Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts

Airway remodeling is a complex process, and controlling it appears to be a key for halting the progression of asthma and other obstructive lung diseases. The aim of this study was to evaluate the anti‐remodeling properties of extracts from transformed and transgenic L. sibiricus roots with overexpression of AtPAP1 transcriptional factor. Our study shows for the first time that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process.

Summary

Bronchial asthma is believed to be provoked by the interaction between airway inflammation and remodeling. Airway remodeling is a complex and poorly understood process, and controlling it appears key for halting the progression of asthma and other obstructive lung diseases. Plants synthesize a number of valuable compounds as constitutive products and as secondary metabolites, many of which have curative properties. The aim of this study was to evaluate the anti‐remodeling properties of extracts from transformed and transgenic Leonurus sibiricus roots with transformed L. sibiricus roots extract with transcriptional factor AtPAP1 overexpression (AtPAP1). Two fibroblast cell lines, Wistar Institute‐38 (WI‐38) and human fetal lung fibroblast (HFL1), were incubated with extracts from transformed L. sibiricus roots (TR) and roots with transcriptional factor AtPAP1 over‐expression (AtPAP1 TR). Additionally, remodeling conditions were induced in the cultures with rhinovirus 16 (HRV16). The expressions of metalloproteinase 9 (MMP)‐9, tissue inhibitor of metalloproteinases 1 (TIMP‐1), arginase I and transforming growth factor (TGF)‐β were determined by quantitative polymerase chain reaction (qPCR) and immunoblotting methods. AtPAP1 TR decreased arginase I and MMP‐9 expression with no effect on TIMP‐1 or TGF‐β mRNA expression. This extract also inhibited HRV16‐induced expression of arginase I, MMP‐9 and TGF‐β in both cell lines (P < 0·05) Our study shows for the first time to our knowledge, that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process. Its effect can be attributed an increased amount of phenolic acids such as: chlorogenic acid, caffeic acid or ferulic acid and demonstrates the value of biotechnology in medicinal research.

Introduction

Despite many scientific advances, asthma remains a major public health concern globally. Asthma is a heterogeneous condition characterized by variable respiratory symptoms and variable airflow limitation [1]. The adult onset of asthma has been linked in some cases to environmental exposure: viral respiratory infections, irritant exposures and newly developed allergies or obesity [1, 2]. Asthma mortality among adults and children has decreased globally during the past 25 years, which is generally due to use of inhaled corticosteroids. Nevertheless, there is still a wide global disparity regarding life years lost because of asthma.

Airway remodeling is one of the pathological features of asthma, characterized by structural changes, involved in worsening lung function and persistent airflow limitation. These changes include goblet cell metaplasia and angiogenesis, increased deposition of extracellular matrix proteins (ECMs) and increased airway smooth muscle mass [3, 4]. Although evidence suggests that airway remodeling is associated with a progressive loss of lung function, no therapies targeting the process have been approved [5, 6].

Airway remodeling in bronchial asthma has been attributed to a chronic inflammatory response involving both permanent airway tissue destruction and chronic tissue repair [5, 7]. Transforming growth factor β (TGF‐β) and T helper type 2 (Th2) cytokines such as, for example, interleukin (IL)‐5, IL‐13, arginase, metalloproteinase 33, disintegrin and matrix metalloproteinase 9 (MMP‐9), have been identified among candidates for airway remodeling markers [8, 9]. Long‐term treatment of asthma focuses currently on controlling airway inflammation, neglecting the airway remodeling process. Pohunek et al. [10] suggest that the use of anti‐inflammatory medications to treat asthma based on early diagnosis may contribute to the development of airway remodeling. Interestingly, the treatment strategy for bronchial asthma consists of ameliorating its symptoms and slowing the inflammatory process [1]. Nevertheless, as asthma presents a complex physiopathology associated with variable symptoms, any treatment can yield varying responses [2, 7].

At present, no effective treatments are known that can halt or reverse the changes of airway remodeling and influence lung function [11]. Longitudinal research has indicated that current therapeutic interventions are relatively ineffective in reversing or inhibiting the development of airway changes [8, 12, 13, 14]. However, an alternative form of treatment may lie in the use of natural products as complementary therapy.

