Biochemical and Biophysical Research Communications 397 (2010) 311–317
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A phosphoinositide 3-kinase-c inhibitor, AS605240 prevents bleomycin-induced pulmonary fibrosis in rats Xin Wei 1, Jing Han 1, Zhi-zhi Chen, Bao-wen Qi, Guang-cheng Wang, Ying-hua Ma, Hao Zheng, You-fu Luo, Yu-quan Wei, Li-juan Chen * State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, China
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Article history: Received 6 May 2010 Available online 26 May 2010 Keywords: Phosphoinositide 3-kinase-c Inflammation Pulmonary fibrosis Bleomycin AS605240
a b s t r a c t Phosphoinositide 3-kinase-c (PI3Kc) has been identified to play the critical roles in inflammatory cells activation and recruitment in multiply inflammatory diseases and it promised to be a prospective target for relevant inflammatory diseases therapy. AS605240, a selective PI3Kc inhibitor, has been proved effective on several inflammatory diseases. In this study, we investigated the protective effect of AS605240 on bleomycin-induced pulmonary fibrosis in rats. Our results showed that orally administration of AS605240 significantly prevented lung inflammation and reduced collagen deposition. AS605240 also inhibited augmented expression of TNF-a and IL-1b induced by bleomycin instillation. Moreover, the mRNA levels of TNF-a and IL-1b in lung were remarkably suppressed. Histological assessment found that AS605240 reduced the expression of TGF-b1 and prevented T lymphocytes infiltration to lung. Phospho-Akt level in inflammatory cells by blocking PI3Kc was down-regulated and the inhibition of Akt phosphorylation was further confirmed by Western blot. Our findings illustrated that AS605240 was effective for preventing pulmonary fibrosis by suppressing inflammatory cells recruitment and production of inflammatory cytokines. These findings also suggest that PI3Kc may be a useful target in treating inflammation diseases and AS605240 may represent a promising novel agent for the future therapy of pulmonary fibrosis. Ó 2010 Published by Elsevier Inc.
1. Introduction Idiopathic pulmonary fibrosis (IPF), which usually results from chronic inflammation, is a progressive and lethal interstitial lung disease. Inflammation, tissue remodeling and repair take place spontaneously in this course [1]. Increased fibroblastic proliferation and deposition of extracellular matrix lead pulmonary functional deficit and eventually failure of respiratory [2]. Clinical investigations suggested that the prognosis of IPF is poor and a median survival is between 2 and 2.8 year [3]. Although the current treatment options for pulmonary fibrosis involve in aspects about corticosteroids and antifibrotic, these possible treatments for pulmonary fibrosis nevertheless have not succeeded in clinical trials [4,5]. Therefore, to develop an effective drug remains an imperative challenge. Abbreviations: IPF, idiopathic pulmonary fibrosis; BALF, bronchoalveolar lavage fluid; BLM, bleomycin; p-Akt, phospho-Akt; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol-(3,4,5)-triphosphate; NF-jB, nuclear factor-kappaB. * Corresponding author. Address: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, 1# Keyuan Road 4, Chengdu, Sichuan 610041, China. Fax: +86 28 85164063. E-mail address:
[email protected] (L.-j. Chen). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Published by Elsevier Inc. doi:10.1016/j.bbrc.2010.05.109
Although the pathogenesis of pulmonary fibrosis remains unclear, it has been widely accepted that abnormal activation of some inflammatory cells and cytokines in the lung play critical roles. In the initiating stage of the disease, many kinds of inflammatory cells, such as macrophages, T lymphocytes, neutrophils and fibroblast are activated and release a variety of cytokines contributing to cell aggregation. Then a lot of inflammatory cytokines such as TNF-a, TGF-b and IL-1b were expressed by these cells. Moreover, these cells and cytokines take responsible for abnormal airway tissue repair, which may lead to lung fibrosis as a result [6,7]. Phosphoinositide 