Abstract
Silicosis as an occupational lung disease has been present in our lives for centuries. Research studies have already developed and implemented many animal models to study the pathogenesis and molecular basis of the disease and enabled the search for treatments. As all experimental animal models used to date have their advantages and disadvantages, there is a continuous search for a better model, which will not only accelerate basic research, but also contribute to clinical aspects and drug development. We review here, for the first time, the main animal models developed to date to study silicosis and the unique advantages of the zebrafish model that make it an optimal complement to other models. Among the main advantages of zebrafish for modelling human diseases are its ease of husbandry, low maintenance cost, external fertilisation and development, its transparency from early life, and its amenability to chemical and genetic screening. We discuss the use of zebrafish as a model of silicosis, its similarities to other animal models and the characteristics of patients at molecular and clinical levels, and show the current state of the art of inflammatory and fibrotic zebrafish models that could be used in silicosis research.
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This review discusses the use of zebrafish as a model of silicosis and its similarities to other animal models, as well as the current state of the art of inflammatory and fibrotic zebrafish models that could be used in silicosis research. https://bit.ly/3XgdDgU
Silicosis and silica crystals
Silicosis is an irreversible occupational lung disease caused by intense and prolonged inhalation of silica dust with crystals up to 10 µm in diameter in various industries, such as cutting, crushing or grinding. It belongs to the group of interstitial lung diseases called pneumoconiosis [1]. Crystallised silicon dioxide (SiO2), which is part of silica dust, is biologically active and the resulting deposition of inorganic solid waste in the bronchi, lymph nodes or pulmonary parenchyma is associated with respiratory dysfunction [2]. SiO2 is found in nature in different forms of quartz as a crystalline deposit. It is also one of the most abundant elements in Earth's crust, accounting for 59%, and is the main constituent of over 95% of all rocks [3]. Silica can occur in two forms, namely crystalline and noncrystalline (amorphous) [4]. Amorphous forms are generally considered to have low toxicity, as there are few studies suggesting adverse health effects [4]. Although inhaled silica crystals are extremely toxic to lung tissue, brief or one-time exposure is unlikely to cause significant health problems in humans [5]. Normally, crystalline particles of different sizes are inhaled together, although larger particles can be captured and expelled by natural defences through the mechanisms of coughing, sneezing and mucus production. Unfortunately, smaller crystalline particles can remain in the lung and accumulate without causing immediate discomfort, ultimately leading to disease [6].
Silicosis is one of the oldest known causes of lung disease, characterised by lung dysfunction, persistent pulmonary inflammation, formation of silicotic nodules and irreversible pulmonary fibrosis leading to respiratory failure and eventually death, with more than 12 900 deaths worldwide in 2019 [5, 7]. The prevalence and incidence of this disease have been rising, especially in developing countries, although it is also a high-profile occupational health problem in developed countries [8, 9]. The duration and intensity of exposure to crystalline silica dust are not the only determinants of its pathogenicity; host factors may also influence the susceptibility of an individual. Smaller particle size may increase the fibrogenicity of the dust. Depending on the severity of the disease, its onset and its speed of progression, silicosis is classified into chronic silicosis, accelerated silicosis, complicated silicosis and acute silicosis. The most common form is chronic silicosis, which occurs after 20 years of exposure to silica dust and is characterised by the presence of silicotic nodules that first develop in the hilar lymph nodes and then in the lungs, reaching 3 mm in diameter [10]. Each nodule has a well-demarcated central area of whorled hyalinised collagen fibres, with aggregates of dust-laden macrophages at the periphery. Over time, these lung nodules become confluent, erasing the intervening normal lung parenchyma and resulting in progressive massive fibrosis.
Accelerated silicosis occurs after 10 years of silica dust exposure and, although the changes are similar to those present in chronic silicosis, the clinical features appear sooner and the nodules develop earlier and are more cellular than fibrotic [11]. Finally, acute silicosis is characterised by different pathology from chronic silicosis, as it shows severe alveolitis and alveolar proteinosis. In addition, alveolar architecture may be preserved in this case, but with minimal fibrosis and rare small silicotic nodules [12, 13]. Patients with acute silicosis may become disabled within months of exposure.
