Important Role For Mineralocorticoid Receptor In Bleomycin-induced Pulmonary Fibrosis

Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal,fibrosing interstitial pneumonia of unknown aetiology and limited treatmentoptions, resulting in a median survival of2.5to3.5years. At present, themechanisms involved in the pathogenesis of IPF remain unclear[1]. There hasbeen quite a lot of research focusing on the pathogenesis and treatment of IPFin recent years. Mineralocorticoid receptor (MR) is a classic nuclearsteroid-receptor, the selective ligand is aldosterone. MR are expressed intissues such as kidney, lung, heart, blood vessels, brain, and immune cells[2]. Inrecent years, it has been shown that MR involve macrophage polarization[3]and development of fibrosis[4]. But weather MR contributes to thedevelopment of pulmonary fibrosis and the underlying molecular mechanismare not fully understood yet.In this study, we firstly investigated the expression of MR in the lungs ofbleomycin-induced experimental fibrotic mice both in mRNA and proteinlevels to confirm role of MR in pathogenesis of pulmonary fibrosis; then,studied whether MR antagonist spironolactone treatment influencespulmonary fibrosis; in the end, we explored the molecular mechanisms for theeffects of MR in pulmonary fibrosis by detecting the expression level ofα-SMA (α-smooth muscle actin), MCP-1(Monocyte chemoattractantprotein-1), TGF-β (Transforming growth factor-β).We also aim to establish stable silencing of MR expression through shorthairpin RNA (shRNA)-mediated silencing in murine RAW264.7macrophages,and to investigate proliferation and apoptosis in this cell line.Methods:1)126C57BL/6male mice (6–8weeks old) were randomlydivided into three groups: control group (Control), bleomycin-treated group(Bleo) and bleomycin-spironolactone-treated group (Bleo+Spiro). For the induction of pulmonary fibrosis, mice were administered bleomycin(2.5mg/kg) dissolved in50μL of saline by the intratracheal (i.t.) route or givensterile saline (50μL) as a control. The (Bleo+Spiro) mice were treated with20mg/kg spironolactone or vehicle daily by oral gavage throughout theexperiment. Mice were sacrificed on hours12, day1,2,3,7,14, and28afteradministration of bleomycin and lungs were removed for further analysis.Bronchoalveolar lavage (BAL) was performed in the half mice of each groupby using the sterile saline. After BAL was performed, the lung was fixed in4%buffered formaldehyde for24h. Sections (5-μm) were stained with H&Eand Masson’s trichrome for histopathologic examination. Forimmunofluorescence staining, sections were stained with a mouse monoclonalantibody (mAb) to mice α-SMA to detected the level of α-SMA expression.The lung tissues without BAL were used for mRNA and Western blot analysis.The level of expression of Col1(Collagen1), Col3(Collagen3), TGF-β,MCP-1, and MR mRNA were examined by Real-time PCR. The level ofexpression of MR protein was examined by Western Blot analysis.2) Stable MR silencing in RAW264.7cells was achieved by recombinantshRNA plasmid targeting murine MR gene via liposome mediated transfection,followed by G418selection. Cells were divided into three groups: wide-typegroup (WT), negtive control group (NC), MR-silencing group (shMR). Theefficacy of plasmid transfection and MR silencing in G418resistant cells wasverified by immunofluorescent microcopy and Real-time PCR, respectively.Proliferative activity of MR silencing cell line was analyzed by CCK-8assay.Cell cycle and apoptosis were evaluated by flow cytometry.Statistical analysis was performed using SPSS19.0. Data shown wereexpressed as mean±standard deviation. Differences in measured variablesbetween groups were analyzed using Student’s2-tailed t test or one-wayANOVE analysis. Statistical significance was set at P<0.05.Results:1) The results of H&E and Masson’s trichrome staining revealedclassical pathological stages of bleomycin induced lung fibrosis, includingacute inflammation phase (from12hours to day3post bleomycin), progressive fibrotic phase (day14), as well as late fibrosis phase (day28postbleomycin). Compared with the Bleo-treated group, the inflammationresponse of the lungs of Bleo+Spiro-treated mice was attenuated in the acuteinflammation phase and the degree of fibrosis was significantly reduced on14days after administration of bleomycin (P<0.001).2) The results of immunofluorescence staining for α-SMA showed theexpression of α-SMA was significantly higher in the lung of Bleo-treated miceon14days after administration of bleomycin.3) Real time-PCR analysis showed that the levels of MR mRNAexpression of Bleo-treated mice in the phase of acute inflammation (12h-3d),compared with the control group, were significantly increased (P<0.05), thendecreased gradually. On14days after bleomycin instillation, the expressionlevels of MR were almostly the same as the control group. Then increasedagain and was significantly higher than control group in the late fibrosis phase(28d)(P<0.05).Spironolactone treatment could effectively down regulate geneexpression of MR. The levels of Col1and Col3mRNA expression in the lungsof Bleo+Spiro-treated mice was significantly down-regulated on14daysafter bleomycin instillation (P<0.05).The levels of MCP-1mRNA expression of Bleo-treated mice in the phaseof acute inflammation (12h-3d), were significantly increased compared withthe control group (P<0.05), then decreased gradually. Spironolactonetreatment could effectively down regulate gene expression of MCP-1.On3days after bleomycin instillation, the levels of TGF-β mRNAexpression in the lungs of Bleo+Spiro-treated mice was significantlyincreased compared with Bleo-treated group (P<0.05); while significantlyup-regulated in Bleo-treated group on day14(P<0.05).4) The results of Western-Blot analysis revealed the levels of MR proteinexpression in the phase of acute inflammation (12h-3d) were significantlyincreased in Bleo-treated mice compared with the control group (P<0.05),then decreased gradually. On7days after bleomycin instillation, the expression levels of MR were significantly down-regulated, and almostly thesame as the control group on day14. In the late fibrosis phase (28d) the levelsof MR protein expression increased again and was significantly higher thancontrol group (P<0.05). Spironolactone treatment could effectively downregulate the expression of MR protein.5) MR gene expression could be down-regulated by70%compared withnegative control (NC) plasmid transfection.6) MR-silencing cells exhibited lower proliferative activity comparedwith NC and wide type RAW264.7cells (P<0.05), along with reducedproliferation index being31.0%±1.3%(all P<0.05), compared with wide typecells (37.2%±0.5%) and NC cells (37.5%±1.6%).7) In resting state, apoptosis rate in wide type, NC and MR-silencingcells were2.18%±0.36%,6.65%±0.81%and7.70%±1.34%, respectively, andno statistical difference was observed between NC and MR-silencing cells(P>0.05).Conclusion:1) MR exhibits a dynamic expression pattern inbleomycin-induced pulmonary fibrosis, and involves in the development ofpulmonary fibrosis;2) Using spironolactone to Block MR could significantly attenuate thedegree of bleomycin-induced pulmonary fibrosis in the progressive fibroticphase (14d).3) MR may contribute to the development of pulmonary fibrosis byinducing the production and secretion of MCP-1, which promotes theinflammatory response, in the acute inflammation phase and promoting theexpression of TGF-β in the progressive fibrotic phase. In addition, MR mayplay an important role in the pathogenesis of pulmonary fibrosis also thoughdirectly triggering the differentiation of fibroblasts into myofibroblasts(expression of α-SMA).4) We created stable MR-silenced RAW264.6macrophage cells by usingshRNA-mediated gene silencing. MR gene silencing could inhibit RAW264.6macrophage proliferation, but has no obvious effect on apoptosis in resting state.