3-kinase-c (PI3Kc), one member of the class IPI3 kinase family, is of great importance in inflammatory cell activation and recruitment. PI3Kc was mostly activated by G-proteincoupled receptors or receptor tyrosine kinase such as the insulin receptor [8]. Once activated, PI3K leads to the formation of PIP3 and consequent phospho-Akt. The interaction of phospho-Akt with PIP3 at the cell membrane regulates downstream targets involving several inflammatory and immune functions, including recruitment of macrophages, activation of neutrophils and T cells. Therefore, blocking the PI3Kc signal pathway is a prospective and innovative therapeutic strategy for addressing inflammatory diseases [9]. Recent studies have shown that proliferation of T cells and production of inflammatory cytokines were inhibited when
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stimulated by Con A in PI3Kc defect mice [10]. This also suggests that using a potent PI3Kc inhibitor to retard PI3Kc pathway may effectively alleviate intensity of inflammation and serve as a promising treatment for relevant diseases. AS605240, a proved PI3Kc inhibitor, inhibits human recombinant PI3Kc, a, b, and d in an ATP-competitive manner and has been reported to suppress joint inflammation in murine rheumatoid arthritis [11]. In addition, other studies found that AS605240 was also effective on treatment of autoimmune diseases including systemic lupus, arteriosclerosis and hepatic injury and fibrosis via inhibiting recruitment of inflammatory cells and suppressing progression of inflammation [12–14]. These findings prompt us to assume that AS605240 may have possible effect on preventing bleomycin-induced pulmonary fibrosis. 2. Methods 2.1. Reagent and animals Specific pathogen free female Sprague–Dawley rats aged 3–4 weeks and with the body weights of 200 ± 30 g were purchased from Western China Experimental Animal Center. During the whole course of experiment, the protocol was approved according to the hospital Animal Care and Use Committee. AS605240 (5-quinoxalin-6-ylmethylene-thiazolidine-2,4-dione) was synthesized by our lab according to the patent (PCT/EP2003/ 050302) with minor modification. The purity and structural identification of AS605240 were analyzed by high performance liquid chromatography and a Q-TOF Premier Mass Spectrometer (Waters Micromass, Milford, MA, USA) and a Bruker Avance 400 NMR system. The final purity achieved more than 99.5%. AS605240 was dissolved in a vehicle (5% Tween-80) (Sigma-Aldrich, USA).
sequent biochemical measurement. Hemocytometer and microscope were used to count total cells and different types of cells through the Wright–Giemsa staining method. 2.5. Histopathological and immunohistochemical examination of lung tissues Lungs were harvest when rats were euthanized on day-28. Paraformaldehyde-phosphate (4%) was perfused in the lungs until lungs were well inflated and then the lungs were immersed in the fix buffer. Lung tissue was embedded with paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) for histological examination. The Ashcroft score was used to determine the extent of fibrotic changes and to indicate severity of pulmonary inflammation [17]. To prevent deviation of observation, this measurement was accomplished by two experienced observers in a blinded fashion. In immunohistochemistry assay, the protocol was carried out as early paper described [18]. The anti-phospho-Akt (dilution 1:100, Cell Signaling Technology, USA) was used as the primary antibody for detecting the level of phospho-Akt (p-Akt) in lung tissue. Besides, anti-TGF-b1 (dilution 1:150, Cell Signaling Technology, USA) and anti-CD4 antibody (dilution 1: 500, Cell Signaling Technology, USA) was used to determine the expression of TGF-b1 and CD4-positive cells in lung tissue, respectively. 2.6. ELISA for cytokines assay in BALF The supernatant of BALF collected on day-14 above was immediately stored at 80 °C. The concentrations of TNF-a and IL-1b in BALF were analyzed by ELISA using commercially available kits (R&D Biotechnology, China) according to the manufacturer’s instructions.