In the early stages of the disease there may be no symptoms, meaning that the patient may be asymptomatic, with no pain and normal spirometric test results, but in the more advanced form of silicosis the person may tire easily with any kind of effort due to breathing difficulties and these symptoms may not appear until years after exposure. Over time, silica dust causes fibrous alterations in the lungs, decreasing respiratory capacity and loss of lung elasticity [14]. If the disease reaches the progressive massive fibrosis stage, exertional dyspnoea may occur [15].
The association between silicosis and tuberculosis has been known for a long time and patients with silicosis show a susceptibility to mycobacterial infections, called silicotuberculosis. The threefold increased risk of incidence is still not well understood. A possible reason may be damage to alveolar macrophages caused by silica crystals, which leads to inhibition of their ability to kill mycobacteria. Even prolonged exposure of silica, without causing silicosis, similarly increases the risk of tuberculosis [16, 17]. In addition to higher susceptibility to mycobacterial infections, patients with silicosis may present chronic bronchitis and airflow limitation, nontuberculous Mycobacterium infection, fungal lung infection, compensatory emphysema and pneumothorax as a pulmonary complication [18]. In addition, an association between silicosis and some autoimmune diseases, such as nephritis, scleroderma and systemic lupus erythematosus, especially in acute or accelerated silicosis, has been demonstrated [19]. Finally, in 1996, the International Agency for Research on Cancer classified silica crystals as a group 1 carcinogen with a strong potential to cause lung cancer in humans [20, 21].
To be diagnosed with silicosis, three key elements must be met. Initially, patient history should reveal exposure to enough silica dust to cause the disease. Next, characteristic radiological features such as chest imaging are necessary to reveal findings compatible with silicosis, usually by radiography or high-resolution computed tomography. There are standard approaches to the recognition and description of chest radiographic changes used in clinical practice. A widely accepted scheme was published by the International Labour Office, which provides a stepwise method for assessment and description of the shape, size, location and profusion of opacities that may have resulted from dust exposure; the patient's radiographs are classified after comparison with standard radiographs [22]. Finally, other conditions such as tuberculosis, sarcoidosis and histoplasmosis need to be excluded in order to determine that there are no other underlying diseases that are more likely to be causing the abnormalities [23]. Lung biopsy is rarely necessary but may be required to distinguish between progressive massive fibrosis and lung cancer. Physical examination adds nothing new but is a standard procedure. Although spirometry has most often been used as the method of first choice to assess pulmonary alterations in patients exposed to silica, it has limited sensitivity in detecting abnormalities before extensive damage occurs [24]. In addition, assessment of alveolar silica crystal load in bronchoalveolar lavage fluid may aid diagnosis.
Apart from the existing direct diagnosis of the disease, the validation of biomarkers by assessing their ability to indicate exposure, effect, disease or susceptibility has been proposed [25]. A large number of biomarkers in plasma or urine with potential to detect silicosis lung disease have already been investigated, including, among others, club cell protein 16 [26], heme oxygenase 1 [27], interleukin-6 [28], copper and zinc [29], selenium [30], and nephonectin [31]. However, it has not yet been possible to establish a single biomarker with the best potential, although it is very clear that there is a need to identify biomarkers to understand the pathogenesis of silicosis and the biological mechanisms underlying its progression.
Recently, exhaled breath condensate (EBC) has been used to assess the respiratory health of silicosis patients. The EBC is collected and then analysed for volatile and nonvolatile macromolecules, including oxidative stress, inflammatory derived biomarkers and other organic compounds [32, 33]. Interestingly, it has been shown that crystalline silica particles can be detected in the EBC of exposed patients [34]. EBC assessment is a noninvasive and highly accurate technique, making it an attractive option for the early detection of silicosis.
