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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.

Inducible nitric oxide (iNO) is produced at sites of vascular inflammation by resident and nonresident vascular wall cells, but its role in the inflammatory process is not known. In this study, we show that a novel function of iNO is to terminate inflammatory processes. We find that iNO produced by murine macrophage-like cells, RAW264.7, can inhibit cytokine-induced endothelial cell activation in a separated and mixed endothelial-RAW264.7 coculture system. Both iNO production and endothelial VCAM-1 expression were induced simultaneously with bacterial LPS and murine-specific IFN-γ. Inhibition of iNO synthase (iNOS) activity with Nω-monomethyl-l-arginine in endothelial-RAW264.7 cocultures, stimulated with murine-specific IFN-γ and LPS, decreased iNO production by 86%, augmented VCAM-1 and iNOS expression in endothelial and RAW264.7 cells, respectively, and increased monocyte adhesion to the endothelial cell surface. Transient transfection studies using various VCAM-1 promoter constructs demonstrated that inhibitory effects of iNO on VCAM-1 gene transcription were mediated, in part, by inhibitory effects of iNO on κB cis-acting elements. Immunofluorescence studies using an Ab to the RelA (p65) subunit of nuclear factor-κB revealed that iNO inhibited the activation of nuclear factor-κB. These studies indicate that iNO attenuates iNOS expression in macrophages and inhibits monocyte adhesion to endothelial cells, and suggest that endogenously derived iNO may be an important autoregulatory inhibitor of vascular inflammation.

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.

To investigate the effect and regulation of extracellular signal-regulated kinase (ERK1/2) signaling pathway on the expression of transforming growth factor-beta1 (TGF-beta1) in human embryonic lung fibroblasts induced by SiO2.Human alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and in the presence or absence of SiO2 (50 ug/ml) exposition for 18h, and then the conditioned supernatants were used to incubate HELF. The expressions of TGF-beta1, of the HELF acted with the conditioned AM supernatant fluid were detected with the immunocytochemistry method after treatment with PD98059 of inhibitor of ERK.The expression of TGF-beta1 in HELF of the SiO2 treatment group (OD value is 0.322 7 +/- 0.023 8) exceed blank group (OD value is 0.163 7 +/- 0.019 6) and AM control group (OD value is 0.240 6 +/- 0.022 5) by the immunocytochemistry method. But the expression of TGF-beta1 had reduction in some extent in the PD98059 intervention group (OD value is 0.271 1 +/- 0.022 9). The values were statistically different (P0.05).ERK inhibitor PD98059 have inhibition effect on the expression of transforming growth factor-beta1 and expression of cytokine of human embryonic lung fibroblasts stimulated by SiO2. The study indicate that the proliferation and collagen production of HELF activated by SiO2 are mediated by ERK/MAPK signal pathway in some extent. PD98059 may antagonizes silica-induced lung fibrosis by inhibiting the expression of transforming growth factor-beta1.

To study the proliferation effect of the AM supernatant incubated activation of p38 mitogen activated protein kinases (p38MAPK) signal transduction pathway in human embryonic lung fibroblasts, and to participate in the development of fibrosis in silicosis.The silicotic alveolar macrophages were collected by bronchoalveolar lavage and incubated in vitro in the DMEM medium containing SiO₂ (50 µg/ml) and DMEM medium without SiO₂ for 18 h. Then the AM supernatant incubated for 18 h was collected. HELFs were isolated by organize paste block method, and incubated with AM supernatants. HELFs were divided into four groups: blank control groups, AM groups, SiO₂ + AM groups, SB203580 + SiO₂ + AM groups. The proliferation in the HELF was detected with MTT method and Flow cytometry.The proliferation in the HELF acted with the conditioned AM supernatant fluid were more than blank control groups, AM groups and SB203580 + SiO₂ + AM groups [average optical density: (0.48 ± 0.03) vs (0.29 ± 0.01), (0.38 ± 0.02), (0.33 ± 0.03)], the values with MTT method were statistically different (P0.05); Proliferous index with flow cytometry in SiO₂ + AM groups (18.12 ± 0.82) was bigger than blank control groups (9.24 ± 0.48), AM groups (14.76 ± 0.43) and SB203580 + SiO₂ + AM groups (11.71 ± 0.70) and the values were statistically different(P0.05).The AM supernatant stimulated by silicon dioxide can accelerate the proliferation in the HELF by activation of p38MAPK signal transduction pathway.

To observe the effect of ERK1/2 signal pathway activated by SiO₂ in the proliferation of human embryonic lung fibroblast mediated by silicotic alveolar macrophages.The alveolar macrophages (AM) harvested from silicotic sufferers by bronchoalveolar lavage (BAL) were interacted with SiO₂ suspension once more. HELF, pretreated with the inhibitor PD98059 (50 µmol/L) for 1 hour, were stimulated by conditional supernatant fluid of silicotic sufferers. The experimentation have been classificated four group: blank group, AM control group, SiO₂ treatment group, PD98059 intervention group. The proliferation activity and expressions of Phospho-ERK1/2 of lung fibroblast activated by AM supernatant fluids of silicotic are detected with the MTT assay, flow cytometry and Western blot method after being pretreatmented with PD98059.The A values of cell proliferation in SiO₂ treatment group and AM control group are 2.6 and 2.0 times that of blank group, in which the difference was statistically significant (P0.05). Comparing with SiO₂ treatment group, the A values of every concentrations of PD98059 intervention group decreased with a dose-response relationship, after 10, 25 and 50 µmol/L PD98059 intervention. The 25 and 50 µmol/L PD98059 intervention group were 72.1% and 48.5% of SiO₂ treatment group, which the difference is statistic (P0.05). The expression of phospho-ERK1/2 in SiO₂ treatment group was up, which appeared in 15 min and apparent activated in 30 min (A value is 0.4653 ± 0.0265), and then still in the higher state afterwards declined after 60 min. In addition to 15 min, the expression of phospho-ERK1/2 protein in SiO₂ treatment group at each time point are 1.25, 1.23, 1.25 times over the same period AM control group respectively, the differences were statistically significant (P0.05).The silicotic supernatant of alveolar macrophages have promote proliferation of HELF and activation of ERK1/2, which may involve in the development of silicosis pathogenesis by ERK1/2 signal pathway.

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.