Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disorder of unknown etiology Cottin et al., 2014Kondoh et al., 1993Raghu et al., 2006. IPF primarily occurs between 60 and 70 years of age, and is slightly more predominant in males Gribbin et al., 2006Raghu et al., 2006. Data from around the world demonstrates that IPF favors no particular race or ethnic group. IPF is considered a complex disease where both genetic and environmental factors are believed to contribute to disease susceptibility Kondoh et al., 1993. The disease appears to be driven by abnormal and/or dysfunctional alveolar epithelial cells (AECs) that promote fibroblast recruitment, proliferation, and differentiation, resulting in scarring of the lung, architectural distortion, and irreversible loss of function King et al., 2011. Exerciseinduced breathlessness and chronic dry cough are the prominent symptoms. The onset of symptoms is slow, but symptoms become progressively worse over time. The initial presentation of breathlessness is commonly attributed to aging, cardiac disease, or emphysema which results in typical delays of diagnosis. At the cellular level, IPF is characterized by alveolar epithelial injury, initiation of inflammatory cascades, exaggerated pro-fibrotic cytokine expression, increased extracellular matrix (ECM) deposition, and the development of fibrotic lesions known as fibroblast “foci” Selman et al., 2001. Injured epithelium could release growth factors, cytokines and matrix metalloproteinase, which caused the activation or proliferation of mesenchymal cell, deposition of extracellular matrix and the accumulation of fibroblasts (Yan et al., 2014). The disease progresses towards chronic restrictive respiratory failure and death Cottin and Cordier, 2012Raghu et al., 2011a. IPF is associated with a median survival of only 3–5 years following diagnosis Collard et al., 2003. In their study, Japanese physicians were the first to describe acute, unexpected deterioration in patients with IPF. This phenomenon has been called the "acute exacerbation" or, more euphemistically, the "terminal complication" of IPF Kondoh et al., 1993.

IPF belongs to a family of lung disorders known as the interstitial lung diseases (ILD) or, more accurately, the diffuse parenchymal lung diseases (DPLD). Within this broad category of diffuse lung diseases, IPF belongs to the subgroup known as idiopathic interstitial pneumonia (IIP). During IPF, airway remodelling occurs which can be defined as changes in the composition, content, and organization of the cellular and molecular constituents of the airway wall (Barratt and Millar, 2014;Kondoh et al., 1993).

The pathogenesis of the disease remains poorly understood, although current paradigms focus on the importance of alveolar epithelial cell injury as a critical initiating event with subsequent dysregulated wound healing and fibrosis, resulting in distortion of the lung architecture. The activation of cell-signaling pathways through tyrosine kinases such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) has been implicated in the pathogenesis of the disease Chaudhary et al., 2007Coward et al., 2010Wollin et al., 2014. IPF not only destroys the normal lung parenchyma but also affects the pulmonary vasculature with aberrant microvascular and macrovascular remodeling (Barratt and Millar, 2014). Previous studies showed that IPF develops from chronic epithelial cell injury and aberrant activation of progressive fibrosis Selman et al., 2001. Therefore, the therapeutic strategy against IPF has shifted from corticosteroids and/or immunosuppressants to antifibrotic agents King et al., 2008Taniguchi et al., 2010.

Current medical therapy for IPF is poorly effective. However, IPF is a progressive, ultimately fatal disorder for which substantive medical therapy is desperately needed. The only care options endorsed by guidelines published in 2011 were pulmonary rehabilitation, long-term oxygen therapy, lung transplantation, and enrolment in a clinical trial Raghu et al., 2011b.

Animal models play an important role in the investigation of diseases, and many models are established to examine pulmonary pathobiology. Chronic diseases such as IPF are more difficult to model, since the etiology and natural history of the disease is unclear and no single trigger is known that is able to induce IPF in animals. Different models of pulmonary fibrosis have been developed over the years. Common methods include radiation damage, instillation of bleomycin, silica or asbestos and transgenic mice or gene transfer employing fibrogenic cytokines. So far, the standard agent for induction of experimental pulmonary fibrosis in animals is bleomycin Moeller et al.,2008.