Treatment for silicosis
As silicosis is a chronic disease, there is no cure and once the damage is done it cannot be reversed [15]. Currently, the treatment options available are very limited and focus solely on relieving symptoms, preventing further progress of the disease and improving general condition, as well as reducing the risks of associated disorders. These include cessation of exposure to silica dust, cough suppressants, antibiotics for bacterial lung infection, chest physiotherapy to aid bronchial drainage of mucus, oxygen administration to treat hypoxaemia, bronchodilators to facilitate breathing and many others. Unfortunately, to date, pharmacological approaches have not shown a beneficial effect in halting or reversing the progression of fibrosis. The antifibrotic drugs pirfenidone and nintedanib, both currently in phase 2 of clinical trials for silicosis, are used in other fibrotic lung diseases such as idiopathic pulmonary fibrosis and are known to be effective in a wide range of progressive fibrotic lung diseases, by downregulating the production of growth factors and procollagens I and II, or inhibiting both nonreceptor tyrosine kinases (nRTKs) and receptor tyrosine kinases (RTKs), respectively [35–38]. Whole-lung lavage to remove retained dust is being investigated as a therapy for people with moderate-to-severe silicosis [39, 40]. In addition, lung transplantation, which essentially involves replacing damaged lung tissue, may be the most effective treatment. It has already been performed in some patients with advanced severe silicosis, but it is a limited resource. It is associated with serious risks inherent to lung transplant surgery, availability of ideal donor match, the consequences of long-term immunosuppression as well as the high cost of the whole procedure [41].
Several novel experimental approaches have been explored for pulmonary silicosis. Most of them consist of suppressing inflammasome activation, reducing oxidative stress by antioxidants, limiting caspase-1-driven inflammation, restoring the function of the autophagy–lysosomal system or blocking pro-inflammatory cytokines or pro-fibrotic factors [41, 42]. In addition, treatment with microRNA [43, 44], mesenchymal stem cells [45–47] or extracellular vesicles from mesenchymal stem cells [48, 49] has also shown favourable effects. Improvement of silicosis-dependent lung fibrosis has also been observed with agents increasing cyclic adenosine monophosphate (cAMP) or guanosine monophosphate (cGMP), such as inhibitors of phosphodiesterases [50, 51] or for corticosteroids [52–54]. As the importance of metabolic reprogramming of arachidonic acid, especially its derivatives prostaglandin D2 and thromboxane A2, in the progression of silicosis has been demonstrated, blocking its receptors by ramatroban may be a novel potential therapeutic therapy to inhibit the progression of silicosis [55]. In addition, several experimental studies have demonstrated a wide potential of natural plant-based compounds that may influence several of the pathological mechanisms of silicosis and thus may mitigate silica-induced inflammation as well as the process of fibrosis (table 1) [56–64].
Plant-based compounds used in different animal models of silicosis
Several methods are used to prevent exposure to respirable silica crystals and avoid the development of silicosis. First, the implementation of appropriate dust control measures in work environments is essential for prevention of the disease. Next, crystalline dust should be captured at the point of generation by enclosure and extracted by an extractor through ducts to suitable bag filters. In addition to ventilation, water-integrated tools should be used. The use of personal protective equipment as well as personal respirators that provide adequate respiratory protection must be used continuously to ensure protection at all times [65].
Animal models of silicosis
An animal model is a nonhuman species that is widely studied to understand biological process or characteristics, with the expectation that the findings obtained in this organism will allow understanding the action of other individuals. At present, animal models are widely used for the reproduction of all different types of human diseases, allowing the deeper study of physical and pathological mechanisms and the search for new therapies and treatments. Silicosis can occur spontaneously in nature and many cases have been reported in numerous mammalian species such as pigs [66], dogs [67], camels [68], horses [69, 70], nonhuman primates [71], river otters [72] and marsupials [73]. It has also been found in various species of birds like ground dwelling kiwi (Apteryx spp.) [73, 74]. Unfortunately, although this lung disease is common in many different species, not all of them can be used as experimental organisms to model silicosis in the laboratory because of difficulties in handling, manipulation and/or genetic modification, among others.
