1- INTRODUCTION

Cystic Fibrosis (CF) results from cystic fibrosis transmembrane conductance regulator (CFTR) mutation found in epithelial cells of those organs adjusted to the transepithelial transport of salt and water. It is also the most common deadly disease in Caucasian populations (1). CF prevalence is 1 in every 2000 to 3000 live births (2). CF may cause severe complications in different organs- i-e., azoospermia in males, intestinal blockage, and defective exocrine pancreatic function, due to dysfunction of CFTR protein and its shortage in the patients (1, 3). CFTR dysfunction leads in decreased transepithelial chloride secretion and an imbalance in the epithelial electrolyte and water homeostasis (4, 5). The thick and sticky mucus deposited in airway lumens forms following the improper transport of salt and water through epithelial cells of airways. Bacteria get stuck in it over time that leads to a persistent infection and inflammation in the airways. Tissue damage and fibrosis caused by chronic inflammation finally lead to pulmonary hypertension and exhaust most patients (1, 3). Though it is generally a multiorgan disorder, the major cause of morbidity and mortality in CF is the progressive pulmonary involvement (6-8), which results from infection caused by Pseudomonas aeruginosa (PA) and chronic pulmonary infections (9, 10).

Although there is difficulty in treating recurrent PA-related infections and other pathogens in CF patients, but different treatment strategies have been developed over the past few decades that have significantly a positive impact on the prognosis of this disease (11). Today, for treating the symptoms of CF-related infections and inflammations, antibiotics and anti-inflammatory drugs are widely used. Furthermore, bronchodilators, mucolytics, and osmotic agents are prescribed to improve the clearance of the airways and sputum (11-15). However, this therapeutic approach treats the symptoms of the disease without affecting its underlying causes (16).

The complexity of the drug regimen in the chronic diseases have led to about 50% compliance for treatments in patients (17), and in recent years, the use of traditional and complementary medicine to reduce the symptoms of CF has been considered by health care providers and about 66% of patients with CF have used one of the alternatives and complementary methods (18).

Complementary and traditional medicine develop new drugs usually origined in natural products. Iranian Traditional Medicine (ITM) is an ancient health practice with worthy roots (19, 20), which date back to thousands of years (21). Iranian Muslim physicians, like Avicenna (980–1037AD) and many others in the early medieval, played a pivotal role in its flourishment (20, 22) so that their manuscripts- e g, the Canon by Avicenna, were the medical textbook in European universities from 11th to 17th centuries (19). These manuscripts were written and used in Iran till the 18th century.  The issues and medications suggested in these manuscripts written from the 10th to the 18th century benefit from an 800-year support by Iranian physicians through examinations and trials (23).

Currently, the use of complementary and Traditional medicine as a therapeutic factor for chronic diseases is being accepted in WHO strategies (24). The present hypothesis aimed at investigating an Iranian Traditional herbal medicine, Flaxseed, as the complementary treatment of CF, cited frequently by many Iranian old medical texts.

2- MATERIAL AND METHODS

In this hypothesis, in order to investigate the effects of Flaxseed on pulmonary symptoms caused by thick and sticky airway secretions of patients with cystic fibrosis, acceptable information was extracted from ITM textbooks such as Qanoon fi al-teb, Zakhire-Kharazmshahi, and Exir-Azam. Then, data sources including (Pub Med and Google Scholar) without time restriction, from inception up to September 2020, were searched by using the combination of keywords such as (cystic fibrosis, Pseudomonas aeruginosa, CFTR, flavonoids, kaempferol, quercetin, inflammation, alpha-linolenic acid, omega 3, ferulic acid, anti-quorum sensing (QS) activity, the type III secretion systems (TTSS)) and Flaxseed were searched thoroughly for the probable mechanisms of action of this herbal medicine "Flaxseed" on CF; in addition, the present study hypothesized that it is a new drug developed based on the suggested formula.

3- RESULTS

In this hypothesis, we attempted to introduce the potential effects of Flaxseed on CFTR, Pseudomonas aeruginosa and pulmonary inflammation in CF patients, described one by one in the following section.

3-1. Cystic fibrosis in Iranian Traditional Medicine

There is no cystic fibrosis disorder in Iranian Traditional Medicine (ITM) sources, but due to the production of thickened secretions in the body ducts, it is classified in the group of diseases caused by the production of thick and sticky secretions. In this assay, we have mentioned Flaxseed as a single herb, recommended in many ITM text books and some recent studies for thick and sticky airway secretions management, as well as its related effects. Flaxseed is one of the most recommended herbs for these types of diseases in ITM documents (25-27).