Bleomycin (bleo) is a glycopeptide-derived antibiotic, isolated from the soil fungusStreptomyces verticillus. It is a chemotherapeutic agent with side effects especially on skin. The most serious complication of bleomycin is pulmonary fibrosis and impaired lung function. It has been suggested that bleomycin induces sensitivity to oxygen toxicity and some studies support the role of the proinflammatory cytokines IL-18 and IL-1β in the mechanism of bleomycin-induced lung injury Hoshino et al., 2009. Animal models are often used to investigate pulmonary fibrosis, and they play an important role in understanding the pathogenesis of this disease. Bleomycin model is the most widely used animal model of pulmonary fibrosis. It is widely used as an inducer in the animal models Manali et al., 2011Moeller et al., 2008. Lung fibrosis induced by bleomycin delivered to animals via different routes has different pattern of foci distributions. Using the bleomycin-induced pulmonary fibrosis model, it has been previously reported that pulmonary inflammation and fibrosis are mediated by secretion of the proinflammatory and pro-fibrotic cytokine IL-1β through Nlrp3 inflammasome activation and IL-1R1/MyD88 signaling Chen et al., 2015Francois et al., 2015Gasse et al., 2007. Fibrosis associated with bleomycin treatment has also been linked to toxic reactive oxygen and nitrogen species produced by infiltrating inflammatory cells Yamazaki et al., 1998. Thus, agents that depress oxidative stress are also of potential clinical value, and could have additional protective effects against bleomycin-induced pulmonary fibrosis.

Plant polyphenols are a class of molecules characterized by the presence of multiple phenol groups in their structural moiety. Over the past several years, polyphenols have been studied for their potential to modulate the production and activity of inflammatory molecules Quideau et al., 2011. Fisetin (3, 7, 3′, 4′- tetrahydroxyflavone) is a flavonol, a structurally distinct chemical substance that belongs to the flavonoid group of polyphenols found in many plants fruits and vegetables, such as strawberries, apples, persimmons, onions and cucumbers. Fisetin inhibits the activity of several pro-inflammatory cytokines, including tumor necrosis factor alpha, interleukin 6, and Nuclear factor kappa B Gupta et al., 2014Sahu et al., 2014. In addition, fisetin is a potent natural anticancer agent.

The polyphenol curcumin is the active ingredient in the herbal remedy and dietary spice turmeric. Chemically, curcumin exhibits keto–enol tautomerism having a predominant keto form in acidic and neutral solutions and stable enol form in alkaline medium (Anand et al., 2007). This vibrant yellow spice, derived from the rhizome of the plantCurcuma longa, has a long history of use in traditional medicines of China and India, where it is used to treat inflammatory diseases, abdominal disorders, and a variety of other ailments (Ammon and Wahl, 1991). Curcumin has been shown to exhibit antioxidant, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activities and thus has a potential against various malignant diseases, diabetes, allergies, arthritis, Alzheimer’s disease, and other chronic illnesses Aggarwal,2007maManolova et al., 2014. Extensive scientific research over the past decade has shown the ability of this compound to modulate multiple cellular targets and hence possesses preventive and therapeutic value against a wide variety of diseases (Anand et al., 2007). Curcumin has been shown to suppress TNF expression in in vitro, in vivo,and human studies. It can suppress TNF expression induced by numerous stimuli and in numerous cell types. Recent work has suggested that curcumin acts as a cancer chemopreventive and chemotherapeutic agent Chuang et al., 2000Goel et al., 2008Hatcher et al., 2008Jiao et al., 2009. Curcumin inhibits activation of nuclear factor κB through blockade of IκB kinase, and inhibits activation of cyclooxygenase 2 (COX2). It also alters activator protein 1 (AP1) complexes and inhibits Akt. In addition to the effects on transcription and cell signaling, curcumin possesses chemical features that may further modulate its chemopreventive activity Deeb et al., 2007Jobin et al., 1999Woo et al., 2003.

