Experimental Study Of Curcumin Anti-mouse Pulmonary Fibrosis And Its Mechanism

BackgroundPulmonary fibrosis is a heterogeneous progressive group of disorders characterized by the remodeling of the alveolar architecture, notably the loss of normal alveolar epithelial and endothelial cells and their replacement by activated fibroblasts that induce alterations in the extracellular matrix and excessive deposition of interstitial collagens. This process ultimately results in the irreversible loss of the lung's ability to transfer oxygen into the bloodstream. Until now, anti-inflammatory agents and immune modulators have proved to be minimally effective in limiting alveolitis and modifying the course of the disease, thus underlining the urgent need for novel therapeutic intervention strategies。Lysosomal cysteine proteases have been involved in the turnover or degradation of ECM, They could apply to play a role in the development of lung fibrosis. One of them, cathepsin K, is the most potent mammalian collagenase compared to other cysteine proteases(CathepsinsB,L and S). In a murine model of lung fibrosis induced by bleomycin, these cathepsins was found to be induced in the lung. Recently, it was suggested to exert a protective role against matrix deposition during pulmonary fibrosis, since lungs of Cathepsin K deficient mice accumulated more collagen than wild type animals in response to bleomycin.Curcumin, the yellow pigment from the rhizomes of the Curcuma longa plant commonly known as turmeric, has long been used as a medicinal agent in many Asian countries. This compound has been shown to exhibit a variety of potentially beneficial effects, including antioxidant and anti-inflammatory activities and promotion of wound healing. Studies to date have suggested possible benefits in the prevention or treatment of numerous diseases, including atherosclerosis, cancer, neurodegenerative diseases including Alzheimer's dementia, pancreatitis and rheumatoid arthritis. In particular, curcumin has been shown to inhibit processes essential to development of lung,liver and pancreatic fibrosis. Objective The purpose of this work was to identify the influence of Curcumin on the lysosomal cysteine proteases cathepsins B,K,L,S expression and the potentially important role in the development of pulmonary fibrosis in a mice model induced by the instillation of bleomycin. We also identified the effect of Curcumin on the change of alveolar macrophages activation, expression of TGF-β1 and apoptosis and to find the mechanism of the antifibrotic effect of Curcumin related to cathepsins.Methods & Results1 The effect of curcumin on Lung histopathology and degree of lung fibrosisC57BL/6 female mice (18±3 g) were randomly divided into 4 groups (30 mice in each group):sham-model control, Bleomycin model, Curcumin group. After anesthetized mice were sterilized with ethanol, the trachea was minimally exposed via a ventral midline neck incision. Bleomycin sulfate (0.1ml,0.025U/mouse, bleomycin diluted in sterile saline) or same volume of saline control was slowly instilled intratracheally. Mice were posttreated (one day) with curcumin (200mg·kg-1·d-1) in 0.5%carboxymethylcel lulose via oral gavage, or saline alone. Mice were treated once daily and then euthanized at various time points(7,14 and 28 days) after the Bleomycin instillation. Lungs were removed, Lung tissue was either flashfrozen and stored at liquid nitrogen for further analysis or vacuumized-fixed with 10%formalin in PBS, pH7.4,for at least 24h at room temperature and paraffin-embedded for histological analysis.Lung sections were stained with H&E, Mallory for pathological and fibrosis analysis.Results show that Curcumin can reduce lung inflammation induced by bleomycin, improve lung architecture repaire, inhibit collagen secretion, and downregulate collagen deposition.2 Experimental study on mechanism of curcumin on mice pulmonary fibrosis induced by bleomycin2.1 Effect of curcumin application on CathepsinsB, K,L, S expression in response to bleomycin-induced mice pulmonary fibrosisReal time RT-PCR, Immunohistochemistry and Western blot were used to measure