Search

Oleanolic acid (OA), a natural pentacyclic triterpenoid, has been reported to have several benefits and medicinal properties. However, its protective effects against silica‑induced lung injury and fibrosis remain to be elucidated. The aim of the present study was to investigate the effects of OA on oxidative stress, and the expression of cytokines and collagen in silicotic rats. Male rats were induced by intratracheal instillation of silicosis (250 mg/kg), with the exception of the control group (NS). The rats in the OA group were intragastrically administered with OA (60 mg/kg/d). The rats in the solvent control group were gavaged daily with 0.6% sodium carboxymethyl cellulose (10 ml/kg) solution for 56 consecutive days. The data showed that OA significantly attenuated the extent of silicosis fibrosis by histopathologic analysis of the lung tissues. In addition, oxidative stress activated by silica exposure, as evidenced by increasing of malondialdehyde content, and activities of superoxide dismutase and glutathione peroxidase in the lung, was regulated by treatment with OA. Furthermore, enzyme‑linked immunosorbent assay analysis showed that OA significantly decreased the levels of tumor necrosis factor‑α and transforming growth factor‑β1. Immunohistochemistry analysis showed that OA significantly decreased collagen types I and III. In investigating the mechanisms underlying the action of OA, it was found that OA decreased the level of phosphorylated AKT1, which in turn inactivated the transcriptional of nuclear factor (NF)‑κB in the development and progress of silicosis. In conclusion, these results suggested that the protective effects of OA were due, at least in part, to its anti‑oxidant activity and its ability to decrease the expression of cytokines and collagen by modulating the AKT/NF‑κB pathway.

Objective To investigate the changes of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) and angiotensin converting enzyme (ACE)/angiotensin Ⅱ(Ang Ⅱ)/angiotensin Ⅱtype 1 receptor (AT1R) axis and their impact on the development and progression of silicotic fibrosis. Methods Sixty male Wistar rats were randomly divided into 6 groups (n=10). The rats inhaled dust for 0, 2, 4, 8, 12, and 16 weeks (w) respectively to reproduce silicosis model. HE staining was performed to observe the pathological changes of the lung tissue. The expression of αsmooth muscle actin (α-SMA), collagen Ⅰ(Col Ⅰ), fibronectin (Fn), ACE and AT1R were measured by Western blotting. The levels of Ac-SDKP and Ang Ⅱin lung tissue were detected by ELISA. Results Macrophage infiltration and widened alveolar wall were observed by HE staining in the silicosis 2-w group. Isolated cell lesions which composed of macrophages were seen in silicosis 4-w group. The alveolar wall widened obviously and the number of silicotic nodules increased at 8w and 12w. The fusion of silicotic lesions, interstitial fibrosis and fibrous lesions were observed at 16w. Compared with control group, the expressions of α-SMA, Col Ⅰand Fn protein in silicosis 4-, 8-, 12-, and 16-w group increased gradually. In silicosis 2-, 4-, 8-, 12-, 16-w group, the expressions of ACE, Ang Ⅱand AT1R gradually increased compared to the control. The level of Ac-SDKP in the lung tissue of silicosis 2-w group was significantly higher than that in control group, and then decreased gradually, and significantly lower in the silicosis 12-and 16-w group than the control (P

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a natural tetrapeptide that is released from thymosin β4 by prolyl oligopeptides. It is hydrolyzed by the key enzyme of the renin-angiotensin system, angiotensin-converting enzyme (ACE). The aim of the present study was to investigate the alterations in Ac-SDKP and the ACE/angiotensin II (Ang II)/angiotensin II type 1 (AT1) receptor axis and its impact on the pathogenesis and development of silicotic fibrosis. For in vivo studies, a HOPE MED 8050 exposure control apparatus was used to establish different stages of silicosis in a rat model treated with Ac-SDKP. For in vitro studies, cultured primary lung fibroblasts were induced to differentiate into myofibroblasts by Ang II, and were pretreated with Ac-SDKP and valsartan. The results of the present study revealed that, during silicosis development, ACE/Ang II/AT1 expression in local lung tissues increased, whereas that of Ac-SDKP decreased. Ac-SDKP and the ACE/AT1/Ang II axis were inversely altered in the development of silicotic fibrosis. Ac-SDKP treatment had an anti-fibrotic effect in vivo. Compared with the silicosis group, the expression of α-smooth muscle actin (α-SMA), Collagen (Col) I, Fibronectin (Fn) and AT1 were significantly downregulated, whereas matrix metalloproteinase-1 (MMP-1) expression and the MMP-1/tissue inhibitor of metalloproteinases-1 (TIMP-1) ratio was increased in the Ac-SDKP treatment group. In vitro, pre-treatment with Ac-SDKP or valsartan attenuated the expression of α-SMA, Col I, Fn and AT1 in Ang II-induced fibroblasts. In addition, MMP-1 expression and the MMP-1/TIMP-1 ratio were significantly higher in Ac-SDKP and valsartan pre-treatment groups compared with the Ang II group. In conclusion, the results of the present study suggest that an imbalance between Ac-SDKP and ACE/Ang II/AT1 molecules promotes the development of silicosis and that Ac-SDKP protects against silicotic fibrosis by inhibiting Ang II-induced myofibroblast differentiation and extracellular matrix production.

