Antagonistic Effect Of Relaxin On DQ12-induced Pulmonary Fibrosis

Silicosis is one of the most prevalent occupational lung diseases in the world and the most serious occupational diseases in China. It is initiated by inhalation of crystalline silica dusts, pathologically and chronologically characterized by inflammatory cells infiltration, alveolitis, fibroblast proliferation, excessive extracellular matrix deposition, formation of silicotic nodule, and subsequent regional or global pulmonary fibrosis. At present, the understanding to the pathogenesis of silicosis fibrosis is still limited and there is also no effective cure for silicosis.Relaxin (relaxin) was first identified in 1926 and subsequently regarded as a hormone that could remodel excessive extracellular matrix in the female reproductive tract to facilitate parturition. Some recent studies broadened its status from a reproductive hormone to an important part of connective tissue homeostasis, and demonstrated anti-fibrotic effects of exogenous relaxin in non-reproductive tissues including heart, kidney, brain, dermal and lung. In vitro, when applied to human lung fibroblasts, RXL decreased matrix accumulation by inhibiting collagen secretion and/or depositions and stimulating matrix metallopeptidase expression, and inhibited TGF-β-induced collagen and/or fibronectin secretion. In vivo, either relaxin or its receptor gene-knockout mice demonstrated an increase in interstitial collagen in the lung. Furthermore, relaxin was shown to inhibit bleomycin-induced fibrosis in a murine model, which shares some histopathological features of silica-induced pulmonary fibrosis. These studies highlighted relaxin's potential as a drug that inhibits silica-induced pulmonary fibrosis. But so far, the effect of relaxin on silica-induced lung fibrosis is not clear.In the present study, we estaboloshed the in vitro and in vivo models of silicosis, and investigated the effect of endogenous and exogenous relaxin on the models by using the techniques including the hematoxylin-eosin stain, acidic hydrolysis, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical staining zymography and enzyme-linked immunosorbent assay (ELISA). The study is composed of the following three parts:Part I Expression of Relaxin and RXFP1 and Cellular Localization in the Silicotic Process in RatsObjective:To investigate the potential role of relaxin and RXFP1 in the silicotic process in rats.Method:Total of 64 wistar rats were randomized into two groups:control group and silicosis group. After building a animal model of silicosis by intratracheal infusion of silica dust suspension,8 rats from each group were sacrificed at 1,7,14 and 28 d. Histopathological examination was performed for lung tissue stained with hematoxylin-eosin stain. The content of hydroxyproline (HYP) and gene and protein expression of relaxin and RXFP1 in lung tissue were determined by acidic hydrolysis, QRT-PCR and immunohistochemical staining, respectively.Results: 1. The content of HYP in lung tissue of ratsNo changes in the HYP content were observed in both two groups at 1,7,14 d (P>0.05), but the HYP content in model group was significantly higher than control group at 28 d (P< 0.05).2. The results of histopathologyThe normal lung structure was observed in the rats of control group. The lung tissues of rats in silicosis group showed acute inflammation at 1,7 d and fibroblasts hyperplasia, chord-shaped fibrous tissues and fibrotic nodules at 28 d after instillation.3. The results of quantitative real-time polymerase chain reaction (qRT-PCR)The mRNA expressions of Rlnl and Rxfpl were observed in both two groups. Compared with control group, the transcriptional levels of Rlnl were significantly up-regulated at 7 d (P<0.05), went down at 14 d (P>0.05) and decreased markedly at 28 d (P<0.05), and the transcriptional levels of Rxfpl were significantly up-regulated at 1 d (P<0.05), went down at 7 d (P>0.05) and decreased markedly at 28 d (P<0.05) in silicosis group.4. Immunohistochemical results of relaxin and RXFP1The protein expression of relaxin in silicosis group showed a downward trend after rising and located primarily in pulmonary alveolar type I cells and alveolar macrophages. Compared with controls, relaxin protein expression was significantly increased at 7 d (P<0.05) and decreased at 28 d (P<0.05). RXFP1 protein expression was significantly increased at 1 d (P<0.05) and decreased at 28 d (P<0.05) and located primarily in pulmonary alveolar macrophages and fibroblast.Conclusion:The result of HYP content and histochemistry (HE staining) proved a successfull building of silicosis model in rats. The relaxin and RXFP1 expression in lung tissue of rats changed with silicosis development in model group and located primarily in pulmonary