2.2. Animal treatment 2.7. Analysis of hydroxyproline content In this study, rats were bred for one week to affirm body weight and then randomly divided into four experimental groups: (a) control group (rats were given vehicle only); (b) BLM group (rats were induced with BLM); (c) BLM + 25 mg/kg AS605240 group (rats were induced with BLM and then administrated with 25 mg/kg AS605240); (d) BLM + 50 mg/kg AS605240 group (the same protocol as the former group except a different dose of 50 mg/kg AS605240). In addition, five rats were given 50 mg/kg AS605240 only to detect whether AS605240 had any side effect simultaneously as the previous four groups. Rats in (c), (d) and AS605240-given-only group were administered orally 25, 50 and 50 mg/kg AS605240 prepared as above-described by gavage while rats in control group and BLM group were given only equivalent saline at day-1 (the day rats were given BLM was marked as day0). The same dosage was maintained once everyday for 28 days. 2.3. Establishment of pulmonary fibrosis animal models
Paraffin sections of lungs were stained with Masson trichrome to observe accumulation of collagens on day-28. The commercial hydroxyproline detection kit (Nanjing Jiancheng Bioengineering, China) was used to examine hydroxyproline content in lungs, according to the manufacturer’s instructions. The absorbance at 550 nm of each group was read by a Spectramax M5 Microtiter Plate Luminometer (Molecular Devices, USA) and the results were calculated as lg hydroxyproline per mg wet lung weight.
2.8. RT-PCR Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The concentration of RNA was determined and cDNA was generated using total RNA with the Reverse Transcriptase kit (TaKaRa, Japan). The sequences of primers for the cytokine genes are as follows:
2.4. Bronchoalveolar lavage fluid (BALF)
TGF-b1: sense 50 -CTACTACGCCAAAGAAGTCACC-30 and 50 -GAAAGCCCTGTATTCCGTCTC-30 ; IL-1b: sense 50 -TCAAGGCATAACAGGCTCATC-30 and 50 -CCACGGGCAAGACATAGGTAG-30 ; TNF-a: sense 50 -CGGGGGCCACCACGCTCTTC-30 and 50 -GGCAAATCGGCTGACGGTGTG-30 ; GAPDH: sense 50 -GTGGAAGAATGGGAGTTGCTGT-30 sense 50 -GTGCTGAGTATGTCGTGGAGTCT-30 .
Rats were euthanized on day-14, BALF was obtained as previous article described [16]. After centrifugation (1000 g, 10 min, 4 °C), the supernatant of BALF was collected and stored at 80 °C for sub-
PCR amplification was carried out for 30 cycles as follows: denaturation at 94 °C for 1 min, primer annealing for 1 min (the
The protocol was carried out according to previous article with a little modification [15]. Briefly, the experimental rats were anesthetized and instilled intratracheally with 0.2 ml saline containing bleomycin (5 mg/kg) (Nihonkayaku, Osaka, Japan) while normal rats received an intratracheal injection of an equal volume of normal saline instead.
antisense antisense antisense and anti-
X. Wei et al. / Biochemical and Biophysical Research Communications 397 (2010) 311–317
annealing temperatures were 55, 52, 59, and 60 °C for TNF-a, IL-1b, TGF-b1 and GAPDH, respectively), extension at 72 °C for 30 s. 2.9. Western blotting An anti-phospho-Akt antibody (dilution 1:750, Cell Signaling Technology, USA) was used for phospho-Akt determination. AntiAkt (dilution 1:750, Cell Signaling Technology, USA) and b-actin antibody (dilution 1:8000, Sigma-Aldrich) were used for determining the total Akt and b-actin, respectively. 2.10. Statistical analysis SPSS16.0 was used to determine the significance of differences between control and experimental groups. Parametric data were statistically analyzed by the Student’s t test or one-way ANOVA followed by post hoc tests when appropriate. The results were considered significant if P < 0.05. 3. Results 3.1. AS605240 decreases collagen accumulation and improves lung histology As shown in Fig. 1A–E, severe lung alveolitis and injury in BLM group was manifested with interalveolar inflammation, thickening alveolar wall, bronchial erosion and large fibrotic area inconsis-
