Effects Of Rosiglitazone On Pulmonary Fibrosis Induced By Bleomycin In Rats And Its Mechanisms

Idiopathic pulmonary fibrosis(IPF) is a progressive and interstitial lung disease of unknown etiology that is characteri- zed with diffuse alveolitis and alveolar structure destruction which finally lead to fibrosis. Diseases characterized by IPF cause significant morbidity and mortality.The pathogenesis of IPF is not yet clarified clear now. Sadly,there are few if any effective therapies.The current therapeutic drugs such as glucocorticoids, immunosuppressive agent or cytotoxic drugs are not satisfactory. Therefore the development of successful strategies to prevent pulmonary fibrosis remains an urgent challenge in the clinic.Thiazolidinedione rosiglitazone(RSG), a potent synthetic agonist of peroxisome proliferator-activated receptor-gamma (PPAR-γ), is involved in metabolic regulation and insulin resistance therapy. Recent studies have demonstrated that activation of PPARgamma and PPAR-γagonists have anti- inflammatory activities and may have potential as antifibrotic agents. RSG is recognized as a highly selective synthetic ligand for the PPAR-γ,but the effects of RSG on pulmonary fibrosis are not reported until now.In the present study,the model of pulmonary fibrosis(PF) was established by a single intratracheal instillation of bleomycin(BLM) in rats.We observed the effects of RSG on BLM-induced PF. Then we also investigate the possible mechanisms about the effects of RSG on the processes of lung fibrosis in rats by deterning mast cells (MCs) and the expression of profibrotic cytokines transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) in lung tissues.This research aims to provide theory foundation for elucidating pathogenic mechanisms of PF and seek novel and more precisely targeted therapies.1 The effect of rosiglitazone on pulmonary fibrosis induced by bleomycin in rats .Aim: The inflammatory response is the initial response following many injury challenges in the progression of lung fibrosis.The response induces an aberrant remodeling of the alveolar epithelial cells and ultimately causes the exaggerated accumulation of extracellular matrix (ECM) components in the interstitial. It has been reported that RSG can reduce acute lung injury in endotoxemic rats. we asked whether RSG is also attenuating lung fibrosis through suppressing lung inflam- mation and reducing collagen deposition. In this study, we extensively investigate the effects of RSG on BLM-induced pulmonary fibrosis in rats determined by observing histomorphology stain and the measurement of hydroxypro- line(HYP) content.Methods: Sixty-two male Sprague-Dawley (SD) rats were randomly divided into two groups: 14-day group and 28-day group. 14-day group was re-divided into 4 subgroups: Control group, B+NS14 group, B+R14 group and R14 group. 28-day group was re-divided into 6 subgroups: Control group, B+NS28 group, B+R14N14 group , B+N14R14 group and R14 group.(6 rats in each group).The model of pulmonary fibrosis was established by a single intratracheal instillation of BLM-A(55 mg·kg^-1)except Control group and R group,which were injection the same amount of normal saline (NS).After intratracheal instillation of BLM for 24 h, rats received RSG (3mg·kg^-1)or NS 2ml by gavage per day on different time respectively. B+NS groups were model groups and treated with NS after instillation of BLM. B+R groups were therapy groups and treated with RSG after instillation of BLM.B+R14N14 group was given RSG from 1st day to 14th day,and then given NS from 14th day to 28th day,whereas B+N14R14 group was treated in the opposed way. B+R28 group was treated with RSG for 28 days. R groups were given RSG 14 days or 28 days after instillation of NS. The control group injected and treated with NS.On the 14^th , and 28^th day after intervention,6 rats of each group were killed and their left lungs were collected to undergo light microscopy, right lungs were taken out to measure the content of HYP. Sections were stained with H&E and Masson's trichrome for collagen identification.Data were entered into a database and analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogeneitic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference (LSD). The statistical significance level was set at P<0.05.Results: As for living state and lung tissue shapes of rats during the study period, RSG therapy groups were better than the model groups. The histopathologic findings showed: the control group displayed normal structure and no pathologic changes under a light microscope.In the model group on 14th day, severe edema, large amounts of inflammatory cells including neutrophils and lymphocytes in both alveoli and interstitium and fibroblasts proliferation were observed.On 28th day after treatment of BLM, fewer inflammatory changes and foci of collagen or large fibrous areas deposition were observed. Furthermore, marked histopathologic changes, such as, collapsed alveolar spaces were seen.The grades of alveolitis and fibrosis on 14th day and 28th day model groups were upregulated, compared with control group(P<0.01). RSG therapy groups had shown more obvious attenuation than model group(P<0.01). Masson stains showed that the area of collagen in model group was increased obviously(P<0.01)and B+R groups displayed less fibrotic lesions compared to it(P <0.01).R groups were similar to the control group.The content of lung HYP of rats increased more significantly in model group than that in control group(P<0.01). B+R14N14, B+N14R14 and R28 group decreased obviously versus model group respectively(P<0.01).These data show that the model of PF was successfully established by a single intratracheal instillation of BLM in rats.RSG (3 mg·kg^-1·d^-1)may have protective effects during the different development of PF induced by BLM.2 The effect of rosiglitazone on MCs in pulmonary fibrosis of rats induced by bleomycin .Aim: Mast cells(MCs) have been traditionally thought to be involved in IgE-associated immediate the allergic