3-2. Flaxseed

Flax (Linum usitassimum) is the member of Lineaceae family (28). The Flaxseed is a medicinal herb, any part of which is used for a certain medical purpose (29, 30). The consumption of Flaxseed has an ancient history (31). Nowadays, more than 50 countries, mostly in the Northern hemisphere, cultivate this plant (32). Countries involved in the cultivation and production of flaxseed include Canada, China, United States, India and Ethiopia (33). Flaxseed is widely used in Iranian Traditional Medicine and it is cited as a treatment for thick and sticky secretions in many Iranian Traditional manuscripts (25-27). Flaxseed contains fat, protein, soluble and insoluble fiber, phenolic, trypsin inhibitor, linatine, lignans (phytoestrogens), minerals, vitamins and selenium (34). Furthermore, Flaxseed has anti-inflammatory effect (35-40). Approved pharmacological effect of flaxseed include decreasing the progression of atherosclerosis (41-43), reducing blood glucose in type 2 diabetic patients (44, 45), alterating the bacterial flora in the animals intestines (46, 47) and having anti-cancer effects (48, 49).

3-2-1. Expected mechanisms of action of Flaxseed in cystic fibrosis pulmonary disorder

According to the main theories regarding CF, it seems that Flaxseed can be suggested as a novel drug with natural origin enjoying an advance formula for the treatment of CF, considering of the following reasons:

Flaxseed is rich in alpha-linolenic acid )ALA) (32) that is the omega-3 fatty acid source in the vegetarian diets (29, 30, 50). Also Flaxseed has anti-inflammatory effect (51-55) and possesses phenolic acids (ferulic acid) (56) which affect Pseudomonas aeroginosa (57) by having anti-quorum sensing (QS) activities, and also contains flavonoid compound which affects CFTR (56).

3-2-1-1. Flaxseed and CFTR

Since CFTR regulates salt and water transepithelial transportations, it exists in a variety of epithelial cells (1).

Flavonoids are categorized as a class of secondary plant metabolites having protective effects against immune and inflammatory reactions. They play a pivotal role in the growth and development of plants (56).

Biochemical analysis of the seedcake extracted from transgenic Flaxseeds demonstrated significant increase in flavonoids (kaempferol). High-performance liquid chromatography (HPLC) analysis of the methanolic extract of the seeds from W92.72 and W92.40 transgenic lines showed higher amounts of quercetin (90% and 46%, respectively); in addition, kaempferol accumulation was also observed in the lines W92.40 and W92.72 by 70% and 83%, respectively (56).

Genistein is a plant-derived isoflavone, which its biological activity was first described in 1940s and early 1950s. In 1980s, it was found that quercetin, a flavone from this group, can inhibit the activity of protein kinase C (PKC), phosphorylase kinase, casein kinase, and the tyrosine kinase associated with the Rous sarcoma virus gene product pp60src (58). Now it is evident that genistein and some related flavones and isoflvones can affect transportation of ions in epithelial cells. This finding was first reported by Nguyen et al. They indicated that quercetin and kaempferol can affect CFTR-medicated ion transport and stimulate secretion of Cl^- across T84 cells (58, 59). A study on IB3-1 (F508/W1282X) bronchial epithelial cells, for examining the effect of flavonoids on CFTR, showed that apigenin, genistein, kaempferol, and quercetin (1 or 5 µmol) can alter the immunofluorescence distribution. Intracellular CFTR was demonstrated during 24-hour treatment with apigenin, genistein, and kaempferol. Finally, this study found combination therapy with selective flavonoids to be useful in the treatment of cystic fibrosis (60). One study showed that flavonoid Quercetin stimulates CFTR in respiratory epithelial in vivo and in vitro (6). Another study showed that an extract from the Phyllanthus acidus (medicinal plant) and its isolated compounds, like kaempferol, provoke airway chloride secretion by activating CFTR (32).