Plant products have been used as medicines for centuries. One such plant is Leonurus sibiricus, of the family Lamiaceae. This medicinal herb is a widely distributed in Asia and South America [15, 16]. Phytochemical analysis has revealed the presence of a range of alkaloids, flavonoids, iridoids, diterpenes or phenolic acids I ntis secondary products [17]. It has been reported that extracts obtained from L. sibiricus may demonstrate a range of anti‐oxidative, anti‐inflammatory and anti‐microbial activities [18, 19, 20, 21]. In turn, extracts from L. sibiricus root have been found to induce apoptosis in various cancer cells via DNA damage or decreased mitochondrial membrane potential, altering the expression of apoptotic genes or cell cycle arrest [20, 22]. Additionally, essential oils derived from the normal and transformed L. sibiricus roots present anti‐inflammatory properties, by decreasing production of IL‐1β, IL‐6, interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α in normal human astrocytes stimulated by lipopolysaccharide [18].

The transcriptional factor from Arabidopsis thaliana (transformed L. sibiricus roots extract with over‐expression of transcriptional factor AtPAP1) inserted by Agrobacterium rhizogenes‐mediated genetic transformation of L. sibiricus roots was found to enhance the production of phenolic acids and may improve its biological properties [20]. This is in agreement with Tuan et al., who demonstrated that the AtPAP1 transcriptional factor improved the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum. In turn, Qui et al. reported that AtPAP1 factor enhanced the production of chlorogenic acid and quercetin glycosides in the transgenic Taraxacum brevicorniculatum [23, 24].

The present study examines transformed (TR) and transgenic over‐expressing AtPAP1 (AtPAP1 TR) root extracts. Transformed roots (TR) were obtained by transformation of wild‐type Agrobacterium rhizogenes (A4). In turn, transgenic roots (AtPAP1 TR) were obtained by introducing the T‐DNA from recombinant vector pCAMBIA1305.1‐AtPAP1 into genomic DNA, with the resulting cultures grown in a bioreactor. Both strategies were aimed at increasing the production of phenolic acids, including chlorogenic acid, ferulic acid and caffeic acid in the roots of L. sibiricus; such production has been confirmed in earlier studies, where the TR demonstrated stronger biological properties compared to non‐transformed roots [25, 26].

As asthma cases are increasing worldwide, more effective anti‐remodeling therapies are desperately needed; researchers have sought potential alternatives of treatment. Phenolic acids, especially chlorogenic acid, appear to be promising compounds regarding airway remodeling inhibition. They are known to be strong natural anti‐oxidants due to their hydroxyl groups and phenolic rings. Moreover, these compounds present a wide range of anti‐inflammatory, anti‐cancer, anti‐microbial, anti‐allergic, anti‐viral, anti‐thrombotic and hepatoprotective activities, and act as signaling molecules [27, 28, 29, 30, 31, 32, 33].

Therefore, the aim of our study was to evaluate the influence of transformed (TR) and transgenic (AtPAP1 TR) root extracts from L. sibiricus on the expression of the genes involved in the airway remodeling process. We hypothesized that AtPAP1 TR extract with increased content of phenolic compounds would significantly affect the expression of the studied genes, and thus might influence the development and maintenance of airway remodeling.

Materials and methods

Cell cultures

Wistar Institute‐38 (WI‐38) and human fetal lung fibroblast (HFL1) cells were purchased from Sigma‐Aldrich (St Louis, MO, USA) and grown in Eagle’s minimum essential medium (EMEM) medium (WI‐38) and HAM’s12 medium (HFL1) with 10% fetal bovine serum, 2 mM L‐glutamine, 1% non‐essential amino acids and standard penicillin–streptomycin solution (Sigma‐Aldrich, St Louis, MO, USA). Wi‐38 and HFL1 fibroblasts, both from normal embryonic lung of females, have very broad human virus spectra, especially useful for isolation and research of rhinoviruses. Additionally, HFL1 is used in preparation of human virus vaccines. All the experiments (n = 6), were performed after reaching 80–90% confluence (passages 3–9).

Virus preparation and cell infection

Human rhinovirus 16 (HRV16) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Ohio HeLa cells were infected [multiplicity of infection (MOI) of 1] until cytopathic effects were seen. HRV specimens were exposed to the temperature of 58°C for 1 h in order to inactivate the virus particles, which was subsequently confirmed by lack of HRV replication.

The target fibroblast cells were infected by the addition of 50 μl vehicle (medium) or HRV16, and the effectiveness of the infections was confirmed by identification of virus RNA utilizing real‐time polymerase chain reaction (PCR). The cells were incubated for 24 h (33°C, 5% CO₂) in order to induce remodeling changes [4].