response. However, recent studies suggest that MCs play a vital role in pulmonary fibrosis by releasing many cytokines and chemokines.The aim of this study was to investigated the effect of MCs and role of RSG on PF induced by BLM.Methods: Animal groups and lung tissue samples collection in this part were same as that in part 1. MCs were displayed by toluidine blue specific stain.Results: In normal rat lungs there were a few MCs distributed in the loose connective tissue areas close to the vessel and along pleural membrane. In the 14th day model group the number of MCs in the lungs increased significantly (P<0.01), and they distributed densely in the lung interstitium areas and around the vessels. On 28th day, the body of MC became bigger and basophilic stippling filled the intracytoplasm.We also observed degranulation of mast cells.The number of MCs maintained a higher level and was distributed densely in fibrous areas. B+R groups decreased the number of MCs obviously in lung tissues (P<0.01), compared with model group.There were no statistical differences in MCs between R group and Control group.These results demonstrate that the number of MCs in the lung fibrotic tissues induced by BLM increased significantly. Our findings confirm the active participation of mast cells in the progression of pulmonary fibrosis in rats. Administration of RSG could inhibite lung fibrotic progression by preventing infiltration and degranulation of mast cells.3. The effect of rosiglitazone on TGF-β₁,CTGF,α-SMA in pulmonary fibrosis of rats induced by bleomycin .Aim: Cytokines disequilibrium is one of important mechanisms involved in pulmonary fibrosis genesis.It is well recognised that TGF-βplays a crucial role in promoting fibrosis among numerous cytokines.It contributing to the ECM pro- duction and deposition. CTGF, a more recently identified potent profibrotic mediator, acts downstream and in concert with TGF-beta to drive fibrogenesis. Significant upregulation of CTGF has been reported in fibrogenic diseases, including IPF, and is partly responsible for associated excessive fibroblast proliferation and extracellular matrix deposition. TGF-βand CTGF may stimulate the differentiation of myofibroblasts(MFb) during fibrosis. MFbs are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. MFb express alpha-smooth muscle actin (α-SMA) de novo which can be as a marker of MFb or MFb differentiation .It is reported that RSG can inhibit TGF-beta induced pulmonary MFb differentiation and collagen production. We hypothesized that the effect of RSG on PF is associated with TGF-βand CTGF. The study in part 2 has been demonstrated that treatment with RSG inhibits lung MCs,so we asked whether MCs can express TGF-βand CTGF and mediate them to promote fibrogenic effects in a murine lung fibrosis model.In following study, serial sections were cut and stained with immunohistochemistry and toluidine blue.We examined the expression of TGF-β₁,CTGF,α-SMA,MC on BLM-induced lung fibrosis to better elucidate their correlation and interaction.Methods: The expression of TGF-β₁,CTGF,α-SMA and MCs in lung tissues was observed by immunohisto- chemistry and toluidine blue stain. The expression of TGF-β1,CTGF,α-SMA will be analyzed by JEDA-801D image analytical system.Results: TGF-β₁:In Control group and R group,TGF-β₁protein expressed weakly in bronchial epithelial cells and vascular endothelial cell(VEC) in lung tissues. In model group, TGF-β₁ showed a high level expression which distributed in the endochylema of bronchial epithelial cells,Fb,MCs, alveolar macrophage(AM) andVEC(P<0.01),whereas that in B+R groups decreased significantly(P<0.01). R group was similar to control group. CTGF: In the normal lung, expression of CTGF was sparsely distributed. But in model group CTGF expressed highly which localized in bronchial epithelial cells, Fb, MCs, typeâ…¡alveolar epithelial cells, AM and VEC(P<0.01). The B+R therapy groups were similar to model group on the expression place, but expression level decreased significantly compared with i(tP<0.05,P<0.01). R group no difference than control group.α-SMA: In Control group and R group,α-SMA expressed in smooth muscle of bronchial wall and vessel wall whereas lung parenchyma had nowhere to express.In model groupα- SMA expressed highly in alveolar wall and incrassate alveolar septum and increased obviously in comparison with that in control group(P<0.01). B+RSG therapy groups decreased compared with model group(P<0.05 or P<0.01). R group no difference than control group.These data show that MCs express TGF-β₁ and CTGF in BLM-induced rats,suggesting TGF-β₁, CTGF, MFb and MCs may be important mediators in lung fibrogenesis. RSG downregulate the expression of TGF-β₁ and CTGF protein and reduce myofibroblast differentiation in rat lungs induced by BLM.Thus the production of exaggerated accumulation of ECM components is lessened. These findings may be one of the possible mechanisms of RSG attenuat pulmonary fibrosis in different development period .Conclusion 1. The model of pulmonary fibrosis was successfully established by a single intratracheal instillation of BLM in rats.RSG (3 mg·kg^-1·d^-1)may have protective effects during the different development of pulmonary fibrosis induced by BLM.2. The number of MCs in the lung fibrotic tissues induced by BLM increased significantly.Our finding confirm the active participation of MCs in the progression of pulmonary fibrosis in rats. Administration of RSG could inhibite lung fibrotic progression by preventing infiltration and degranulation of mast cells.3. The study shows that MCs express TGF-β₁ and CTGF in BLM-induced rats,suggesting TGF-β₁, CTGF,MFb and MC may be important mediators in lung fibrogenesis.RSG downregulates the expression of TGF-β₁, CTGF protein and reduce myofibroblast differentiation in rat lungs induced by BLM.Thus the production of exaggerated accumulation of ECM components is lessened. These findings may be the possible mechanisms of RSG prevention and cure PF in different development period .