3-2-1-2. Flaxseed and inflammation

CF patients’ airways are normal at birth except for the plugged and dilated submucosal gland ducts, which indicate prenatal changes in the airways. Pulmonary diseases start in childhood. Newborns who are diagnosed by newborn screening, following diagnosis showed inflammation and infection with bacteria, particularly with Staphylococcus aureus. Broncho alveolar lavage fluid at high levels was found with inflammatory markers, like interleukin 8 (IL-8), peroxidases, and their oxidants, even among newborns with normal lung function and with no evident bacterial colonization (61, 62). One study revealed that dietary Flaxseed reduced lung inflammation and lipid peroxidation that indicate protective effect upon pro-oxidant-related tissue destruction in vivo (63). There are some studies that show the anti-inflammatory effect of flaxseed and Alpha-linolenic acid, some of which are reported in the following: A study found that flaxseed had significant effects on reducing C - reactive protein in obese people (64). Another study found that the combined use of L. Plantarum probiotics with flaxseed-rich flakes of n-3 PUFAs had anti-inflammatory effects by stimulating immune cells associated with Th1 and phagocytosis (65). Yet another study found that flaxseed and quercetin improved the levels of anti-inflammatory cytokines (66). A further study showed that concentration of IL-6, IL-1Beta and TNF-alpha produced by peripheral blood mononuclear cells and serum TNF-alpha were lower in ALA diet than in the other studied diets (67). The only study on CF and Flaxseed revealed that Flaxseed modulates inflammatory oxidative stress biomarkers and inflammation in cystic fibrosis (68).

3-2-1-3. Flaxseed and Pseudomonas aeruginosa

Infection is the leading cause of morbidity and mortality in these patients due to Pseudomonas aeruginosa and chronic respiratory infections (9, 69). To react, lung infections with organisms, like P. aeruginosa and neutrophils (white blood cells) accumulate in the airways, producing proteolytic enzymes and oxidants that intercede the inflammatory reaction (70, 71). The thick and viscous secretions are caused by such neutrophils leading to mucus plugs in the smaller airways and consequently infection and inflammation. Treating with anti-inflammatory agents, such as corticosteroids (71, 72) and non-steroidal anti-inflammatory agents (71, 73) is effective (71).

 In addition, P. aeruginosa is the human opportunistic pathogen, which prefers to infect cystic fibrosis patients. The pathogen generates many extracellular productions such as pyocyanin (74, 75). Such extracellular virulence factors seem essential in competing Pseudomonas aeruginosa for establishing and maintaining infection. Their production is managed by quorum sensing system (QS). Pseudomonas aeruginosa also engages QS to control the organization of biofilms (74, 76). Biofilm generation can protect the microorganisms against host defenses and supply elevated resistance against antibacterial agents (74, 77). Inactivation of the QS system in a pathogen had significantly decreased virulence factor generation (74, 78).

The main mechanism that lets P. aeruginosa to increase in affected hosts and overcome host defense strategies is secreting different protein factors by specific secretion apparatus. The Pseudomonas aeruginosa genome can harbor genes for the type II and III secretion systems (TTSS) (79). The latter is a specific cytotoxin-translocating apparatus including gram-negative bacteria related to septic shock, lung injury, and inappropriate patient outcomes (80). Although Pseudomonas aeruginosa TTSS genes are available in almost all environmental and clinical isolates (81), TTSS expression is linked to more serious infections (82). The TTSS-expressing phenotype rate in 108 clinical isolates of P. aeruginosa was remarkably more in acutely infected cases in comparison to CF patients with chronic infection; and it was associated with a higher mortality rate (83).

Plants as promising sources of molecules inhibiting bacterial QS have been long applied in medicine(57, 84, 85). Flaxseed has Phenolic compounds (56) that are the plants’ secondary metabolites, extensively found in plant kingdom with antioxidant and antimicrobial effects (57, 86). Nonetheless, their anti-QS effects on microorganisms have been not widely considered (57). Biochemical analysis of the seeds’ extracts of transgenic flax plants showed a significant elevation in the levels of phenolic acids (coumaric, ferulic, and synapic acids) (56). Additionally, one study showed that flaxseed oil has antibiofilm activity against methicillin-sensitive S. aureus, Methicillin-resistant S. aureus, Klebsiella pneumonia and Staphylococcus epidermidis (87), and another study showed that flaxseed extract has positive antibacterial effects against Streptococcus mutans, Streptococcus pyogenes, Pseudomona aerigenosa; and it was comparable in efficacy to that of Streptomycin (88).

3-2-1-4. Flaxseed and omega 3

ALA and linoleic acid (LA) are two fatty acids, respectively, belonging to the omega-3 and omega-6 families of polyunsaturated fatty acids (PUFAs), which are essential for humans and should be obtained from food. During the PUFAs metabolism, long chain fatty acids are derived that have significant biological activities (89).

Omega-3 fatty acids, by applying anti-inflammatory responses, play a pivotal role in the cell membrane integrity. Regarding their effect on inflammatory complications, omega-3 fatty acids can reduce the conversion of the omega-6 fatty acids to pro-inflammatory metabolites and increase the production of those omega-3 end products that are biologically less-active (90). ALA is converted to docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, and LA to arachidonic acid (AA). No mechanism has been recognized thus far for the low levels of LA in CF; however, a potential mechanism can be the elevated metabolism of the omega-6 fatty acids (89).