In the light of the promising properties and broad spectrum of activities of fistine and curcumin, the aim of our study was to evaluate the beneficial effect of fistine, curcumin and mesoporous carbaon nanoparticle (MCN) loaded fisetin as an anti-inflammatory agents against bleomycin-induced changes in mice with idiopathic pulmonary fibrosis.

Figure 1 showing comparison between control and treated sample with respect of their hydroxyproline concentration. We found that there is 19 fold increase in collagen concentration for bleo treated group, 6 fold increase for bleo+fisetin treated group , 3 fold increase for bleo+fisetin+ MCN treated group and 9 fold increase for bleo+curcumin treated group. Here we observed that bleomycin is highly effective in causing fibrosis in mice as compared to control groups and the palnts flavonoids also showed their antifibrotic action.

The average weights of different groups of mice were significantly different at the 0thday. During the treatment mainly from the day 7 ( Figure 2 ) we observed that the weight of bleo treated groups were significantly reduced. Weight reduced by 45%, 42% and 19 % on 7th ,14th and 21st day respectively as compared with control groups. When we challenged the bleo treated group with flavonoids (curcumin and fisetin) the weight of the mice were significantly increased 39%, 37% and 5% at day 7,14 and 21strespectively for the curcumin treated group; 55%, 33% and 9% at the respective days for fisetin treated groups and 46%, 33% and 14% at the respective days for MCN loaded fisetin groups. Significant weight loss indicates development of fibrosis after treating with bleomycin.

We found that, total cell count in peripheral circulating blood increases 1.7 fold in the bleo treated group as compared to control. We also found that, the count decreases 1.43 fold with fisetin, 1.40 fold with MCN+fisetin, and 1.5 fold with curcumin when compared to only bleo. This indicates 1.3 fold increase with fisetin, and 1.2 fold increase with MCN+fisetin, and 1 fold increase as compared to control treated groups ( Figure 3 ). These findings indicate that after the development of fibrosis during bleomycin treatment the total cell count was greatly increased which was decreased significantly after challenging with the plant flavonoids.

The polymorphonuclear (PMN) cell count was increased by 5.6 % and the mononuclear (MN) cell count by 2% for bleo treated as compared to placebo groups. Both PMN and MN cell counts decreased after treatment with fisetin, curcumin and MCN loaded fisetin. PMN cell count decreased 5% by fisetin treatment and 4% by both curcumin and MCN loaded fisetin treatment, and the MN cell count decreased by 3% for fisetin and 5% by both curcumin and MCN loaded fisetin as compared to only bleo treated samples. Here we found that the inflammatory cell count was greatly increased for bleo treated individuals and effectiveness of fisein was increased after addition of MCN with it, curcumin also showed anti-inflammatory effects ( Figure 4 ).

The clonogenic potential in spleen decreases 1.94 fold in the bleo treated as compared to control. The count decreases 1.94 fold with fisetin, 1.07 fold with MCN+fisetin, and 1.4 fold with curcumin as compared to control. 1.80 fold increase with MCN+fisetin, and 1.38 fold increase with curcumin as compared to only bleo. Bleomycin effectively inhibits the cellular recruitment to the spleen and treatment with fisetin, and curcumin increase the cellular recruitment in spleen. Colony count was also increased in MCN+F treated groups, and it shows a statistical significance as compared both with bleo and bleo + fisetin ( Figure 5a ).

The clonogenic potential in lung decreases 2 fold significantly (p < 0.05) than the untreated control groups after challenge with bleomycin, whereas the total number of lung progenitor cell increase 1.35 fold with fisetin 1.30 fold with MCN loaded fisetin and 1.66 fold with curcumin as compared to the bleo treated groups. This denotes that bleomycin effectively decreasing the clonogenic potential of the lung cells and fisetin, MCN loaded fisetin and curcumin also shows its regenerative effect on it among them curcumin showed the best results ( Figure 5b ).