The induction of vascular cell adhesion molecule-1 (VCAM-1) expression by tumor necrosis factor (TNF)-alpha requires the activation of nuclear factor-kappaB (NF-kappaB) via a process involving the phosphorylation and degradation of its cytoplasmic inhibitor, IkappaBalpha. We have shown that nitric oxide (NO) decreases VCAM-1 expression via inhibition of NF-kappaB activation. To determine how NO inhibits NF-kappaB, we studied the fate of IkappaBalpha following TNF-alpha stimulation in the presence of NO donors S-nitrosoglutathione and sodium nitroprusside. Activation of NF-kappaB by TNF-alpha occurred within 15 min and coincided with rapid degradation of IkappaBalpha. Co-treatment with NO donors did not prevent IkappaBalpha phosphorylation or degradation. However, after 2 h of TNF-alpha stimulation, NO donors inhibited NF-kappaB activation and augmented IkappaBalpha resynthesis and nuclear translocation by 2.5- and 3-fold, respectively. This correlated with a 75% reduction in TNF-alpha-induced VCAM-1 expression. In a time-dependent manner, NO donors alone caused the nuclear translocation of IkappaBalpha. To confirm that NO donors have similar effects as endogenously derived NO, murine macrophage-like cells, RAW264.7, were co-cultured with endothelial cells. Induction of RAW264.7-derived NO inhibited lipopolysaccharide-induced endothelial VCAM-1 expression, which was reversed by the NO synthase inhibitor Nomega-monomethyl-L-arginine. These findings indicate that NO inhibits NF-kappaB activation and VCAM-1 expression by increasing the expression and nuclear translocation of IkappaBalpha.

Atherogenesis involves cellular immune responses and altered vascular smooth muscle cell (SMC) function. Cytokines such as interleukin (IL)-1α and interferon-γ (IFN-γ) may contribute to this process by activating SMC. To determine whether the anti-atherogenic mediator, nitric oxide (•NO), can modulate cytokine-induced SMC activation, we investigated the effects of various •NO-generating compounds on the expression of intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1). Induction of ICAM-1 expression by IL-1α and VCAM-1 expression by IFN-γ was attenuated by •NO donors but not by cGMP analogues. Nuclear run-on assays and transfection studies using various VCAM-1 promoter constructs linked to the chloramphenicol acetyltransferase reporter gene showed that •NO repressed IFN-γ-induced VCAM-1 gene transcription, in part, through inhibition of nuclear factor-κ B (NF-κB). Electrophoretic mobility shift assay revealed that SMC possess basal constitutive NF-κB activity, which was augmented by treatment with IL-1α. In contrast, IFN-γ induced and activated interferon regulatory factor (IRF)-1 but had little effect on basal constitutive NF-κB activity. •NO donors had no inhibitory effect on IRF-1 activation but did inhibit basal and IL-1α-stimulated NF-κB activation. These findings suggest that the induction of ICAM-1 and VCAM-1 expression requires NF-κB activation and that •NO attenuates IFN-γ-induced VCAM-1 expression primarily by inhibiting basal constitutive NF-κB activity in SMC.

To test the hypothesis that nitric oxide (NO) limits endothelial activation, we treated cytokine-stimulated human saphenous vein endothelial cells with several NO donors and assessed their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1). In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55% as determined by cell surface enzyme immunoassays and flow cytometry. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. Nuclear run-on assays, transfection studies using various VCAM-1 promoter reporter gene constructs, and electrophoretic mobility shift assays indicated that NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. We propose that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall.

To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-κB, we stimulated human vascular endothelial cells with tumor necrosis factor-α in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-κB activation by tumor necrosis factor-α. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-κB, suggesting tonic inhibition of NF-κB under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-κB inhibitor, IκBα, by preventing its degradation from NF-κB. NO also increased the mRNA expression of IκBα, but not NF-κB subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the IκBα promoter suggested transcriptional induction of IκBα by NO. We propose that the induction and stabilization of IκBα by NO are important mechanisms by which NO inhibits NF-κB and attenuate atherogenesis.

Oxygen (O2) may regulate pulmonary vascular resistance through changes in endothelial nitric oxide (NO) production. To determine whether constitutive NO synthase (cNOS) is regulated by O2, we assessed cNOS expression and activity in bovine pulmonary artery endothelial cells exposed to different concentrations of O2. In a time-dependent manner, changes in O2 concentration from 95 to 3% produced a progressive decrease in cNOS mRNA and protein levels resulting in 4.8- and 4.3-fold reductions after 24h, respectively. This correlated with changes in cNOS activity as determined by nitrite measurements. Compared with 20% O2, cNOS activity was increased 1.5-fold in 95% O2 and decreased 1.9-fold in 3% O2. A decrease in O2 concentration from 94 to 3% shortened cNOS mRNA half-life from 46 to 24 h and caused a 20-fold repression of cNOS gene transcription. Treatment with cycloheximide produced a threefold increase in cNOS mRNA at all O2 concentrations, suggesting that cNOS mRNA expression is negatively regulated under basal condition. We conclude that O2 upregulates cNOS expression through transcriptional and post-transcriptional mechanisms. A decrease in cNOS activity in the presence of low O2 levels, therefore, may contribute to hypoxia-induced vasoconstriction in the pulmonary circulation.

Macrophage-colony stimulating factor (M-CSF) contributes to atherogenesis by regulating macrophage-derived foam cells in atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits the expression of M-CSF in human vascular endothelial cells independent of guanylyl cyclase activation. The induction of M-CSF mRNA expression by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis factor-alpha (TNF alpha) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and 3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also increased M-CSF expression in control and TNF alpha-stimulated cells. Nuclear run-on assays and transfection studies using M-CSF promoter constructs linked to chloramphenicol acetyltransferase reporter gene indicated that NO repressed M-CSF gene transcription through nuclear factor-kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrated that activation of NF-kappa B by L-NMA, ox-LDL, and TNF alpha was attenuated by GSNO and SNP, but not by glutathione or cGMP analogues. Since the induction of M-CSF expression depends upon NF-kappa B activation, the ability of NO to inhibit NF-kappa B activation and M-CSF expression may contribute to some of NO's antiatherogenic properties.