Hydrogen Sulfide And Propargyl Cysteine Cardiovascular Inflammation Resistance Research

Objectives:Inflammatory reponse plays a pivotal role in cardiovascular disease, such as myocardial ischemia, atherosclerosis. The gasotransmitter hydrogen sulfide (H2S) plays an important role in cardiovascular disease. Our present work investigated whether exogenous H2S exerts anti-inflammatory effects in tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVEC), and then studied the effects and possible mechanisms of exogenous H2S on inflammatory reponse, remodeling, and angiogenesis in myocardial infraction rats, and studied the effects and possible mechanisms of S-propargyl-cysteine (SPRC), a endogenous H2S modulator, on inflammatory reponses in lipopolysaccharide (LPS)-stimulated H9c2cells and in TNF-α-stimulated HUVEC.Methods:1. To explore the effects and mechanisms of sodium hydrosulfide (NaHS, a H2S donor) on TNF-α-induced HUVEC dysfunction, dexamethasone (Dex) and N-acetyl-L-cysteine (NAC) served as positive controls. The effects of NaHS on U937cells adhesion onto TNF-α-activated endothelial cells was measured by adhesion assay. Expression of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), P-selectin, and E-selectin mRNA was analyzed by realtime reverse transcription-polymerase chain reaction (realtime RT-PCR). The expression of ICAM-1, VCAM-1, heme oxygenase-1(HO-1), mitogen-activated protein kinases (MAPK) phosphorylation, IκBα degradation, and phosphorylation and translocation of nuclear factor-KB (NF-κB) p65was detected by Western blot. The translocation of NF-κB p65and intracellular reactive oxygen species (ROS) productin were detected by immunofluorescence.2. Myocardial infarction (MI) was induced by a ligation of the left coronary artery (LAD) in male rats. NaHS or DL-propargylglycine (PAG) was administered via intraperitoneal injection (i.p.) to rats after LAD and was continued afterward. During treatment, oxidative stress (HO-1, inducible nitric oxide synthase [iNOS]), inflammation (CD68+, nuclear factor-KB [NF-κB], MAPK, TNF-α, VCAM-1, and ICAM-1), remodeling (matrix metalloproteinases [MMP]-9, type Ⅰ and type Ⅲ collagen), and neovascularization (vascular endothelial growth factor [VEGF], protein kinase B (Akt), CD34) were analyzed from the peri-infarcted area at admission or/and after treatment period. The LV morphometric features and echocardiographic parameters were also investigated after42days treatment.3. To explore the effects and mechanisms of SPRC on TNF-α-induced HUVEC dysfunction, Dex and NAC served as positive controls. The effects of SPRC on U937cells adhesion onto TNF-α-activated endothelial cells was measured by adhesion assay. The expression of ICAM-1, VCAM-1, cystathionase-γ-lyasis (CSE)(an enzyme that synthesizes H2S), MAPK phosphorylation, IκBα degradation, and phosphorylation and translocation of NF-κB p65was detected by Western blot. The translocation of NF-κB p65and ROS productin were detected by immunofluorescence.4. To investigate the effects of SPRC on LPS-induced inflammatory response in H9c2cardiac myocytes, the expression of imflammatory mediators was measured by RT-PCR or enzyme-linked immunosorbent assay (ELISA). The phosphorylation of Akt, ERK1/2, NF-κB p65, and IκBα degradation were detected by Western blot. Intracellular ROS production was analyzed by immunofluorescence.Results:1. Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and protein expressions of ICAM-1and VCAM-1, mRNA expression of P-selectin and E-selectin as well as U937monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, HO-1, was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.2. At day3, NaHS-treatment reduced the expression of proinflammatory mediators (iNOS, TNF-α, ICAM-1, TLR2and VCAM-1), and inflammatory cells (CD68+) infiltration and upregulated HO-1expression accompany with activation of Akt and inhibition of p38and NF-κB activation. NaHS-treatment significantly increased HO-1, CSE, and VEGF expression at days14,42. NaHS-treatment but not PAG resulted in a decrease in myocardial fibrotic area in association with decrease levels of type Ⅰ, type Ⅲ collagen and MMP-9, improvement cardiac function and an increase capillary density at days42. NaHS downregulated Ang Ⅱ-induced MMP-2、MMP-9、type Ⅰ collagen, and Nox4expression and inhibited Ang Ⅱ-induced p38signaling pathway in primary myocardial fibroblasts in vitro.3. Application of SPRC concentration-dependently suppressed TNF-α-induced expressions of ICAM-1and VCAM-1as well as U937monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of H2S-producing enzyme, CSE, was induced and coincident with the anti-inflammatory action of SPRC. SPRC significantly inhibited TNF-α-induced intracellular ROS production. Furthermore, SPRC attenuated TNF-α-induced JNK1/2phosphorylation, IκBα degradation and phosphorylation of NF-κB p65, accompanied by decreased NF-κB translocation.4. SPRC prevented NF-κB activation assessed by NF-κB p65phosphorylation and IκBα degradation; suppressed LPS-induced extracellular signal-regulated kinase1/2(ERK1/2) phosphorylation and intracellular ROS production. Furthermore, incubation of H9c2cells with SPRC induced phosphorylation of Akt in a time-and concentration-dependent manner. In addition to this, SPRC attenuated LPS-induced mRNA and protein expression of TNF-α, and mRNA expression of ICAM-1and iNOS. The effects of SPRC were abolished by CSE inhibitor, DL-propargylglycine (PAG), SPRC-induced Akt phosphorylation and TNF-α release were also abolished by the phosphoinositide3-kinase (PI3K) inhibitor LY294002. Furthermore, SPRC also increased LPS-induced down-regulation expression of CSE and H2S level in H9c2cells. PAG abolished SPRC-induced up-regulation of H2S level.Conclusion:1. H2S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1and inhibition of NF-κB signaling pathway.2. H2S ameliorates inflammatory responses and contributes to improve LV function and remodeling by inhibiting fibrosis and by promoting neovascularization.3. SPRC has vascular anti-inflammatory and potentially anti-atherosclerotic properties partly through the modulation of endogenous H2S production and inhibition of NF-κB signaling pathway.4. SPRC produced an anti-inflammatory effect in LPS-stimulated H9c2cells partly through the CSE/H2S pathway by impairing IκBα/NF-KB signaling and by activating PI3K/Akt signaling pathway.