Nutraceuticals as potential therapeutics for vesicant-induced pulmonary fibrosis

Abstract

Exposure to vesicants, including sulfur mustard and nitrogen mustard, causes damage to the epithelia of the respiratory tract and the lung. With time, this progresses to chronic disease, most notably, pulmonary fibrosis. The pathogenic process involves persistent inflammation and the release of cytotoxic oxidants, cytokines, chemokines, and profibrotic growth factors, which leads to the collapse of lung architecture, with fibrotic involution of the lung parenchyma. At present, there are no effective treatments available to combat this pathological process. Recently, much interest has focused on nutraceuticals, substances derived from plants, herbs, and fruits, that exert pleiotropic effects on inflammatory cells and parenchymal cells that may be useful in reducing fibrogenesis. Some promising results have been obtained with nutraceuticals in experimental animal models of inflammation-driven fibrosis. This review summarizes the current knowledge on the putative preventive/therapeutic efficacy of nutraceuticals in progressive pulmonary fibrosis, with a focus on their activity against inflammatory reactions and profibrotic cell differentiation.

Nutraceuticals

Nutraceuticals represent a cross between a nutritional supplement and a pharmaceutical; they are substances present in foods or plant extracts that are thought to be capable of providing health benefits and countering the progression of diseases, especially chronic-degenerative diseases connected to oxidative stress and inflammation. Much interest has focused on the active components of nutraceuticals, especially since the observation that a Mediterranean diet rich in plant foods provides significant health benefits relative to the Western diet.^1–3 In Southern European and Mediterranean countries, important dietary sources are nuts, olive oil, fruits, and vegetables.^4,5 Among the most investigated nutraceuticals, at least in the Mediterranean area, are polyphenols characterized by one or more phenolic rings. These are obtained mainly from the skins of red grapes and from the leaves and fruit of olive trees; they are also present in berry fruits, such as blueberries, and in green tea and coffee. Oleuropein is the major phenolic compound in the olive tree, Olea europaea L.; it has demonstrated antioxidant, anti-inflammatory, and cardio- and neuroprotective activities and its consumption has been directly related to increased longevity.^6,7 In 2006, the PREDIMED trial was the first to demonstrate anti-inflammatory effects of the Mediterranean diet supplemented with extra virgin olive oil for 3 months (compared with a low-fat diet) in a group of 727 participants.⁸ The ability of the diet to reduce inflammation has been traced to a derivative secoiridoid species bound to tyrosol, such as oleocanthal, which is similar in structure to ibuprofen. This compound inhibits the synthesis of numerous proinflammatory cytokines and chemokines via downregulation of key regulators of inflammatory pathways, such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, NF-κB, and JNK.⁹

Another polyphenol extensively studied for its beneficial properties is resveratrol, which naturally occurs in foods, such as blueberries and peanuts, as well as grapes and red wine.¹⁰ Resveratrol protects against oxidative stress by promoting the upregulation of mRNA expression of antioxidant enzymes, such as catalase and superoxide dismutase (SOD),¹¹ and downregulation of extracellular signal–regulated kinase (ERK), which is activated by reactive oxygen species (ROS).¹² Resveratrol has potent anti-inflammatory activities, as demonstrated by its ability to inhibit tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6 activity, downregulate high mobility group box 1, and suppress NF-κB and Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways.^13,14

Another substance with nutraceutical properties that has recently aroused considerable interest is curcumin, a spice obtained from Curcuma longa that exerts antioxidative and anti-inflammatory properties through the suppression of numerous cell signaling pathways, including NF-κB, STAT3, Nrf2, ROS, and COX-2.¹⁵ Additionally, curcumin inhibits histone acetyl transferase (HAT) activity, with specificity for the p300/CREB-binding protein, which may also contribute to its anti-inflammatory activity.^16,17 In this regard, the inhibition of HAT by curcumin and several other phytochemicals leads to the suppression of monocyte production of IL-6 and TNF-α.¹⁸ HAT inhibition and IL-6 release have been linked to epithelial-to-mesenchymal transition, a key step in fibrogenesis.¹⁹ Curcumin has been shown to be active against various chronic diseases, including cancer, diabetes, obesity, cardiovascular, pulmonary, neurological, and autoimmune diseases; it also synergizes with other nutraceuticals, such as resveratrol, piperine, catechins, quercetin, and genistein.¹⁵ Curcuminoids also suppress systemic inflammation in patients with chronic pulmonary complications induced by sulfur mustards (SMs).²⁰ This anti-inflammatory effect was due to its inhibitory effects on IL-6, IL-8, TNF-α, TGF-β, substance P, hs-CRP, CGRP, and MCP-1. Curcuminoids were also found to be safe and well tolerated throughout the trial. These and other nutraceuticals may find use in the treatment of the main chronic complications that arise after exposure to mustard vesicants, specifically, pulmonary fibrosis.