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tently located in lung tissue. In contrast, lung was effectively protected by AS605240 and interalveolar inflammation and tissue damage was markedly alleviated in rats receiving AS605240. In addition, no obvious difference was observed between control group and the group given only AS605240, indicating that administration of AS605240 was safe and had no side effect on normal lung tissue. Masson staining is regarded as a method for collagen determination in locally defined tissue areas. As shown in Fig. 1F–I, BLM group displayed an increased grade of collagen deposited and large fibrotic areas, compared with the control group. Collagen accumulation was remarkably decreased in two prevented groups, compared with the BLM group. This result was further confirmed by Ashcroft examination (Fig. 1J) and analysis of hydroxyproline content in lung tissue (Fig. 1K). The Ashcroft score on day-28 of BLMinduced group increased to 4.09 ± 0.70 compared with 0.55 ± 0.40 from control group (mean ± SD, P < 0.05). However, Ashcroft scores on day-28 of 25 and 50 mg/kg AS605240-treated groups were 2.64 ± 0.64 (P < 0.05, vs. BLM group) and 2.77 ± 0.58 (P < 0.05, vs. BLM group), respectively. Hydroproxyline content, defined as a representative parameter to indicate grade of collagen deposition. Bleomycin evidently raised the level of hydroxyproline content to approximately 0.74 mg/g in lung tissue, which was nearly 1.8-fold compared to rats receiving vehicle only (P < 0.01, control group vs. BLM group). In sharp contrast, administration of AS605240 significantly reduced hydroxyproline content compared to BLM group (28.3%
Fig. 1. A–E, H&E staining (200 original magnification) of lung tissue. Alveolitis was induced by bleomycin and AS605240 could attenuate the inflammation. The lung physiological architecture of AS605240 only group (E) did not show any difference from normal lungs. F–I, Masson staining (200 original magnification) of lung tissue. The blue color indicates stained collagens. Arrows show collagens at airway. Ashcroft score on day-28 (J) and hydroxyproline content (K). Both fibrotic degree and hydroxyproline content were increased by bleomycin instillation and statistically reduced by AS605240. , P < 0.05, , P < 0.01, vs. BLM group; #, P < 0.05, ##, P < 0.01, vs. control group. Column, mean. Bar, SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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reduction for 25 mg/kg AS605240 + BLM group and 24.3% for 50 mg/kg AS605240 + BLM group). 3.2. AS605240 alleviates bleomycin-induced cell count in BALF and inflammatory infiltration In BLM-induced group, the total cell count and numbers of macrophages, neutrophils and lymphocytes in BALF on day-14 were significantly increased compared with those in rats not exposed to bleomycin (as shown in Fig. 2). Twenty-five and 50 mg/kg AS605240 administration markedly reduced total cell count and numbers of macrophages, neutrophils and lymphocytes to 84.3%, 73.8%, 85.0%, 79.2% and 82.4%, 70.0%, 85.6%, 72.9% of those in BLM group, respectively. Immunohistochemical assay with CD4 antibody revealed a conspicuous decrease of CD4-positve cells in lungs of rats prevented by AS605240 (Fig. 2E). 3.3. AS605240 reduces inflammatory cytokines and down-regulates TNF-a and IL-1b mRNA expressions in bleomycin-induced pulmonary fibrosis Inflammatory cytokines play important roles in the pathogenesis and development of pulmonary fibrosis. Bleomycin instillation significantly increased the level of such relevant inflammatory cytokines as TNF-a (195.3 ± 14.6 pg/ml) and IL-1b (73.4 ± 17.3 pg/ ml) in BALF on day-14 compared with 77.9 ± 9.45 pg/ml (P < 0.05) and 39.8 ± 6.9 pg/ml (P < 0.05) of control group, respectively.