Plant material

Establishment and confirmation of transformation in TR and AtPAP1 TRroot cultures of L. sibiricus

Briefly, transformed (TR) and transgenic (AtPAP1 TR) roots were induced on 5‐week‐old shoots grown in vitro. Both A. rhizogenes (wild‐type and harboring recombinant vector) cultures were used to TR and AtPAP1 TR roots induction, respectively. Shoot fragments were inoculated with bacterial suspensions under sterile conditions and the plant material was grown in the dark until transformed and transgenic roots appeared at the wound site (i.e. approximately 2 weeks). Elimination of bacteria was carried out by supplementing the culture medium with 500 mg/l ampicillin for TR and 250 mg/l cefotaxime for AtPAP1 TR roots, respectively. Finally, antibiotics were eliminated from the liquid culture medium after a few passages. The culture was carried out under strictly controlled aseptic conditions. The TR used for this study were grown in liquid medium in Erlenmeyer flasks. The transgenic natures of the transformed and transgenic roots were confirmed by PCR using hptII gene‐specific primers (hygromycin B resistance) for AtPAP1 TR roots and rolB and rolC genes for TR clones according to our previous studies [25, 26, 34]. The most productive AtPAP1 transformed root clone was chosen for culture in a 5‐l sprinkle bioreactor [35].

Preparation of TR and AtPAP1 TR extracts for testing

Dried plant material from AtPAP1 TR and TR root line was lyophilized into powder and used for analysis. The analysis was performed as described previously [26].

High‐performance liquid chromatography (HPLC) and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis of phenolic acids

Briefly, chromatographic analysis was carried out using an HPLC system (Dionex, Sunnyvale, CA, USA) equipped with a photodiode array detector. Separation of the compounds was achieved on an RP column (aQ Hypersil GOLD; 250 × 4·6 mm, 5 μm) linked with a guard column (GOLD aQ Drop‐In Guards; 10 × 4 mm, 5 μm; Polygen, Gliwice, Poland) at 25°C using a mobile phase composed of (a) water and (b) methanol, both with 0·1% formic acid. LC‐MS/MS was carried out using API LC/MS/MS system (Applera, Foster City, CA, USA) with electrospray ionization (ESI) source equipped with the Dionex (Lohmar, Germany) HPLC system. Separation was achieved on aQ Hypersil GOLD column (C18, 2·1 × 150 mm, 5 μm) at 30°C using a gradient as described above for HPLC and a flow rate of 0·2 ml/min [25]. The phenolic acid content of both tested root extracts (TR and AtPAP1 TR) was determined by HPLC, as described previously [35].

Experimental procedure

The cultures were exposed to the HRV16 virus for 24 h (33°C, 5% CO₂). Following this, WI‐38 and HFL1 cells were incubated with L. sibiricus TR or AtPAP1 TR extracts at concentrations of 0·07, 0·156, 0·312, 0·625, 1·25, 2·5, 5 and 10 mg/ml appropriate medium for 24 h (37°C, 5% CO₂). The controls were treated with medium only. After assessing viability, 0·156, 0·312 and 0·625 mg/ml of the extracts were selected, and added to HRV16‐infected cells to assess their effect on virus‐induced remodeling. Afterwards, RNA was isolated.

RNA isolation and cDNA synthesis

Total RNA was isolated from the cells utilizing the total RNA mini kit (A&A Biotechnology, Gdynia, Poland). The RNA was subsequently, purified and stored at −80°C. Reverse transcription (RT) using 1 μg of total RNA was performed using high‐capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). The procedures were performed according to the producer’s protocols.

Gene expression analysis

The changes in the expression of MMP‐9, tissue inhibitors of metalloproteinases (TIMP‐1), arginase I and TGF‐β1 were assessed utilizing quantitative polymerase chain reaction (qPCR). TaqMan gene expression assays were used for the selected genes: TIMP‐1, Hs00171558_m1, MMP‐9, Hs00957562_m1, arginase I, Hs00968979_m1 and TGF‐β1, Hs00998133_m1 (Life Technologies, Carlsbad, CA, USA). Each sample was measured in triplicate using the TaqMan analyzer and the 2^−ΔΔCt method was used to calculate gene expression. The results were normalized to an endogenous reference gene (β‐actin, Hs99999903_m1). By comparing RQ (2^−ΔΔCt), the fold change of mRNA expression was calculated.