Pulmonary fibrosis

Pulmonary fibrosis develops after exposure to drugs such as bleomycin and amiodarone, and following inhalation exposure to mustard vesicants, as well as toxicants such as silica and asbestos.^21–27 The development of fibrosis induced by all these agents depends on the recruitment of immune and inflammatory cells, including macrophages, neutrophils, T lymphocytes, and fibrocytes, to the lung. Repeated injury, abnormal wound healing, and a failure of alveolar re-epithelialization lead to progressive replacement of lung tissue by fibrotic elements originating from mesenchymal stem cells (MSCs) induced to differentiate into myofibroblasts. These cells secrete extracellular matrix proteins, causing scarring and impairing respiratory functions.

A multitude of inflammatory mediators, including ROS and RNS, proteases (e.g., MMPs and TIMPs), and cytokines (e.g., TNF-α, IL-1, chemokines, and growth factors), regulate the fibrogenic process. In fibrotic foci, recruited inflammatory and immune cells and resident macrophages and epithelial cells not only respond to mediators in their microenvironment, but also release these mediators, perpetuating the proinflammatory and/or profibrotic response.^28,29

Cytokines and growth factors play a central role in the development of fibrosis. Their contributions, however, vary with the stage of the fibrogenic process. Thus, while early in the process proinflammatory cytokines such as TNF-α, IL-1, and various chemokines promote inflammation, injury, and oxidative stress, in later stages of fibrosis growth factors such as TGF-β, CTGF, and PDGF become prominent, stimulating fibroblast recruitment, their differentiation into myofibroblasts, and the production of extracellular matrix proteins.^28,29 Of particular importance in fibrogenesis in many tissues is TGF-β. In the lung, it is produced by macrophages, epithelial cells, endothelial cells, fibroblasts, and airway smooth muscle cells in response to reactive oxygen and reactive nitrogen species generated by inflammatory cells.^28–30 Through activation of SMAD signaling, TGF-β stimulates epithelial–mesenchymal transition (EMT), fibroblast proliferation, and the release of collagen and fibronectin.^31,32 TGF-β also stimulates fibroblasts to release CCL2, a potent macrophage chemoattractant, promoting inflammatory cell accumulation at sites of injury.

Dysregulated type 2 immune response mediators involved in wound repair, including IL-4 and IL-13, also contribute to the development of pathological fibrosis.³³ In this connection, it has been reported that IL-4 and IL-13 activate macrophages toward a profibrotic phenotype, which is thought to be key in pulmonary fibrogenesis.^28,29

Mustard vesicant–induced pulmonary fibrosis: potential therapeutics

SM and nitrogen mustard (NM) are cytotoxic vesicants synthesized and stockpiled for use as chemical warfare agents. The major cause of morbidity and mortality following exposure to mustards is pulmonary toxicity.^21,22,24 In rodent models and humans, the initial damage to the upper respiratory tract consists of loss of cilia from the airway epithelium, accumulation of fibrin in the lumen, and focal ulcerations in the upper airways; an accumulation of inflammatory cells at the alveolar level and thickening of the alveolar septa are also observed.^21–24 The injury progresses over time, culminating in the irreversible development of chronic diseases, including chronic obstructive pulmonary disease and pulmonary fibrosis. The main feature of both acute and chronic lung pathology is the presence of inflammatory cells in the lung parenchyma, which release reactive oxygen and nitrogen species, cytokines, chemokines, proteases, and growth factors; these inflammatory mediators promote oxidative stress, tissue injury, and the development of pulmonary fibrosis.^21–23

Several studies have been performed aimed at elucidating mechanisms underlying the development of pulmonary fibrosis following mustard exposure, in order to develop therapeutics capable of blocking the progression of the disease. The focus has mainly been on inflammatory cells and mediators. One mediator of interest as a potential target for therapeutic intervention is the proinflammatory/profibrotic cytokine TNF-α. Derived mainly from macrophages, TNF-α has been shown to promote inflammation and acute injury as well as fibrosis following SM or NM exposure.^21,22 Moreover, in models of mustard vesicant–induced lung injury, inhibition of TNF-α reduced inflammation and oxidative stress and downregulated TGF-β1 expression, a response associated with blunted fibrogenesis.²³