The clonogenic potential of bone marrow (aspirated from the femur) the site of poiesis decreases 2 fold than the untreated control groups after bleomycin challenge whereas in fisetin treated groups, total cell number in bone marrow increases 1.36, in MCN+Fisetin treated groups, total cell number increases 4 fold and for curcumin treated 1.83 fold when compare to only bleo. This indicates a 126.43 fold decrease with Fisetin, and a 2.82 fold decrease with MCN+Fisetin, as compared to only Ova. This shows that the synthesis of cells in the bone marrow, which decreases after challenge with bleomycin, is increased due to the treatment ( Figure 5c ).

Clonogenic potential of peripheral blood was also decreased (2 fold ) for bleo treated groups, treatment with the plant flavonoids trying to show its regenerative affect against bleomycin, among the 3 groups here MCN loaded fisetin showed the best affect, it increases the clonogenic potential by 2 fold as compared to bleo treated groups, fisetin and curcumin also showed its regenerative affect by increasing the clonogenic potential 1.9 fold and 1.7 fold respectively compared to bleo treated groups ( Figure 5d ).

Figure 6a demonstrated that, after challenge with bleomycin, there was an decrease in the levels of IL-4 (1.06 fold), IL-5 (1.16 fold), IFN-γ (1.01 fold) and TNF- α (1.08 fold) as compared to control except IL-2 (1.06 fold increase), there was an increase in the level of cytokines with fisetin treatment (1.03 fold for IL-2, 1.04 fold for IL-4, 1.11 fold for IL-5, 1.11 fold for IFN-γ and1.03 fold for TNF-α),with curcumin treatment (1.03 fold for IL-2, 1.06 fold for IL-4, 1.14 fold for IL-5, 1.29 fold for IFN-γ and1 fold for TNF-α) and with MCN +F treatment (1.03 fold for IL-2, 1.01 fold for IL-4, 1.04 fold for IL-5, 1.15 fold for IFN-γ and1.15 fold for TNF- α) as compared to the bleo treated samples.

After challenge with bleomycin, there was an decrease in the levels of IL-2 (1.36 fold), IL-4 (1.39 fold), IL-5 (1.04 fold), IFN-γ (1.67 fold) and TNF-α (1.41 fold) as compared to control , there is an increase in the level of cytokines with curcumin treatment (1.31 fold for IL- 2, 1.30 fold for IL-4, 1.45 fold for IL-5, 1.35 fold for IFN-γ and1.17 fold for TNF-α) ,with fisetin treatment (1.29 fold for IL-2, 1.35 fold for IL-4, 1.29 fold for IL-5, 1.46 fold for IFN-γ and1.42 fold for TNF-α) and with MCN +fisetin treatment (1.38 fold for IL-2, 1.41 fold for IL-4, 1.45 fold for IL-5, 1.81 fold for IFN-γ and1.44 fold for TNF-α) as compared to the bleo treated sample ( Figure 6b ).

For the first time, our study presents evidence that fisetin, curcumin and mesoporous carbaon nanoparticle (MCN) loaded fisetin could be a promising therapeutic option against idiopathic pulmonary fibrosis (IPF).