Protein isolation and immunoblotting

RIPA protein extraction buffer (Sigma‐Aldrich), supplemented with protease inhibitor cocktail (Sigma‐Aldrich), was used to extract total protein, determined subsequently by the bicinchoninic acid (BCA) Protein Assay Kit (Pierce ThermoScientific, Waltham, MA, USA). Electrophoresis was performed utilizing 10 µg protein in denaturing polyacrylamide 4–20% NuPage gel (Invitrogen, Carlsbad, CA, USA) for 60 min (140 V and 110 mA). The specimens were then transferred into a nitrocellulose membrane with the eBlot Protein Transfer System (Genscript, Piscataway, NJ, USA).

The membrane was incubated for 1 h at room temperature with 5% non‐fat milk dissolved in Tris‐buffered saline–Tween 20 (TBST). Subsequently, the membrane was incubated with primary antibodies (mouse) for 12 h at 4°C and then with secondary anti‐mouse immunoglobulin (Ig)G goat antibodies, conjugated with alkaline phosphatase for 90 min at room temperature. All the antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The bands on the membrane were developed using BCIP/NBT alkaline phosphatase substrate (Merck Millipore, Darmstadt, Germany), and then analyzed with Image J version 1.49 software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). Densitometric image analysis was performed and % optical density (OD) was presented over the background.

Statistical analyses

The obtained results were analyzed with software Statistica software (StatSoft, Tulsa, OK, USA). The Shapiro–Wilk and Levene’s tests, respectively, were utilized to check the distribution of data and the equality of variances. Significant changes were calculated with the analysis of variance (ANOVA) test with the appropriate post‐hoc tests as a multiple comparison procedure. P‐values < 0·05 were considered to be statistically significant.

Results

Cytotoxicity of L. sibiricus root extract

As illustrated in Fig. 1, the assessment of cell viability revealed that the AtPAP1 TR and TR root extracts decrease fibroblast viability in a dose‐dependent manner. Therefore, three concentrations were chosen to utilize in qPCR experiments: 0·156, 0·312 and 0·625 mg/ml. The two cell lines demonstrated similar responses to the extracts, and none of the extracts showed cytotoxic effects at the concentrations applied.

Fig. 1.

Viability of Wistar Institute‐38 (WI‐38) and human fetal lung fibroblasts (HFL1) after exposure to varying concentrations (0–10 mg/ml) of Leonurus sibiricus‐transformed (TR) and transcriptional factor from transformed L. sibiricus roots extract with transcriptional factor over‐expression‐transformed (AtPAP1 TR) root extracts. Cell viability was assessed after 24 h. The values represent mean ± standard deviation (s.d.).

AtPAP1 TR root extracts from L. sibiricus modify remodeling‐mediated gene expression

While AtPAP1 TR reduced arginase I expression in both cell lines at 0·312 mg/ml, it only reduced this expression in WI‐38 at 0·625 mg/ml (P < 0·05); this result was confirmed at the protein level (P < 0·05). In contrast, the TR extracts did not change arginase I expression at any concentration (P > 0·05, Fig. 2).

Fig. 2.

Arginase I expression on mRNA (a) and protein (b) levels. (a) mRNA expression presented as relative quantity levels in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblasts (HFL1) (right panel) after exposure to three chosen concentrations of transcriptional factor from transformed Leonurus sibiricus roots extract with transcriptional factor over‐expression‐transformed (AtPAP1 TR) and L. sibiricus TR extracts. The results of quantitative polymerase chain reaction (qPCR) are presented as relative expression in relation to β‐actin. *P < 0·05. Data presented as relative quantity ± standard deviation (s.d.). (b) The results of immunoblotting are presented as % optical density (OD) over background levels. Data presented as mean ± s.d. after incubation with AtPAP1 TR and TR extracts at a concentration of 0·312 mg/ml.

Similar results were obtained in the case of MMP‐9. Reduced mRNA concentrations were observed in WI‐38 following 0·312 and 0·625 mg/ml AtPAP1 TR treatment, and in HFL1 cells after 0·312 mg/ml AtPAP1 TR (P < 0·05). However, these results were not statistically significant for protein levels. Similarly, TR extract did not appear to have any significant influence on mRNA or protein expression (P > 0·05, Fig. 3).

Fig. 3.