AS605240 significantly reduced the levels of TNF-a and IL-1b in BALF to 132.7 ± 11.2 pg/ml and 49.2 ± 11.3 pg/ml in 25 mg/kg AS605240 + BLM group and 131.3 ± 10.7 and 49.6 ± 8.8 pg/ml in 50 mg/kg AS605240 + BLM group, respectively (Fig. 3). To further elucidate the effect of AS605240 on ameliorating lung inflammation and pulmonary fibrosis, we assessed TNF-a and IL-1b mRNA expressions in the lung. As shown in Fig. 3G, the augmented levels of TNF-a and IL-1b mRNA expression by BLM in lung tissue were remarkably inhibited by AS605240. 3.4. AS605240 inhibits prefibrotic cytokines production in bleomycininduced pulmonary fibrosis TGF-b1, regarded as one of a variety of fibrogenic cytokines in the development of pulmonary fibrosis, was also analyzed through immunohistochemistry and RT-PCR. In Fig. 3A–D, the expression of TGF-b1 was obviously higher in BLM group meanwhile the degree of TGF-b1 expression was alleviated in protected groups by AS605240. Further study examining TGF-b1 mRNA by RT-PCR (Fig. 3G) concurred with the immunohistochemical assay. Bleomycin up-regulated the expression of TGF-b1 and AS605240 administration ameliorated the situation. 3.5. AS605240 inhibits phosphorylation of Akt of inflammatory cells in bleomycin-induced pulmonary fibrosis model In order to explicate the effect of AS605240 on the activation of Akt in bleomycin-induced pulmonary fibrosis, we examined the
Fig. 2. Cell counts in BALF including numbers of total cells (A), neutrophils (B), macrophages (C) and lymphocytes (D) on day-14. , P < 0.05, , P < 0.01, vs. BLM group; #, P < 0.05, ##, P < 0.01, vs. control group. Data in A–D, mean ± SEM. CD4-positive T cell infiltration in lung tissues by immunohistochemistry (E) (400 original magnification). Arrows indicate CD4-positive T cells.
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Fig. 3. Fibrogenic cytokine TGF-b1 expression in lung tissue by immunohistochemical methods (200 original magnification), containing control group (A), BLM group (B), 25 mg/kg AS605240 + BLM group (C), 50 mg/kg AS605240 + BLM group (D). Levels of TNF-a (E) and IL-1b (F) in BALF on day-14 via ELISA and RT-PCR assay about mRNA expressions of IL-1b, TNF-a on day-14 and TGF-b1 (G) on day-28. TGF-b1 was also detected by immunohistochemical staining (arrows show TGF-b1 with yellow). , P < 0.05, , P < 0.01, vs. BLM group; #, P < 0.05, ##, P < 0.01, vs. control group. Column, mean. Bar, SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
phospho-Akt expression in lung tissue. Immunohistochemical analysis (Fig. 4A–D) revealed that bleomycin induced an enhanced phospho-Akt in areas where inflammatory cells recruited. However, up-regulated expression of phospho-Akt induced by bleomycin was decreased by AS605240 prevention. Western blot analysis (Fig. 4E) testified that phosphorylation of Akt in lung tissue from the bleomycin-induced rats potentialized and this enhancement was significantly inhibited by AS605240. 4. Discussion It is well known that pulmonary fibrosis is a chronic inflammation related to proliferation of the fibroblasts, activation of extracellular matrix syntheses, as well as the collagen unusually
increased. Further mechanism remains not clear. However, it has been widely accepted that inflammatory cells recruitment, inflammatory cytokines production and abnormal tissue repair contribute greatly to this inflammation and fibrogenesis process. Moreover, PI3Kc plays an essential role in regulating inflammation procedure. In this study, we illustrated that AS605240, a defined PI3Kc inhibitor, had good preventive function on bleomycin-induced pulmonary fibrosis in rats. AS605240 administration significantly inhibited inflammatory cells accumulation in BALF. Besides, fewer CD4-positive T cells were found to infiltrate into the lung. The inflammatory cytokines levels of TNF-a and IL-1b and the prefibrotic cytokine TGF-b1 expression were also reduced. Down-regulation of Akt phosphorylation in inflammatory cells via interfering PI3K/Akt signaling pathway by AS605240 might be responsible for
Fig. 4. Immunohistochemical staining (400 original magnification) with anti-phospho-Akt. Arrows show phospho-Akt in lung tissue. Western blotting was also used to determine expressions of p-Akt, total Akt and b-actin (E).