There are many different animal models used for their resemblance to human lung silicosis, as summarised in table 2; however, recently, the murine model has been widely used. The choice of experimental animal depends on the characteristics of the disease to be studied as well as the economical and organisational capacity of the experimental unit. Importantly, it has been shown that gender influences the severity of lung injury as well as the development of fibrosis, which is why male animals are often chosen for the experiments whenever possible [75, 76]. In addition, studies have shown a direct correlation between the dose and time of exposure to the silica crystalline dust and the severity of the silicotic lung disease [77, 78]. In addition, the presentation and severity of the disease has been shown to depend on the genetic predisposition of the experimental animal chosen for the study [79, 80]. Furthermore, it is important to bear in mind while performing an experiment that the artificial induction of silicosis will also depend on the form of silica and the route of exposure, as well as whether the administration of crystals is single or repeated in the experimental animal [5, 81–83]. It has also been found that the volume of material instilled can influence the severity of the disease, with animals receiving the same dose in a larger volume showing more diffuse inflammation and more severe pathology [84–86]. Finally, when the method of silica administration requires a liquid carrier, PBS is often used in experiments as a control [84, 87] and, when administered in a gaseous state, pure air is used [88].
Animal models of silicosis
Zebrafish models of silicosis
In 1995, the World Health Organization initiated a campaign with the goal of eliminating silicosis from the world by 2030; however, unfortunately, over the years, silicosis remains a major health problem internationally [89]. Therefore, there is a continuous need to generate new tools as well as to implement new experimental animal models for the study of lung silicosis.
Since 1960, zebrafish (Danio rerio) has emerged as an experimental model organism to study vertebrate developmental biology, cancer, toxicology, drug discovery, molecular genetics and, most importantly, it is nowadays widely used in biomedicine to model many different human diseases [90]. As a small tropical freshwater fish, it is ideal for use as an experimental model in the laboratory, as it ensures ease and low cost of husbandry. In addition, its physical and developmental characteristics as a vertebrate and the development and function of zebrafish organs closely resemble those of humans. There is a long list of the advantages of using zebrafish to model lung silicosis in vivo. One very important advantage is that adult female fish can produce up to 200–300 offspring per week, which makes them very suitable for high-throughput genetic studies with high efficiency and robustness. What differentiates them from mammalian models is that their embryonic development is external and the larvae are completely transparent without any feeding requirements for up to 5 days post-fertilisation [91]. During the first weeks of life, all major organ systems are formed and start to function, which can be observed under low-power microscopy due to their transparency [92]. Zebrafish embryos and larvae are relatively small, reaching millimetres in length, allowing hundreds of individuals to fit into a single Petri dish. This makes them more economically profitable at a reasonable cost than maintaining bigger experimental animals that require more space and resources. Importantly, genetic conservation between zebrafish and humans is very high, as they share 70% of all genes and 84% of all human disease genes [93]. The zebrafish genome is fully sequenced and allows for easy genetic modification. Many new genome-editing technologies have emerged and are used for creating new transgenic and mutant lines. However, as a nonmammalian species, zebrafish also have certain disadvantages for modelling human diseases. Most importantly, fish lack some mammalian organs, such as lungs. Hence, modelling lung diseases may seem impossible in this model, but fortunately it has been overcome. For example, the hindbrain ventricle situated in the back of the zebrafish head, a cavity into which immune cells can be easily recruited, mimics the alveolar environment of the human lung. It has already been used to model mycobacterial infections, coronavirus disease 2019 and silicosis in zebrafish [94–97]. In addition, gene duplication can lead to gene subfunctionalisation and neofunctionalisation, and phenotypic characteristics of diseases caused by ortholog genes may be different in zebrafish models.
Hindbrain crystalline silica dust injection
The zebrafish model of silicosis has been established by injecting crystalline silica dust resuspended in sterile PBS into the hindbrain of zebrafish larvae. This neuroepithelium-lined cavity has been used to mimic the alveolar environment that can be found in human lungs and, as a convenient injection site, allows the study of the interaction between silica crystals and host immune cells (figure 1). Normally, the hindbrain lacks macrophages and can be used to assess the recruitment of those cells to the injection site. In the case of neutrophils, although they may be present in the cavity, additional stimuli are able to enforce neutrophil recruitment as well. Because of the rapid development of zebrafish larvae and changes in larval physiology, which basically include shrinking of the hindbrain ventricle over time as well as increased rigidity of the larval epithelium, needle penetration becomes difficult or even impossible. The optimal time for this procedure is around 30–48 h post-fertilisation in zebrafish larvae. In detail, at that age zebrafish larvae need to be dechorionated, anesthetised and placed on a Petri plate to be pulled by the suction device, which is a microprobe called a VAMP (Vacuum-Assisted MicroProbe) [98]. One by one, the larvae can be immobilised and the crystalline silica dust can be injected several times into the hindbrain until the cavity is filled. Hindbrain injection can be done by directly injecting the needle into the cavity (however, this can generate a hole through which the injected liquid and crystals can escape) or it can be performed by penetration through the forebrain [95].