Expression of metalloproteinase 9 (MMP‐9) mRNA (a) and protein (b) in fibroblast cell lines. (a) mRNA expression presented as relative quantity (RQ) levels in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblasts (HFL1) (right panel) after exposure to three chosen concentrations of transcriptional factor over‐expression‐transformed (AtPAP1 TR) and Leonurus sibiricus‐transformed (TR) extracts. The results of quantitative polymerase chain reaction (qPCR) are presented as relative expression in relation to β‐actin. *P < 0·05. Data presented as RQ ± standard deviation (s.d.). (b) The results of immunoblotting are presented as % optical density (OD) over background values. Data are presented as mean ± s.d. after incubation with 0·312 mg/ml AtPAP1 TR and TR extracts.

TIMP‐1 gene expression was not changed in the cell lines; however, TR extracts did not increase mRNA TIMP‐1 expression in HFL1 cells (P > 0·05, Fig. 4). Interestingly, although AtPAP1 TR root extract increased TGF‐β expression in WI‐38 and HFL1 cells at concentrations of 0·625 mg/ml, these results were insignificant and not confirmed at protein level (P > 0·05, Fig. 5).

Fig. 4.

Tissue inhibitor of metalloproteinases 1 (TIMP‐1) expression profile represented by mRNA (a) and protein (b) levels in fibroblasts. (a) mRNA expression presented as relative quantity (RQ) levels in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblasts (HFL1) (right panel) after exposure to three chosen concentrations of transcriptional factor over‐expression‐transformed (AtPAP1 TR) and Leonurus sibiricus‐transformed (TR) extracts. The results of quantitative polymerase chain reaction (qPCR) are presented as relative expression in relation to β‐actin. *P < 0·05. Data presented as RQ ± standard deviation (s.d.). (b) The results of immunoblotting are presented as % optical density (OD) over background values. Data are presented as mean ± s.d. after incubation with AtPAP1 TR and TR extracts at a concentration of 0·312 mg/ml.

Fig. 5.

Expression of transforming growth factor (TGF)‐β mRNA (a) and protein (b) levels. (a) mRNA expression presented as relative quantity (RQ) levels in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblasts (HFL1) (right panel) after exposure to three chosen concentrations of transcriptional factor over‐expression‐transformed (AtPAP1 TR) and Leonurus sibiricus‐transformed (TR) extracts. The results of quantitative polymerase chain reaction (qPCR) are presented as relative expression in relation to β‐actin. *P < 0·05. Data presented as RQ ± standard deviation (s.d.). (b) The results of immunoblotting are presented as % optical density (OD) over the background. Data are presented as mean ± s.d. after incubation with AtPAP1 TR and TR extracts at a concentration of 0·312 mg/ml.

Inhibitory effect of AtPAP1 TR root extracts from L. sibiricus on rhinovirus‐16‐induced changes in gene expression in fibroblasts

AtPAP1 TR root extract significantly inhibited the increase in arginase I expression induced by rhinovirus‐16 in both WI‐38 and HFL1 fibroblasts (Fig. 6). In this extract, 43·9% inhibition of arginase mRNA expression was observed at 0·312 mg/ml (P < 0·05) and 42% at 0·625 mg/ml (P < 0·05) in WI‐38 cells. Similarly, 27·3% (0·312 mg/ml) and 32·6% (0·625 mg/ml) reductions were observed in HFL1 cells (P < 0·05).

Fig. 6.

A. Inhibition of rhinovirus 16‐induced arginase I expression by Leonorus sibiricus extracts in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblast (HFL1) (right panel) fibroblast cell lines. Rhinovirus elevated the expression of arginase I; however, it was inhibited by transcriptional factor over‐expression‐transformed (AtPAP1 TR) in both cell lines. Data presented as relative quantity ± standard deviation (s.d.). *P < 0·05. (b) Inhibition of arginase I protein expression in WI‐38 and HFL1 fibroblasts. Inhibition by 0·312 mg/ml AtPAP1 TR was calculated as % decrease following rhinovirus 16‐induced expression. Data presented as % optical density (OD) over background values. Data presented as mean ± s.d. *P < 0·05.

Importantly, AtPAP1 TR extracts inhibited protein expression induced by HRV16, causing a significant reduction in content: 26·7% in WI‐38 and 20·7% in HFL1 cells (P < 0·05).

AtPAP1 TR root extract also inhibited MMP‐9 mRNA and protein expression at concentrations of 0·312 mg/ml and 0·624 mg/ml. In WI‐38 fibroblasts, the reduction of HRV16‐induced expression was 35% (0·312 mg/ml) and 27·7% (0·624 mg/ml) (P < 0·05). In HFL1 cells, inhibition was only observed after incubation with 0·312 mg/ml AtPAP1 TR extract (P < 0·05). MMP‐9 protein was inhibited most effectively in WI‐38 cells after incubation with 0·312 mg/ml AtPAP1 TR extract (P < 0·05, Fig. 7).