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these alleviations. Our findings also demonstrated that AS605240 attenuated accumulation of collagen in lung tissue, which was assumed as a possible subsequent result of inhibiting pulmonary inflammation and lung injury. Although bleomycin-induced pulmonary fibrosis model was doubted whether it well mimic IPF, it has been widely used in researches about pulmonary fibrosis in recent years. More researchers regard this animal model as a good one since it can be easily reproduced and it involves many aspects similar to IPF such as alveolitis in early stage of IPF [19]. Intratracheal administration of bleomycin induces acute alveolitis and interstitial inflammation, defined by the sequential recruitment of leukocytes [20]. The leukocytes such as macrophages, neutrophils and lymphocytes play important roles in response to foreign stimuli and involving in inflammation and tissue remodeling. In previous study, bilirubin could ameliorate bleomycin-induced pulmonary fibrosis through weakening recruitment of inflammatory cells [21]. As could be seen from our study, AS605240 significantly reduced the number of total cells, macrophages, neutrophils and lymphocytes in BALF. It may partly explain the reasons for attenuation of pulmonary inflammation. Furthermore, T lymphocytes activation is the major pathology in inflammation. It was found that the proliferation of T lymphocytes was inhibited in PI3K deficient mice [22] and AS605240 prevent joint inflammation and tissue damage via defective neutrophil migration [23]. In this study, CD4-positive cells were apparently decreased by AS605240. We could infer that blocking the PI3K signaling pathway by AS605240 might suppress proliferation of T lymphocytes and retarded infiltration of inflammatory cells. Therefore, the inhibited leukocytes recruitment, which directly impacted inflammation and tissue repair, might partly account for the preventive effect of AS605240 on bleomycin-induced pulmonary fibrosis. Increased expressions of some inflammatory cytokines in the lung are related with recruitment of inflammatory cells and accumulation of extracellular matrix components. The cytokine of TNFa and IL-1b, derived from inflammatory cells such as macrophages and other cells including endothelial cells, has the function of response and induction to inflammation [24]. These two relevant cytokines have been linked to airway fibrosis because of their ability to regulate fibroblast and matrix production [25]. Early studies revealed that expression of TNF-a and IL-1b was up-regulated in bleomycin-induced animal [26,27]. In this observation, AS605240 conspicuously decreased the levels of TNF-a and IL-1b production and reduced severity of inflammation. Moreover, some compelling reasons were given to believe that TNF-a played an important role in the pathogenesis of pulmonary fibrosis, which mediated production of TGF-b1, one of the crucial prefibrotic cytokines [28,29]. This early result can be one of the explanations for the down-regulation of TGF-b1 and subsequent reduction of hydroxyproline content and collagen accumulation in lung tissue in AS605240 prevented groups. TGF-b1, as a link between inflammation and fibrosis, was considered to promote lung structure changes [30]. Our results also manifested that AS605240 evidently attenuated expression of TGF-b1 in both protein and gene level, and then weakened collagen deposition. We therefore speculated that AS605240 might potently ameliorate pulmonary fibrosis partly because it reduced expression and activation of some inflammatory cytokines and fibrogenic cytokines, further it alleviated multi-interactions among them. PI3K-Akt signaling pathway, regarded as one of the most complicated and critical way to transmitting biological and chemical signals, takes responsibility for regulating numerous bio-functions by activating or deactivating many mediators. Phospho-Akt also modulates some targets relevant to inflammation such as recruitment and activation of leukocytes and other downstreams including the fibrogenesis-related nuclear factor-kappaB (NF-jB). Early
