Influence of the interaction between Ac­SDKP and Ang II on the pathogenesis and development of silicotic fibrosis.

N­acetyl­seryl­aspartyl­lysyl­proline (Ac­SDKP) is a natural tetrapeptide that is released from thymosin ß4 by prolyl oligopeptides. It is hydrolyzed by the key enzyme of the renin­angiotensin system, angiotensin­converting enzyme (ACE). The aim of the present study was to investigate the alterations in Ac­SDKP 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 Ac­SDKP. For in vitro studies, cultured primary lung fibroblasts were induced to differentiate into myofibroblasts by Ang II, and were pretreated with Ac­SDKP 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 Ac­SDKP decreased. Ac­SDKP and the ACE/AT1/Ang II axis were inversely altered in the development of silicotic fibrosis. Ac­SDKP treatment had an anti­fibrotic effect in vivo. Compared with the silicosis group, the expression of α­smooth muscle actin (α­SMA), Collagen (Col) I, Fibronectin (Fn) and AT1 were significantly downregulated, whereas matrix metalloproteinase­1 (MMP­1) expression and the MMP­1/tissue inhibitor of metalloproteinases­1 (TIMP­1) ratio was increased in the Ac­SDKP treatment group. In vitro, pre­treatment with Ac­SDKP or valsartan attenuated the expression of α­SMA, Col I, Fn and AT1 in Ang II­induced fibroblasts. In addition, MMP­1 expression and the MMP­1/TIMP­1 ratio were significantly higher in Ac­SDKP and valsartan pre­treatment groups compared with the Ang II group. In conclusion, the results of the present study suggest that an imbalance between Ac­SDKP and ACE/Ang II/AT1 molecules promotes the development of silicosis and that Ac­SDKP protects against silicotic fibrosis by inhibiting Ang II­induced myofibroblast differentiation and extracellular matrix production.

Protective effects of oleanolic acid on oxidative stress and the expression of cytokines and collagen by the AKT/NF­κB pathway in silicotic rats.

Oleanolic acid (OA), a natural pentacyclic triterpenoid, has been reported to have several benefits and medicinal properties. However, its protective effects against silica­induced 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, enzyme­linked immunosorbent assay analysis showed that OA significantly decreased the levels of tumor necrosis factor­α and transforming growth factor­ß1. 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 anti­oxidant activity and its ability to decrease the expression of cytokines and collagen by modulating the AKT/NF­κB pathway.

[The effect of extracellular signal-regulated kinase 1/2 signaling pathway on the expression of transforming growth factor-beta1 in lung fibroblast activated by silicon dioxide].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (P < 0.05). CONCLUSION: 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.

[Effect of ERK1/2 signal pathway on the proliferation of lung fibroblast activated by SiO2].

OBJECTIVE: 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. METHOD: 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. RESULT: 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 (P < 0.05). 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 (P < 0.05). 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 (P < 0.05). CONCLUSION: 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.

[Effect of p38MAPK on proliferation in human embryonic lung fibroblasts in vitro].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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 (P < 0.05); 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(P < 0.05). CONCLUSIONS: The AM supernatant stimulated by silicon dioxide can accelerate the proliferation in the HELF by activation of p38MAPK signal transduction pathway.