Fig. 7.

(a) Reduction of rhinovirus 16 (HRV16)‐induced metalloproteinase 9 (MMP‐9) mRNA expression by Leonurus sibiricus extracts in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblast (HFL1) (right panel) cell lines. Rhinovirus elevated the expression of MMP‐9, which was inhibited by transcriptional factor over‐expression‐transformed (AtPAP1 TR) in both cell lines. Data presented as relative quantity ± standard deviation (s.d.). *P < 0·05. (b) MMP‐9 protein decrease in WI‐38 and HFL1 fibroblasts. The inhibition was calculated as % decrease of rhinovirus 16‐induced expression by 0·312 mg/ml AtPAP1 TR and L. sibiricus‐transformed (TR). Data presented as % optical density (OD) over background values. Data presented as mean ± s.d., *P < 0·05.

Interestingly, neither AtPAP1 TR nor TR root extracts influence TIMP‐1 mRNA or protein expression (P > 0·05, Fig. 8). However, slight changes of TGF mRNA expression were observed as the result of AtPAP1 TR treatment. The dose of 0·312 mg/ml significantly inhibited expression induced by HRV16 (15% in WI‐38, P < 0·05); nevertheless, this was not reflected at protein level (P > 0·05, Fig. 9).

Fig. 8.

(a) Diagrams representing rhinovirus 16 (HRV16)‐induced tissue inhibitor of metalloproteinases 1 (TIMP‐1) expression inhibited by Leonurus sibiricus extracts in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblast (HFL1) (right panel) cell lines. Rhinovirus elevated the expression of TIMP‐1, which was inhibited by transcriptional factor over‐expression‐transformed (AtPAP1 TR) in both cell lines. Data presented as relative quantity ± standard deviation (s.d.). (b) AtPAP1 TR inhibits TIMP‐1 protein expression in WI‐38 and HFL1 fibroblasts. The inhibition is presented as % of decrease of HRV16‐induced expression at a concentration of 0·312 mg/ml. Data presented as % optical density (OD) over background values. Data presented as mean ± s.d.

Fig. 9.

(a) Inhibition of rhinovirus 16 (HRV16)‐induced transforming growth factor (TGF)‐β mRNA expression by Leonurus sibiricus extracts in Wistar Institute‐38 (WI‐38) (left panel) and human fetal lung fibroblast (HFL1) (right panel) cell lines. Rhinovirus elevated TGF‐β mRNA expression, which was inhibited by transcriptional factor over‐expression‐transformed (AtPAP1 TR) in both cell lines. Data presented as relative quantity ± standard deviation (s.d.). *P < 0·05. (b) Inhibition of TGF‐β protein expression in WI‐38 and HFL1 fibroblasts. The histogram presents % inhibition of HRV16‐induced expression by 0·312 mg/ml AtPAP1 TR and L. sibiricus‐transformed (TR). Data presented as % optical density (OD) over background values. Data presented as mean ± s.d. *P < 0·05.

Discussion

The pathogenesis of asthma is complex, with many aspects remaining unconfirmed. Airway remodeling is, next to airway inflammation, one of the major pathological asthma manifestations [36]. The inflammation theory suggests that the major impact, leading to most of the airway remodeling aspects, is chronic airway inflammation.

This is due to the fact that steroid treatment in asthmatic patients, apart from reducing airway inflammation, presents advantageous effects on airway remodeling [5, 37, 38, 39, 40, 41, 42, 43, 44, 45]. However, other studies suggest that the remodeling process may be independent of airway inflammation; however, it can also contribute to the development and persistence of the airway inflammatory process itself [8, 46].

Plant biotechnology methods allow various materials to be obtained in a short time and in large amounts without environmental degradation [47]. The use of plant cell, tissue and organ cultures has overcome several obstacles for the production of secondary metabolites. The most productive in‐vitro cultures are hairy roots derived by A. rhizogenes transformation or by introducing a gene construct that can further increase the production of secondary metabolites by expressing genes involved in the metabolic pathway [48]. One such factor is A. thaliana anthocyanin pigment 1 (AtPAP1), which can significantly increase accumulation of phenolic acids and can activate most of the phenylpropanoid pathway genes [26, 49, 50]. The roots obtained in this manner are also genetically stable, and represent an attractive model for the mass production of high‐value secondary metabolites in a short time. Both transformed (TR) and transgenic (AtPAP TR) roots produced higher levels of secondary metabolites compared to non‐transformed material [26, 47].