MSC therapy has also been used to treat lung injury induced by SM. MSCs are known to migrate to sites of injury where they function to down regulate oxidative stress and inflammation and to promote tissue repair.³⁴ SM has been reported to suppress mesenchymal cell migration, which is thought to contribute to the persistence of its long-term adverse effects.³⁵ Administration of bone marrow–derived MSCs to mice was found to alleviate SM-induced inflammation and to promote wound repair, providing strong experimental basis for further development as an antifibrotic therapeutic.³⁶

A few nutraceuticals have also been examined as potential therapeutics for treating SM poisoning (Table 1). For example, carvacrol, a monoterpenic phenol and the main component of essential oil of various plants, especially Origanum vulgare (oregano) from the Lamiaceae family, was found to reduce inflammatory cytokines and chemokines and increase anti-inflammatory cytokines in veterans after SM poisoning; this was associated with improved respiratory function.³⁷ Another study employed curcuminoid supplementation to treat 89 male subjects who were suffering from chronic SM–induced pulmonary complications. Improvements in spirometric values and reduced proinflammatory cytokine levels (IL-6, TNF-α, and TGF-β) were obtained, with the conclusion that short-term adjunctive therapy with curcuminoids can suppress systemic inflammation in these patients.²⁰ These preliminary findings suggest that further evaluation of nutraceuticals may lead to the identification of promising drug candidates for treatment of chronic disease, in particular, pulmonary fibrosis, caused by mustard poisoning. This is supported by findings using bleomycin as an experimental model. Results from some of these studies are presented below.

Table 1.

Effects of nutraceuticals on pulmonary fibrosis

Nutraceutical (dose)	Model	Specific target	Response	References
Ecliptae herba ethanol extract (0.625–2.5 mg/kg) plus eclipta saponin A (80 mg/kg)	Male ICR mice; bleomycin (5 mg/kg)	Decreased: hydroxyproline; TGF-α, COX-2, and α-SMA	Decreased: alveolitis and fibrosis	43
Nigella sativa oil (1 mL/kg/day)	Male Wistar rats; bleomycin (2 mg/kg)	Decreased: TGF-β; density of lung fibrocytes	Decreased: inflammation and fibrosis	45
Gallic acid derivative (75, 150, and 300 mg/kg) daily	Male Kunming mice; bleomycin (5 mg/kg)	Inhibition: IL-6, TGF-β/Smad2 pathway Increased: SOD and GSH	Decreased: oxidative stress and lung fibroblast proliferation	46
Polyphenols: Resveratrol (50 mg/kg) Mangiferin (10 mg/kg) Quercetin (10 mg/kg) Dihydroquercetin (10 mg/kg)	Male CD-1 mice; bleomycin (1 mg/kg)	Decreased: iNOS, apoptosis, Bid, and COX-2 Increased: BCL-2	Decreased: edema, inflammation, and loss of body weight	48
Chamomile hydroalcoholic extract (400, 600, 800, 1000, and 1500 mg/kg/day)	Male and female N. MARI rats; bleomycin (7.5 IU/kg)	Decreased: IL-1α, IL-6, TNF-α, and COX-2	Decreased: inflammation	59
Various Chinese herbal formulas (75–500 mg, depending on the administration route)	Rodents; bleomycin	Increased: antioxidant and anti-inflammatory activity	Decreased: inflammation and fibrosis	52
PM014 (50, 100, and 200 mg/kg)	Male C57BL/6 mice; bleomycin (5 mg/kg)	Decreased: TGF-β–induced epithelial–mesenchymal transition and fibroblast activation	Decreased: inflammation and fibrosis	51
Hirsutella sinensis mycelium extracts (1–2% v/v)	Male C57BL/6J mice; bleomycin (4 mg/kg)	Decreased: TGF-β–induced differentiation of lung fibroblasts into myo-fibroblasts	Decreased: NLRP3 inflammasome, P2 × 7R, transcription, and cleavage of IL-1β and IL-18	53
Hirsutella sinensis mycelium	Male C57BL/6J mice; bleomycin (4 mg/kg)	Decreased: phosphorylation mTOR/p70S6R; levels of IL-6 and TNF-α	Decreased inflammation Altered: T cell responses	54
PM014 (0.1, 0.4, and 1.0 mg/mL)	Human alveolar epithelial A549 cells treated with TGF-α (48 h)	Increased: E-cadherin expression Decreased: α-SMA expression; and cell migration	Decreased: epithelial–mesenchymal transition; canonical and noncanonical p38 signaling	51
PM014 (0.1, 0.4, and 1.0 mg/mL)	Primary human lung fibroblasts from idiopathic fibrosis patients	Decreased: TGF-β–induced collagen production; and migration	Decreased: deposition of collagen fibers	51
Hirsutella sinensis mycelium extracts (1–2% v/v)	Human THP-1 macrophages treated with LPS (0.5 mg/mL)	Decreased: IL-1α and IL-18 secretion, and ROS production	Inhibition of NLRP1 inflammasome	60
Hirsutella sinensis mycelium extracts (1–2% v/v)	Fetal human MRC-5 lung fibroblasts treated with TGF-β1 (5 ng/mL) or bleomycin (2.5 μM, 24 h)	Decreased: ROS production Increased: SOD in response to TGF-β	Antioxidant activity	53
Carvacrol (1.2 mg/kg/day, 2 months)	SM-exposed victims (n = 21)	Decreased serum levels of TNF-α, EGF, VEGF, inflammatory cytokines, and chemokines Increased anti-inflammatory cytokines	Improved respiratory symptoms	37
Curcuminoids (500mg TID per oral for 4 weeks)	SM-exposed victims (n = 89) male suffering from chronic SM–induced pulmonary complications	Decreased serum levels of IL-6, IL-8, TNF, MCP-1, and TGF-β	Suppression of systemic inflammation	20