Bleomycin-induced pulmonary fibrosis is a well established disease model for IPF and widely used in the investigation of the efficacy and mechanism of therapeutic candidates Song et al., 2010. IPF is a progressive interstitial pneumonia of unknown etiology, while its pathogenesis is not fully understood Raghu, 2011. The risk of developing IPF is likely due to both host and environmental factors, and their elucidation may lead to improved prevention and treatment strategies Ley and Collard,2013. Elevated levels of TNF-α, a cytokine with inflammatory and fibrogenic properties have been detected in the lungs in patients with IPF. IPF remains a major cause of morbidity and mortality around the world, and there is still a large unmet medical need. There is no definitive approach to the treatment of IPF because evidence for effective medical therapy is still lacking. A better understanding of the basic biology of IPF is crucial to enable the development of novel therapeutic agents for this disease. Animal models supported the concept not only of abnormal vascular remodelling as a pathogenic mechanism in pulmonary fibrosis (PF), with reports of newly formed vascular networks within the fibrotic lung, but also of increased capillary irregularity and dilatation Peao et al., 1994. Progressive fibrosis with loss of normal lung tissue leads to restricted ventilation, impaired gas exchange, respiratory symptoms and exercise limitation, poor quality of life, and ultimately death Ley and Collard, 2013. Bleomycin is a chemotherapeutic drug used clinically for a variety of human malignancies, including lymphoma. It has been reported that administration of a high dose of bleomycin often leads to lethal lung injury and pulmonary fibrosis in human patients, as well as in rodent Hoshino et al., 2009. Therefore, it has been widely used in making pulmonary fibrosis animal models Lee et al., 2014. The bleomycin model has the advantage that it is quite easy to perform, widely accessible and reproducible, and therefore fulfills important criteria expected from a good animal model Mouratis and Aidinis, 2011. Bleomycin inhibits the incorporation of thymidine, causing DNA fragmentation resulting in apoptosis with the release of chemical mediators for recruitment of immune cells. On the other hand, the epidermal atrophy could be related to the effect of bleomycin, which has been shown to cause cessation of the epidermal cell cycle, with induction of epidermal cell apoptosis Juniantito et al., 2013Kandeel and Balaha, 2015. The bleomycin model has contributed tremendously to elucidate the roles of cytokines, growth factors and signaling pathways involved in pulmonary fibrosis. For instance, it has helped to determine transforming growth factor (TGF) β as one of the key factors in the development of pulmonary fibrosis Zhao et al., 2002.

Unfortunately, no pharmacologic therapy exists at this time that has been proven to improve survival. Due to the heterogeneity of IPF’s clinical course, lack of complete understanding of the pathogenesis and infrequency of the disease itself, there is a lack of largescale randomized controlled trials. During the last decade, several clinical trials for IPF have been conducted worldwide to determine an effective treatment regimen for IPF, but the results have been disappointing. Clinical trial failures may arise for many reasons, including disease heterogeneity, lack of readily measurable clinical end points other than overall survival, and, perhaps most of all, a lack of understanding of the underlying molecular mechanisms of the progression of IPF (Camelo et al., 2014). Some clinical trials are presently assessing the utility of novel agents in the treatment of IPF. Most medications are not recommended due to their side effects and lack of proven benefit Kekevian et al., 2014.

Fisetin has recently received attention for its beneficial effects against several diseases. In the past years, fisetin was a subject of research because of its antiproliferative Haddad et al., 2006Khan et al., 2008Suh et al., 2009, apoptotic Suh et al., 2009Sung et al., 2007, and antioxidant Hou et al., 2001 activities. Several studies indicate that fisetin is a promising novel antioxidant. Fisetin has been reported to inhibit human low-density lipoprotein (LDL) oxidation in vitro Myara et al., 1993. It induced quinone oxidoreductase activity in murine hepatoma 1c1c7 cells in a time- and dose-dependent manner, and the induction of activity was associated with increase in mRNA expression. It was found that fisetin prevented LDL from oxidation, in part, through reducing CD36 gene expression in macrophages, a possible effect in ameliorating atherosclerosis Lian et al., 2008. Curcumin is the active ingredient in the traditional herbal remedy and dietary spice turmeric. Research has revealed that curcumin has a surprisingly wide range of beneficial properties, including antiinflammatory, antioxidant, chemopreventive and chemotherapeutic activity. These activities have been demonstrated both in cultured cells and in animal models, and have paved the way for ongoing human clinical trials Hatcher et al., 2008. Previous report showed that curcumin inhibits the production of proinflammatory monocyte/macrophage-derived cytokines in PMA- or LPS-stimulated peripheral blood monocytes and alveolar macrophages (Abe et al., 1999). Clinical trials of curcumin in humans have been promising. Phase I studies demonstrated virtually no toxicity in humans consuming up to 8g curcumin per day for 3 months or a single dose of up to 12g Hsu et al., 2002Lao et al., 2006. Based on the encouraging preclinical and phase I clinical data, several additional human trials have been initiated and are currently enrolling patients. This includes trials testing the activity of curcumin in patients with colon cancer, pancreatic cancer, multiple myeloma, and myelodisplasia Jiao et al., 2009. Nanoparticles have promising applications in medicine Jiao et al., 2014. Recently, targeted and triggered drug delivery systems accompanied by nanoparticle technology have emerged as prominent solutions to the bioavailability of therapeutic agents. The factors affecting the immune response are complex, including particle composition, size, surface chemistry, plasma protein binding, and exposure route. Investigation of the relationship between properties of nanoparticles and systemic immune response is crucial for their application in medicine and other areas.