Similarities between human pulmonary alveolus and zebrafish hindbrain ventricle.
The zebrafish model of silicosis has shown similar characteristics at the molecular level to those seen in different experimental animal models, as well as in silicotic patients. SiO2 crystals were able to induce both local and systemic inflammation in zebrafish [95]. The crystals induced significant recruitment of neutrophils and macrophages at the injection site, demonstrating that they can be recognised by the zebrafish immune system. The C-X-C motif chemokine ligand 8/C-X-C motif chemokine receptor 2 axis has been shown to be essential for the spreading of the inflammatory response induced by silica crystals. In addition, induction of proinflammatory cytokines and M1 polarisation of macrophages after silica injection was also observed in the experimental zebrafish model. This model has also revealed that silica crystals activate Toll-like receptor and Nlrp3-inflammasome signalling pathways in zebrafish. Moreover, neutrophilia and monocytosis were found in larvae injected with silica crystals, suggesting that SiO2 crystals can activate emergency myelopoiesis. Interestingly, two signalling pathways have been shown to be activated by silica crystals in zebrafish: a Caspa/Gsdme (caspase a/gasdermin e) axis that regulates myeloid cell pyroptosis and inflammation, and a Caspa/Gata1 (gata-binding protein 1) axis that promotes emergency myelopoesis [95].
Fibrotic processes also appear to be present in the zebrafish model of silicosis as a result of silica crystal injection, which start after resolution of inflammation. Thus, 5 days post-injection of silica crystals, the fibrotic markers tgfb1, acta2 (smooth muscle actin alpha 2), col1a1a (type I collagen alpha 1a), col1a2 (type I collagen alpha 2) and fn1a (fibronectin 1a) were significantly elevated at the site of injection, as were some (acta2, col1a2 and fn1a) in the whole body [95]. In addition, a histological study could be performed on silica injected fish, thus adding information about the pathophysiological mechanisms of fibrosis in this model.
Another interesting observation is the lower motility of the larvae injected with silica dust [95]. They remained in the centre of the Petri dish, in contrast to the control larvae, which were very motile and always explored the edges of the dish. In addition, the silica-injected fish showed an impaired escape response to a tactile stimulus, whereas the control larvae instantly reacted by swimming rapidly away when touched on the tail.
It should be noticed that silicotuberculosis can also be easily modelled in zebrafish by combining the already well-established mycobacterial infection model in zebrafish [94] with the new model of silicosis in zebrafish [95].
Inflammatory zebrafish transgenic reporter lines
The zebrafish model of silicosis generated by injecting silica dust into the hindbrain of zebrafish larvae can be used not only in wild-type fish, but also in many different transgenic or mutant lines, which may help to decipher the mechanisms orchestrating the pathology and constitute a unique platform for chemical screening in the future. An important characteristic of zebrafish as an experimental model is the wide variety of transgenic lines expressing fluorescent genes driven by a lineage-specific promoter, which allows real-time imaging of pathophysiological processes in vivo [99]. Of interest for the study of silicosis and its pathophysiology has been the development of stable transgenic markers for macrophages and leukocytes, as well as pro-inflammatory markers that allow simultaneous and real-time fluorescent detection of the processes occurring during the development of the disease. Here, we summarise the main representative zebrafish transgenic lines labelling different cell populations or molecules that can be used for characterisation of inflammatory events in silicosis (table 3).