Nutraceuticals as therapeutics in an experimental model of pulmonary fibrosis induced by bleomycin

Bleomycin is an antibiotic with antitumor, antiviral, and antibacterial properties. It is also used in chemotherapy for the treatment of Hodgkin lymphoma and testicular germ-cell tumors.³⁸ However, its use has been limited by dose-dependent development of pneumonia, which can progress to interstitial lung fibrosis.^39,40 As bleomycin-induced fibrosis is inflammation driven much like mustard vesicant–induced fibrosis, it represents an excellent model for studying the effects of nutraceuticals with anti-inflammatory activity.⁴¹ Intratracheal administration of bleomycin to mice causes alveolar epithelial cell damage and inflammation within 7 days. This is followed by thickening of the alveolar septa and the collapse of the alveoli, proliferation of myofibroblasts, EMT, and an accumulation of collagen fibers in the lung parenchyma. Several inflammatory mediators have been shown to contribute to bleomycin-induced fibrosis, including ROS, which can trigger NLRP3 inflammasome activation and the release of IL-1β and IL-18. TGF-β1 is also released in response to bleomycin-induced oxidative stress, which stimulates EMT, myofibroblast activation, and collagen production. This is facilitated by the excessive release of type 2 cytokines, including IL-4 and IL-13.^33,42

A variety of nutraceuticals with anti-inflammatory and antioxidant activity have been tested as therapeutics using the bleomycin model (Table 1). When administered early after bleomycin, many are effective in reducing fibrosis by reducing the production of inflammatory mediators, suppressing oxidative stress, and/or blunting the proliferation of fibroblasts, recruitment of mesenchymal cells, and their transition into myofibroblasts and the deposition of collagen in the lung.²⁵

Ecliptae herba

A medicinal herb, widely used in China, India, Thailand, and Brazil to treat inflammatory lung disease, Ecliptae herba and its component ecliptasaponin A are derived from the aerial part of Eclipta prostrata.⁴³ Both compounds have been reported to decrease collagen within the lungs of bleomycin-treated mice. This is associated with increases in antioxidant enzymes and decreases in levels of COX-2 and TGF-β1.

Nigella sativa

Seeds from this traditional Tunisian herbal medicine have long been used in the Middle and Far East, not only as a spice and a food preservative, but also to treat inflammation. In animals treated with bleomycin, Nigella sativa was found to reduce histopathological alterations in the lung; specifically, decreases in inflammation and collagen were observed. A significant decrease in TGF-β1 within inflammatory infiltrates was also noted.^44,45

Gallic acid

A phenolic acid extracted from Chinese herbs, gallic acid derivatives have been reported to decrease the synthesis of hydroxyproline and collagen in bleomycin-treated mice; expression of IL-6 was also reduced, while the production of antioxidants, such as SOD and glutathione (a free radical scavenger), was increased.⁴⁶ Gallic acid has also been shown to inhibit the TGF-β1/Smad2 pathway, which is linked to EMT. This resulted in decreased collagen and α-SMA in the lung.⁴⁶