In our study, we found that bleomycin is highly effective in causing fibrosis in mice as compare to control groups and the plants flavonoids also showed their anti fibrotic action ( Figure 1 ). The average weights of different groups of mice were significantly different at the 0th day, and the weight of bleo treated groups were significantly reduced mainly from the day 7 ( Figure 2 ). We demonstrated that, total cell count in peripheral circulating blood increases 1.7 fold in the bleo treated group as compared to control. The count decreases 1.43 fold with fisetin, 1.40 fold with MCN+fisetin, and 1.5 fold with curcumin as compared to only bleo. This indicates 1.3 fold increase with fisetin, and 1.2 fold increase with MCN+fisetin, and 1 fold increase as compared to control treated groups ( Figure 3 ).These indicates that after the development of fibrosis during bleomycin treatment the total cell count was greatly increased which was decreased significantly after challenging with the plant flavonoids. In this study, the inflammatory cell count was greatly increased for bleo treated individuals and effectiveness of fisein was increased after addition of MCN particles with it, curcumin also showed anti- inflammatory effects ( Figure 4 ). In another experiment, bleomycin effectively inhibits the cellular recruitment to the spleen and treatment with fisetin, and curcumin increases the cellular recruitment in spleen. Colony count was also increased in MCN+fisetin treated groups, and it was statistically significant ( Figure 5a ). Figure 5b demonstrated that, blebleomycin effectively decreasing the clonogenic potential of the lung cells and fisetin, MCN loaded fisetin and curcumin also showed its regenerative effect on it; among them curcumin showed the best results. We also observed the increased level of cytokines with fisetin treatment, with curcumin treatment and with MCN +fisetin treatment as compared to the bleo treated sample ( Figure 6a ). In other experimental assays, fisetin, curcumin and MCN+fisetin showed promising results in bleomycin-induced idiopathic pulmonary fibrosis in mice.

It is important to emphasize that probably no single agent is going to work in this disease and that a combination, including agents fisetin and curcumin will be necessary. Improvement of our knowledge about the biopathological principles of the disease will increase the opportunities of finding new agents.

In conclusion, Idiopathic pulmonary fibrosis is a challenging, terminal disease characterized by progressive dyspnea and cough and the present research suggests that fisetin and curcumin may be a promising therapeutic agent for bleomycin-induced changes in mice with IPF. Additionally, the administration of mesoporous carbaon nanoparticle loaded fisetin enhanced the beneficial effects. This may open up new perspectives for a potential role of these drugs as a molecular target in Idiopathic pulmonary fibrosis.

IPF: Idiopathic pulmonary fibrosis, AEC: alveolar epithelial cell, VEGF: vascular endothelial growth factor, PDGF: platelet derived growth factor, FGF: fibroblast growth factor, ILD: interstitial lung disease, DPLD: diffuse parenchymal lung disease, IIP: idiopathic interstitial pneumonia, BALF: broncho alveolar lavage fluid, MCN: mesoporous carbon nano particle, PB: peripheral blood, BM: bone marrow, TC: total count,DC: differential count, IMDM: iscoves modified dulbecco modified medium, CFU: colony forming unit, BLEO: bleomycin, NIN: national institute of nutrition, RHMP: rodent health monitering programme, COX2: cyclooxygenase 2, AP1: activator protein 1, IL-2: Interleukin 2, IL-4: Interleukin 4, IL-5: Interleukin 5, IFN- γ: Interferon-γ, TNF: Tumour necrosis factor, TGF: transforming growth factor, PMN: polymorphonuclear.