the change of mRNA and protein of lysosomal cysteine proteases Cathepsins B, K, L, S. From the results, we can see that CathepsinsB, K, L, S were significantly upregulated at different time points by Curcumin compare to treated by bleomycin alone. Cathepsins K (14d,P<0.01; 7d,28d,P<0.05) Cathepsins L (7d, P/0.05; 14d,28d, P<0.01); Cathepsins B (7d,P<0.01; 14d,28d, no significan); Cathepsins S (7d,14d, P<0.01; 28d, P<0.05)Results show that Curcumin further upregulated CathepsinB,S and CathepsinK,L expression significantly at early inflammatory stage and later fibrogenic stage respectively, we can conclude that the increased CathepsinK, L may contribute to the degradation of extracellular matrices (ECM) and reduce the collagen deposition.2.2 Curcumin decrease TGF-β1 expression in response to bleomycin-induced mice pulmonary fibrosis.Immunohistochemistry was used to detect TGF-β1 expression in different group. The result show that Curcumin decrease TGF-β1 expression in mice in response to bleomycin challenge at the fibrogenic stage (14d,28d, P<0.05; 7d, no significance)The result demonstrate that Curcumin downregulate the TGF-β1 expression, and rel ieve the suppression of TGF-β1 on Cathepsin K expression, then contribute to the degradation of extracellular matrices (ECM) and reduce the collagen deposition, inhibit the fibrogenic activity of TGF-β1.2.3 Effect of Curcumin on CD68 expression and alveolar macrophages activation in response to bleomycin-induced mice pulmonary fibrosis.Immunohistochemistry result show that Curcumin promote CD68 expression in response to bleomycin challenge at the early inflammatory stage compared with bleomycin application alnoe (7,14d, P<0.01; 28d, P<0.05); suggesting alveolar macrophages activation. Activated alveolar macrophages are also contributed, at least in part, to the upreglated expression of Cathepsins B,K,L,S.2.4 Effect of Curcumin on Apoptosis of mice lung in response to bleomycin challenge.TUNEL result show that Curcumin increased apoptosis significantly in the inflammatory area in response to bleomycin-induced mice pulmonary fibrosis compared with bleomycin application alnoe (7d,14d,P<0.01; 28d, P<0.05); But the caspase-3 expression has not changed at all between the Curcumin application and bleomycin challenge alone; this result demonstrate that caspase-3 activity is not contributor to apoptosis induced by curcumin. Together with the result that Curcumin upregulating the expression Cathepsins B, S at the early inflammatory stage may contribute to apoptosis of lung tissue in the inflammatory area. Conclusion1 Cucumin can downregulate the degree of pulmonary inflammation and fibrosis, and thus prevent pulmonary fibrosis by stimulate the degradation of extracellular matrices (ECM) and reduce the collagen deposition.2 Mechanism of curcumin preventing mice pulmonary fibrosis induced by bleomycin2.1 Overexpression of CathepsinsB,K,L,S induced by Curcumin can reduce lung collagen contents and decrease collagen deposition in response to bleomycin challenge.2.2 Curcumin downregulate the TGF-β1 content increasing in response to bleomycin-induced pulmonary fibrosis, and relieve TGF-βrepressing the expression of Cathepsins B, K, L, S during the development of fibrosis.2.3 Curcumin promote alveolar macrophages activation in response to bleomycin challenge, activated alveolar macrophages engulf inflamatory cells and damaged cells by phagocytosis to reduce inflammatory reaction; activated alveolar macrophages are contributed, at least in part, to the upregulated expression of Cathepsins B,K,L,S.2.4 Curcumin induce apoptosis of lung tissue in the inflammation area,promote the lung structure repairing (or remodeling)by upregulating the expression of CathepsinB, S in response to bleomycin-induced pulmonary fibrosis.Together, this study shows that overexpression of CathepsinsB,K,L,S and Apoptosis induced by Curcumin at least partially protects against lung fibrotic responses and lung resistance upon bleomycin challenge. These data suggest a protective role of Curcumin to antagonize excessive ECM deposition in response to bleomycin-induced pulmonary fibrosis.