Resveratrol

One of several polyphenols derived from red grapes, our group has shown that resveratrol suppresses inflammation by promoting macrophage activation toward an anti-inflammatory phenotype.⁴⁷ Resveratrol also antagonized 7-oxo-cholesterol-triggered proinflammatory signaling in macrophages and prevented the upregulation of TNF-α and IL-6. In addition, it prevented the upregulation of chemokines IL-8, CCL-4, and RANTES and growth factors G-CSF and GM-CSF.⁴⁷ In bleomycin-treated mice, resveratrol, as well as the related polyphenols mangiferin, quercetin, and dihydroquercetin, inhibited inflammatory MAPKs and NF-κB pathways.⁴⁸ This resulted in reduced lung inflammation and edema and decreases in IL-6, TNF-α, IL-1β, COX-2, and collagen. The protective effects of polyphenols were related to a decrease in iNOS, which was associated with reduced fibroblast proliferation.⁴⁹ Resveratrol has also been shown to inhibit TGF-β–induced phosphorylation of ERK in human pulmonary fibroblasts, resulting in reduced cell proliferation.⁵⁰

PM014 extract

Derived from the herbal compound Chung-Sang-Bo-Ha-Tang (CSBHT), PM014 has been widely used for treating lung disorders in traditional Korean medicine. More recently, it has been shown to be effective in reducing fibrosis and other chronic lung diseases. In animals exposed to bleomycin, PM014 suppressed the release of TNF-α, IL-1β, IL-6, TGF-β, and IL-17A from inflammatory cells, especially macrophages.⁵¹ PM014 also blocked the transition of fibroblasts to myofibroblasts and inhibited collagen deposition in the lung. TGF-β1–induced migration of alveolar epithelial cells and fibroblasts was also suppressed. This was shown to be due to the inactivation of Smad-dependent and Smad-independent p38 MAPK signaling. PM014 also blocked TGF-β1–mediated EMT via suppression of transcriptional factors, including Snail and Slug, which are known to be key regulators of this process.⁵¹

Other herbal products

A number of other traditional herbal products have been analyzed for their ability to attenuate various aspects of bleomycin-induced inflammation, oxidative stress, and pulmonary fibrosis in mice.⁵² For example, inflammation, TNF-α, IL-1β, and/or IL-6 levels in lungs of bleomycin-treated mice were reduced by Ginkgo biloba extract, glycosides, and flavonides from Chrysanthemum indicum and polyphenols from grape seed.⁵² Rosmarinus officinalis, Paeonia lactiflora, Rhodiola rosea, or grape seed polyphenols, and Chinese herbs and their active ingredients, including Feitai, Feining, Yupingfeng, Renshen Pingfei, and salvianolic acid, suppressed bleomycin-induced fibroblast proliferation, EMT, the synthesis of TGF-β and/or collagen deposition.⁵² Some of these herbs were found to block TGF-β by suppressing Smad-dependent signaling and/or MAPK signaling.⁵² Bleomycin-induced oxidative stress was also reportedly suppressed by Chrysanthemum indicum, polyphenols extracted from Rhodiola rosea, glycosides, and flavonides, as measured by the presence of oxidizing enzymes, myeloperoxidase, and MDA and antioxidants, such as glutathione and SOD.⁵²

Hirsutella sinensis mycelium

This fungus-derived nutraceutical has been reported to attenuate both inflammation and fibrogenesis induced by bleomycin.⁵³ In the early stages of the disease process, Hirsutella sinensis mycelium blocks NLRP3 inflammasome activation, leading to reduced IL-1β and IL-18 production. Subsequently, it suppressed TGF-β1 production, resulting in reduced differentiation of fibroblasts into myofibroblasts. Hirsutella sinensis mycelium was also found to modulate lymphocyte activity.⁵³ Following bleomycin administration to mice, greater numbers of T_H2 and T_reg cells are observed in the lung, but lower numbers of T_H1 and T_H17 cells. This was associated with overactivation of the mTOR signaling pathway. mTOR overactivation was also observed after stimulation of human epithelial cells with TGF-β1, a response suppressed by Hirsutella sinensis mycelium.⁵⁴

Conclusions and perspectives

From what has been described above, it emerges that the profibrotic factors involved in the metaplastic transformation of the lung parenchyma are different and probably not yet fully understood. What is certain is that it is urgent to identify treatments that restore the morphology and function of the lung. In this context, great interest has focused on natural products that have demonstrated efficacy in experimental models of pulmonary fibrosis. However, these studies are not completely free from bias. Thus, water extracts of plants and mushrooms typically activate immune cells, whereas ethanol extracts inhibit immune cells. Moreover, the evaluation of nutraceutical effectiveness in suppressing fibrosis has mainly been performed in an animal model of fibrosis induced by bleomycin. Further experimental studies using existing models of mustard vesicant–induced pulmonary fibrosis^23,55–58 will be required to more precisely assess their usefulness for treating chronic diseases induced by these chemical threat agents. Going forward, challenges will certainly need to be overcome to move nutraceuticals into advanced development. In addition to human studies, it is essential to ensure that the products are well defined and their biologic activity confirmed prior to clinical evaluation.