Some factors in patients with CF disrupt fatty acids uptake (90) and accordingly, compared to healthy people, the serum essential fatty acids are in lower levels in such patients (89). Therefore, although achieving a certain therapeutic regimen needs further investigations, omega-3 fatty acids supplementation can be a treatment of choice in patients with CF (90). The clinical benefits and feasibility of omega-3 fatty acids supplementation in CF are investigated in about 10 studies. DHA, an omega-3 fatty acid, can normalize LA metabolism, according to recent data; and so, it can help scientists to provide a rationale supplementation regimen (89).

According to recent findings, changes in serum fatty acid concentration contribute to the pathogenesis in lungs. For instance, serum essential fatty acid levels are usually lower in patients with CF than in healthy people. The infection caused by Pseudomonas aeruginosa plays a crucial role in the pathogenesis of CF, although the reason for the vulnerability of CF patients to these bacteria is unclear yet. As already known, by targeting sphingolipid metabolism and ceramide accumulation, omega-3 PUFAs containing bioactive compounds (like DHA ethyl ester) can modulate inflammation and immune responses in CF and chronic inflammatory diseases; hence, they can be suggested as alternatives to supplementation with high-dose LA. It is already known that the expression of a CFTR gene encountered mutations alters the metabolism of sphingolipids and ceramic accumulation that affects the vulnerability of the patient with CF to infections and inflammations. Evidently, the lack of a wild-type CFTR expression intensifies the accumulation of ceramide. Data obtained from studies on mouse models of CF show that if ceramide levels are normalized, the secretion of inflammatory markers and accordingly, the susceptibility to infections decreases (89).

Nutritionists suggest the incorporated consumption of omega-3 fatty acid sources and Flaxseed is highly rich in omega-3 fatty acid and can be the best source of this compound for those who do not eat marine foods (29, 30). Flaxseed is the source of ALA as a functional ingredient. In the vegetarian diets, it is the exclusive omega-3 fatty acid source (29, 30, 50). ALA is a fundamental PUFA that exhibits anti-inflammatory, anti-thrombotic, and anti-arrhythmic activities (91). The amount of omega-3 fatty acids in Flaxseed oil is 6 times higher than it in fish oil (92), also Flaxseed has anti-inflammatory effects (51-55). Flaxseed oil inhibits prostaglandin E2 (PGE2) (51). PGE2 inhibits macrophage, neutrophil, and T helper type 1 (Th1) function and enhances T helper type 2 (Th2), T helper type 17 (Th17), and T regulatory responses (93). Therefore, with this mechanism, it can be effective in modulating the immune response. The oil also inhibits arachidonic acid-induced inflammation by inhibiting the inhibitory capacity of both cyclooxygenase and lipoxygenase in arachidonic acid metabolism (51).

4- Conclusion

CF is also the most common deadly disease in Caucasian populations. Although it is generally a multi-organ disorder, but progressive respiratory involvement is a major cause of mortality and morbidity in most patients and the main cause of mortality and morbidity in these patients is infection caused by airway obstruction because of the thick and sticky secretions. Plant "Flaxseed" for the management of cystic fibrosis presented in this study is supported by classical Iranian traditional texts as well as recent investigations. According to the mentioned studies, Flaxseed is a medical herb for the respiratory diseases with high thick and sticky secretions production and all the ingredients of this herb according to this review, affect the cystic fibrosis pulmonary pathogenesis that by the glance Flaxseed is a good source of ALA, which is the omega-3 fatty acid source in the vegetarian diets. Also Flaxseed has anti-inflammatory and anti-bacterial effects and possesses phenolic acids (ferulic acid) which affects P.aeroginosa by having anti-QS activity, and also contains flavonoid compounds which affect CFTR. This medical herb prepares a proper complementary and alternative to conventional medications, with fewer complications. Systematic validity studies are needed to enhance the traditional drugs as complementary and alternative medications for cystic fibrosis. It seems that some of the components present in this plant can be employed for designing new drugs.

4-1. Acknowledgments

This research was derived from an ongoing PhD thesis at the School of Traditional Medicine, Shahid Beheshti University of Medical Science.

Funding source: There is none to be declared.

Ethical statement: There is none to be declared.

Competing interests: No competing interests to be disclosed.

Authors’ contribution: All authors contributed proportionately to this work