 µM) was given 30 min before the addition of 6-OHDA (50 µM). The results were obtained after the treatment with 6-OHDA for 1 h. (B–C) Effect of ACS84, L-Dopa and NaHS at 10 µM on ROS generation (B) and SOD (C) in SH-SY5Y cells treated with 6-OHDA. SOD activity was measured 4 h after 6-OHDA treatment. Data are presented as mean ± SEM, n = 4–8, ^###<i>P</i><0.001 versus control; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, versus 6-OHDA-treated cells; ^†††<i>P</i><0.001, versus ACS84-treated cells.</p