The phenolic acid content of L. sibiricus TR and AtPAP1 TR root extracts has been analysed previously [22, 35]. The AtPAP1 TR root extract from the 5‐l sprinkle bioreactor was found to demonstrate greater levels of phenolic acids such as chlorogenic acid, caffeic acid, p‐coumaric acid or ferulic acid compared to the TR extracts. One of the main compounds was chlorogenic acid: 21 mg/g DW for the AtPAP1 TR extract and 4·1 mg/g DW for the TR extract.

Chlorogenic acid (CGA) is an ester of caffeic acid and quinic acid. This polyphenol compound occurs abundantly in fruit and vegetables (e.g. apples, bananas, coffee, beans) [51]. Chlorogenic acid has been suggested to alleviate lipopolysaccharide (LPS)‐induced inflammation and down‐regulate cyclooxygenase‐2 expression – the feature attributed to inflammation in many chronic diseases [52, 53, 54]. Most of the natural products containing CGAs demonstrate anti‐inflammatory effects, and it has been proposed that CGA may be a noticeable anti‐oxidative agent [55, 56]. Chlorogenic acid may have anti‐asthmatic effects [57] and inhibit oxidative stress‐induced secretion of IL‐8 and mRNA expression [58].

CGA is also known as important anti‐oxidant and displays multi‐anti‐viral activities against HSV‐1, HSV‐2 and adenovirus [56, 59, 60]. Although no data exist concerning CGA and rhinovirus, it may have been responsible for inhibition of arginase I, MMP‐9 and TGF‐β expression by the AtPAP1 TR extracts in the present study. Conversely, a study by Tan et al. reported that CGA did not influence TGF‐β expression [61]. According to Yang et al., mRNA and protein expression of TIMP‐1 and TGF‐β1 were inhibited by CGA both in vitro and in vivo. Interestingly, CGA elevated the mRNA and protein expression of MMP‐9 in this study [62]. Those studies are in contrast to our research, which might be a result of different cell types (dermal papilla cells and liver fibroblasts) and/or the presence of HRV16 infection. We suppose that the chlorogenic acid may be responsible for modulating the expressions of the above genes. Moreover, the differences between CGA content may account for the variation observed for the effects of AtPAP TR and TR extracts. It has been reported to attenuate allergic airway inflammation and hyper‐responsiveness in a murine model of ovalbumin‐induced asthma [63]. Caffeic acid phenethyl ester was demonstrated to reduce airway inflammation and remodeling in chronic asthma via the mitogen‐activated protein kinase/protein kinase B (MAPK/Akt) pathway [64].

In turn, p‐coumaric acid has been shown to decrease the production of IL‐8 in cigarette smoke extract (CSE)‐stimulated A549 cells [65], and ferulic acid treatment was shown to suppress one of the main features of airway remodeling, indicated by reduction of mucus production [66], and to present anti‐viral properties against RSV and influenza [67, 68, 69]. Therefore, it is possible that the cumulative action these substances may enable complementary remodeling treatment.

Fibroblasts contribute to airway remodeling by increasing the secretion of the extracellular matrix (ECM). Moreover, ECM components may influence cellular migration and proliferation, with a contribution to increased airway wall thickness [70, 71, 72]. Our results indicate that AtPAP1 TR extracts might modulate the expression of the genes that are involved in airway diseases in two fibroblast cell lines: arginase I was inhibited by AtPAP1 TR treatment. Arginase plays a key role in airway remodeling, inflammation and airway hyper‐responsiveness in chronic asthma [73, 74]. Moreover, in airway cells of animal models as well as patients with allergic asthma, arginase I showed increased expression or activity [74, 75, 76]. Genome‐wide association studies (GWAS) studies showed a strong association of asthma and ARG1 and ARG2 gene variants [77, 78, 79], and augmented arginase activity in serum was shown to be associated to airflow limitation, measured by forced expiratory volume in 1 s (FEV₁) [74, 80].