Zebrafish transgenic lines useful for studying silicosis
The contribution of immune cells to the development of silicosis can be easily assessed. To study neutrophil recruitment, their total number or dispersion after silica crystal injection, the transgenic lines Tg(mpx:eGFP) and Tg(lyz:dsRED) have been generated, in which neutrophils express the fluorescent proteins eGFP or dsRED, respectively, under the control of the specific promoter myeloperoxidase (mpx) or lysozyme (lyz) promoters [100, 101]. Similarly, zebrafish transgenic lines Tg(mpeg1:eGFP) and Tg(mpeg1:mCherry) with the promoter of the macrophage-specific marker mpeg1 (macrophage-expressed gene 1) [102] can be used to study macrophage recruitment or their total number in response to SiO2 as a stimulus. From 3 days post-fertilisation, the downregulation of endogenous mpeg1 gene expression in zebrafish was observed and an additional long-term transgenic line with fluorescent macrophages was generated. Thus, the mfap4 (microfibril-associated protein 4) promoter has been used as a macrophage-specific marker leading to long-term expression of the red fluorescent protein mCherry in the line Tg(mfap4:mCherry-F) [103]. In addition, two other transgenic lines driving eGFP expression in macrophages have been generated, although they are not as widely used as the previous ones. The first uses the promoter of the immunoresponsive gene 1 (irg1), which labels macrophages only after activation Tg(irg1:eGFP) [104]. The other, Tg(fms:eGFP), uses a bacterial artificial chromosome transgene with the promoter of the zebrafish fms gene, encoding the macrophage colony stimulating factor 1 receptor, to label macrophages and skin xanthophores with eGFP [105]. Interestingly, the transgenic line Tg(coro1a:eGFP), with eGFP driven by the promoter of coronin 1a, is expressed in a full repertoire of myeloid cells, as it is highly enriched in leukocytes [106]. In addition, the zebrafish transgenic line Tg(lck:eGFP) can be used to assess the role of T-cells in the pathogenesis of silicosis by expressing eGFP under the control of the T-cell-specific tyrosine kinase (lck) promoter [107].
The production of inflammatory markers can also be tracked in vivo by fluorescent proteins. To date, several transgenic reporter lines have been generated using the promoters of pro-inflammatory molecules that drive the expression of fluorescent proteins. The nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is considered a master regulator of inflammation and immune responses. The NF-κB reporter system in vivo, consisting of the expression of the fluorescent protein eGFP under the promoter of the master transcription factor NF-κB, can serve as a useful tool to monitor temporal and spatial patterns of activation to stimuli [108]. In fact, this line has been successfully used to demonstrate the induction of NF-κB following silica crystal injection [95].
The pro-inflammatory cytokine interleukin-1β (IL-1β) is a key mediator of the inflammatory response and can provide an important information about the inflammatory status of the studied process. A Tg(il1b:GFP-F) zebrafish line has been generated, in which the expression of farnesylated GFP, which is membrane-targeted, is driven by the il1b promoter. It has been used to study the spatiotemporal behaviour of the cells that produce the il1b transcripts in resting conditions and after stimulus [109]. Similarly to Tg(NFkB:eGFP), Tg(il1b:GFP-F) has been used to show that il1b increases locally after silica dust injection [95].
Another very useful line is the Tg(tnfa:eGFP-F) line, in which the expression of eGFP is driven after tnfa gene promoter stimuli [110]. This line can be used to track the polarisation of macrophages into M1 in vivo and to track Tnfa+ cells upon stimuli [110]. In particular, silica crystal injection into the hindbrain of larvae caused M1 polarisation of macrophages [95].
Fibrosis zebrafish transgenic reporter lines
Zebrafish transgenic lines used to study the process of fibrosis in vivo have been widely used. The promoter of the zebrafish fibrosis marker acta2 has been used to drive the expression of the green and red fluorescent proteins Tg(acta2:eGFP) and Tg(acta2:mCherry), resulting in the labelling of vascular mural cells and visceral smooth muscle cells [111]. Another useful line for modelling fibrosis in zebrafish models of silicosis is Tg(krt19:col1a2-GFP), which express Col1a2 fused to GFP under the promoter krt19, driving expression in the basal layer of epidermal cells, which are cells previously reported to be responsible for deposition of skin collagen I in early stages of zebrafish development [112]. The expression of Col1a2 is restricted to one cell type in this model and fluorescence became apparent within the skin at 2 days post-fertilisation and increased in intensity until 5 days post-fertilisation, being first observed in the dorsal region of the head. GFP–collagen was detected intracellularly in occasional epidermal cells, although it was also apparent as extracellular wispy fibrils adjacent to these cells [112]. Finally, other zebrafish lines could be designed to drive expression of the fluorescent protein with the promoters of other markers of fibrosis, such as Col1a1a, Col1a2, Fn1 and others. These transgenic lines could be used to assess this pathological process in vivo by and for high-throughput antifibrotic drug screening.