studies about NF-jB showed that when NF-jB was inhibited, pulmonary inflammation severity and fibrotic extent of lung tissue was allayed [31]. Another research regarding to Smad signaling pathway, which was also impacted by PI3K-Akt signaling pathway, discovered that phosphorylation of Smad2 was enhanced and Smad3 was deactivated during development of pulmonary fibrosis [32]. In this study, AS605240 successfully blocked the PI3Kc signaling pathway, reduced the phosphorylation of Akt and thus prevented rats against bleomycin-induced pulmonary fibrosis. These results about NF-jB and Smad mentioned above may provide a possible explication about our observation, since the two downstream targets of PI3K-Akt pathway were probably modulated by decreased p-Akt. However, the detailed mechanism acquired further investigations. Taking together, we have shown for the first time that inhibition of PI3Kc can effectively prevent bleomycin-induced pulmonary fibrosis in rats. It has been proved that PI3Kc as a new target in lung inflammation was able to modulate multiple aspects including leukocytes recruitment, inflammatory cytokines production and tissue repair. PI3Kc inhibitor has already succeeded in addressing several kinds of inflammations or immunological deficient diseases in animals. Although only preventive effect of AS605240 was evaluated, our findings also suggested a potential way to treat pulmonary fibrosis and might promise a clinical strategy on idiopathic pulmonary fibrosis in the future. Acknowledgments This work was supported by grants from National 863 Project of China (2007AA021201) and National Project of China (2009ZX09501-015). References [1] Narayanan Sriram, Srinivasan Kalayarasan, Ganapasam Sudhandiran, Epigallocatechin-3-gallate augments antioxidant activities and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2–Keap1 signaling, Pulm. Pharmacol. Ther. 22 (2009) 221–236. [2] T.J. Gross, G.W. Hunninghake, Idiopathic pulmonary fibrosis, N. Engl. J. Med. 345 (2001) 517–525. [3] Hiroto Matsuoka, Toru Arai, Masahide Mori, Sho Goya, Hiroshi Kida, Hiroshi Morishita, Hiroshi Fujiwara, Isao Tachibana, Tadashi Osaki, Seiji Hayashi, A p38 MAPK inhibitor, FR-167653, ameliorates murine bleomycin-induced pulmonary fibrosis, Am. J. Physiol. Lung Cell. Mol. Physiol. 283 (2002) L103– L112. [4] J.A. Lasky, L.A. Ortiz, Antifibrotic therapy for the treatment of pulmonary fibrosis, Am. J. Med. Sci. 322 (2001) 213–221. [5] D.W. Mapel, J.M. Samet, D.B. Coultas, Corticosteroid and treatment of idiopathic pulmonary fibrosis. Past, present and future, Chest 110 (1996) 1058–1067. [6] P.J. Margetts, P. Bonniaud, L. Linmin, C.M. Hoff, C.J. Holmes, J.A. West-mays, M.M. Kelly, Transient over-expression of TGF-beta-1 induces epithelial mesenchymal transition in the rodent peritoneum, J. Am. Soc. Nephrol. 16 (2005) 425–436. [7] F.S. Martinez, F. Gutierrez, C.S. Sanchez, M.J. Tunon, Quercetin attenuates nuclear factor-kappa activation and nitric oxide production in interleukin1beta-activated rat hepatocytes, J. Nutr. 135 (2005) 1359–1365. [8] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002) 1655–1657. [9] T. Ruckle, M.K. Schwarz, C. Rommel, PI3Kgamma inhibition: towards an ‘aspirin of the 21st century’?, Nat Rev. Drug Discov. 5 (2006) 903–918. [10] T. Sasaki, J. Irie-Sasaki, R.G. Jones, A.J. Oliveira-dos-Santos, W.L. Stanford, B. Bolon, A. Wakeham, A. Itie, D. Bouchard, I. Kozieradzki, N. Joza, T.W. Mak, P.S. Ohashi, A. Suzuki, J.M. Penninger, Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration, Science 287 (2000) 1040–1046. [11] M. Camps, T. Rückle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, C. Ferrandi, C. Chabert, C. Gillieron, B. Francon, T. Martin, D. Gretener, D. Perrin, D. Leroy, P.A. Vitte, E. Hirsch, M.P. Wymann, R. Cirillo, M.K. Schwarz, C. Rommel, Blockade of PI3Kc suppresses joint inflammation and damage in mouse models of rheumatoid arthritis, Nat. Med. 11 (9) (2005) 936–943. [12] D.F. Barber, A. Bartolome, C. Hernandez, J.M. Flores, C. Redondo, C. FernandezArias, M. Camps, T. Ruckle, M.K. Schwarz, S. Rodriguez, A.C. Martinez, D. Balomenos, C. Rommel, A.C. Carrera, PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus, Nat. Med. 11 (2005) 933–935.
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