alveolar type I cells, alveolar macrophages and pulmonary fibroblast cells, which demonstrate relaxin may affect the silicosis development by these cells.PartⅡEffect of Relaxin on the DQ12-induced mRNA expression in THP-1 cellsObjective:, To investigate the effect of relaxin on DQ12-treated THP-1 cells based on the results from the first part of the study.Method:THP-1 cells were primed to differentiation into macrophages by phorbol esters (PMA) and then treated into different 4 treatment groups including MEM only (control), DQ12 (200μg/ml) only, relaxin (10 ng/ml) only and DQ12 (200μg/ml) plus relaxin (10 ng/ml). At 1 h,3 h,6 h,12 h and 24 h after the treatment, the mRNA expression of RLN2, RXFP1 and TGFB1 genes were detected.Results:1. Effect of PMA on induce and differentiation of the THP-1 CellsAfter incubation with PMA (10 ng/mL) for 48 h, more than 90% of THP-1 cells became adherent to the culture disk. The cells were characterized by spindle-like shape and exhibited the morphological phenotype of macrophage.2. RLN2 mRNA expression of THP-1 CellsCompared with control, the expressions of RLN2 mRNA in THP-1 cells increased when exposed to DQ12 for 3 h and 24 h (P<0.05) and decreased when exposed to exogenous relaxin for 24 h (P<0.05). Compared with DQ12 treatment alone, the expressions of RLN2 mRNA dramatically decreased in THP-1 cells exposed to both DQ12 and relaxin for 3 h and 24 h (P<0.05).3. RXFP1 mRNA expression of THP-1 CellsCompared with control, the expressions of RXFP1 mRNA in THP-1 cells increased when exposed to DQ12 for 24 h (P<0.05) and increased first and then decreased markedly after exposed to endogenous relaxin for 1 h (P<0.05) and for 24 h (P<0.05), respectively.4. TGFB1 mRNA expression of THP-1 CellsCompared with control, the expressions of TGFB1 mRN A in THP-1 cells increased when exposed to DQ12 for 12 h (P<0.05). Compared with DQ12 treatment alone, the expressions of TGFB1 mRNA dramatically decreased in THP-1 cells exposed to both DQ12 and relaxin for 3 h (P<0.05).Conclusion:The DQ12 may induce the mRNA expression of RLN2 and RXFP1 in THP-1 Cells. The exogenous relaxin can make this effect weaker. The results demonstrated that macrophages may be one of the main sources of endogenous relaxin in the silicotic process.PartⅢEffect of Relaxin on the cell function of MRC-5 cellObjective:To further investigate the anti-fibrotic mechanism of exogenous relaxin by building in vitro models of silicosis using THP-1 and MRC-5 cell lines.Method:THP-1 cells were primed to differentiation into macrophages by PMA, and then treated with DQ12 quartz, and the culture supernatants (CS) from THP-1 cells (THP-1 CS) were harvested after 12 h. The MRC-5 cells were divided into four groups including MEM only (control), THP-1 CS only, relaxin (1,10 and 100 ng/ml) only and THP-1 CS plus relaxin (1,10 and 100 ng/ml). At 48 h after the treatment, the proliferation of MRC-5 cells was examined. The collagen type-Ⅰsynthesis, matrix metallopeptidase 2(MMP2) mRNA expression and protein activity were detected by qRT-PCR, Egelatin zymography assayResults:1. Effect of relaxin and THP-1 CS on proliferation of MRC-5 CellsCompared to control, the proliferative activity of MRC-5 cells increased after exposure to THP-1 CS (P<0.01). Compared to THP-1 CS treatment alone, the proliferative activity of MRC-5 cells significantly decrease in MRC-5 cells exposed to THP-1 CS plus relaxin (P<0.01). The treatment of relaxin alone showed no effect on proliferation of MRC-5 cells.2. The results from qRT-PCR detectionThe COL1A1 expression increased in MRC-5 cells exposed to THP-1 CS alone compared with control (P<0.05) and decreased in MRC-5 cells exposed to THP-1 CS plus relaxin compared with THP-1 CS alone (P<0.05). The MMP2 expression increased in MRC-5 cells exposed to relaxin alone compared with control (P<0.05) and decreased in MRC-5 cells exposued to THP-1 CS plus 100-ng/ml relaxin comparing with THP-1 CS treatment alone (P< 0.01).3. The results from gelatin zymography assayThe MMP-2 activity in MRC-5 cells increased when exposed to THP-1 CS alone compared with control (P<0.05) and when exposed to THP-1 CS plus relaxin compared with THP-1 CS alone (P< 0.05).4. The results from ELISACompared with control, the level of collagen type-I in culture supernatants of MRC-5 cells increased when exposed to THP-1 CS alone (P<0.05) and decreased when exposed to THP-1 CS plus relaxin (P< 0.01).Conclusion:The present results showed that relaxin could affect the function of silica-induced, macrophage-mediated pulmonary fibroblasts by the following ways:1) inhibiting cell proliferation; 2) antagonizing collagen synthesis; 3) inducing MMP-2 activation. These effects of relaxin implicate its potential as a new therapeutic option for the treatment of sillicosis.