Concluding remarks and future directions
To date, silicosis lung disease has no available cure that can alter the course of the disease and no medications that can reverse lung damage. Strategies to effectively combat crystalline silica lung disease are prevention by reducing and controlling exposure to SiO2 dust. The development of drug therapies requires fundamental knowledge of the biology of the disease and the molecular basis of the pathogenesis. However, despite many inconveniences, drug development strategies to deal with lung silicosis can be classified into the following groups that offer many different drug targets, as follows: 1) targets to prevent the onset of inflammation by anti-cytokine therapy; 2) targets to prevent the onset of inflammation by blocking inflammasome activation; 3) targets to prevent the onset of inflammation by agents enhancing an autophagy–lysosomal system; 4) targets to prevent the onset of inflammation by antioxidants; 5) targets to prevent the onset of inflammation by corticosteroids; 6) use of agents that increase cAMP and cGMP; 7) use of agents that influence TGF-β-signalling; 8) use of microRNAs; and 9) targets to prevent the process of fibrosis, called anti-fibrotic drugs [50, 54, 87, 113–124]. With all these possibilities in mind, several treatments are in different stages of development, giving hope to patients with silicosis. In relation to this, the zebrafish is a great model for human health and can act as an intermediary between in vitro assays performed in cells and mammalian in vivo studies to evaluate efficacy, appropriate dose, toxicity and simultaneous assessment of different drugs [125, 126]. Unfortunately, all tests performed with cell-based methods provide limited information on the aspects of absorption, distribution, metabolism, excretion and toxicity of anti-silicotic compounds, whereas in vivo zebrafish assays often allow discovering these pharmacological aspects. The compounds tested must have the ability to be absorbed, reach the target tissue and avoid rapid metabolism and excretion in the in vivo zebrafish model. The great advantage of testing newly discovered compounds in zebrafish is that they can be rapidly tested in mammalian models in vivo with minimal optimisation of pharmacological aspects.
The entire drug discovery process is very time-consuming and the usual timeline for approval of a new therapeutic agent for any given disease can be as long as 10 years. The most promising and time-saving strategy for the search, identification and development of treatment for lung diseases is the use of drugs that have already been approved by the United States Food and Drug Administration (FDA), especially drugs involved in diseases with inflammatory and fibrotic lung processes [127]. This will shorten the path to clinical research, as the identified compounds have already been tested pharmacokinetically and undergone safety trials to be approved for human use. To date, many drug libraries are available for testing in in vitro and in vivo models [128]. Beyond the design of novel drugs and discovery and the testing of already FDA-approved drugs, screening natural products for anti-inflammatory and antifibrotic activity also seems to be a good approach [56]. Herbal compounds may be beneficial for pulmonary silicosis considering their biological relevance, characteristics and diversity. The “COlleCtion of Open NatUral producTs” (COCONUT) has been created, which contains structures and sparse annotations of more than 400 000 nonredundant natural products that can be tested in experimental animal models of human diseases [129]. In conclusion, all of the above approaches can easily be used in zebrafish models of many human diseases, such as the silicosis model, as they have already shown high efficacy in high-throughput screening of repurposed drugs [130–132] and natural or herbal products [133–135], as well as helping to discover specific therapeutic targets in humans.
Footnotes
Provenance: Submitted article, peer reviewed.
Conflict of interest: All authors have nothing to disclose.
Support statement: This work has been funded by Fundación Séneca (Saavedra Fajardo postdoctoral contract 21118/SF/19 to S. Candel), the Spanish Ministry of Science and Innovation (Juan de la Cierva-Incorporación postdoctoral contract to S.D. Tyrkalska) and ZEBER research grant funded by Consejería de Salud de la CARM.
- Received April 18, 2023.
- Accepted June 8, 2023.
- Copyright ©The authors 2023
This version is distributed under the terms of the Creative Commons Attribution Non-Commercial Licence 4.0. For commercial reproduction rights and permissions contact permissions{at}ersnet.org
References