It is possible that decreasing arginase activity could be a promising target in treating inflammatory airway diseases. It has recently been reported that arginine metabolism controls neutrophilic and eosinophilic pathways which regulate inflammation severity [81]. Moreover, arginase I expression, more than arginase II, was induced in Th2 cytokine‐mediated lung inflammation and allergen‐challenged lungs in mice [82, 83, 84]. It has also been demonstrated that, in bronchoalveolar lavage from asthmatics and in airway epithelium obtained from bronchial biopsies, inflammatory cells show increased mRNA and protein expression of arginase I [83, 84, 85]. Therefore, it seems particularly interesting that the extracts from L. sibiricus roots in the present study significantly reduced the expression of arginase increased by HRV16. As arginase and superoxide dismutase 1 (SOD1) expression are known to be correlated with one another [86], the evaluation of AtPAP1 TR extracts seems promising for further research. Despite this, our results require extension and confirmation, particularly because some of the data concerning arginase I mRNA and protein expression are inconsistent.

The MMPs produced during mesenchymal differentiation are responsible for spreading inflammation, which can take place through the lung tissues and matrix degradation or by chemokine and cytokine processing. This allows immune cell recruitment within the lung [87, 88, 89]. MMP‐9 and TIMP‐1 imbalance plays a role in the inflammatory process and airway remodeling in asthma and expression of MMP‐9 in elevated in asthmatic patients, notably during exacerbations [90, 91, 92]. Therefore, understanding the MMP‐9 expression controlling mechanisms might lead to the design of new therapeutic solutions and/or improve current therapies of asthma. Our present findings indicate that treatment with L. sibiricus root extracts decreased MMP‐9 expression, which was reflected in reduced protein production. Recently, Kim et al. reported that Leonurus sibiricus ethanol extract suppressed the expression of MMP‐9 in osteoblasts [93]. However, our present study provides the first examination, to our knowledge, of the influence of L. sibiricus extracts in fibroblasts in the context of airway remodeling.

Chung et al. [94] reported an increase in MMP‐9 in airways and alveolar macrophages from chronic asthmatics, accompanied by a rapid decline in FEV₁, despite regular treatment with inhaled corticosteroids. This suggests that MMP‐9 could be used as an indicator of chronic airway inflammation. Conversely, increased MMP‐9 levels were also found in acute respiratory tract diseases, including both bacterial and viral pneumonia [95, 96]. Therefore, MMP‐9 level cannot be considered as a specific marker of asthma [89], and its regulation by the extracts tested should be analyzed with a measure of criticism. Nevertheless, based on the literature, it may be assumed that MMP‐9 decrease would be beneficial in the treatment of inflammatory diseases of the respiratory system, in particular airway remodeling.

The expression and activity of MMPs are regulated through the activity of TIMPs and CD147 [97]. By binding to MMPs, TIMPs hinder their enzymatic activity, and TIMP‐1 might be responsible for basement membrane thickening in asthma [94, 98, 99]. Thus, TIMP–MMP imbalance may cause clinical alterations in chronic airway diseases [94]. Interestingly, in our study, TIMP‐1 level was not influenced by exposure to rhinovirus‐16 nor to the TR and AtPAP1 TR extracts (P > 0·05). In turn, TGF‐β1, a cytokine promoting the pathogenesis of several airway diseases involving inflammation and remodeling, appeared to be decreased by the AtPAP1 TR extracts (P < 0·05). As TGF‐β is elevated in the airway of patients with asthma and contributes to airway structural cell remodeling in asthma [100, 101, 102, 103], this effect of AtPAP1 TR appears promising.

TGF‐β1 promotes the differentiation of fibroblasts into myofibroblasts, the proliferation of myofibroblasts and the accumulation of the extracellular matrix by activating the Smad protein family [104]. Recently, Walker et al. presented data showing that a large number of genes are modulated in an airway fibroblast on transdifferentiation to myofibroblasts [105]. Recently, it has been shown that TGF‐β treatment of an airway fibroblast cell line induces fibroblast‐to‐myofibroblast transdifferentiation accompanied by alterations in the TGF‐β signaling pathway: the cells demonstrate decreased Smad3 expression and altered glucocorticoid responses due to changes in the expression of the GR isoforms [106]. Chakir et al. [107] found increased levels of TGF‐β and types I and III collagens to be associated with severe asthma, and suggested that the failure of corticosteroids to decrease collagen deposition might be due to persistently elevated TGF‐β expression. Therefore, the decrease of TGF‐β might represent one of the promising features of L. sibiricus.

We are aware of the limitations of our work, one of which is the inconsistency of some of the data from gene and protein expression. Gene expression is controlled at many different stages and in many different ways; for example, RNA secondary structure, regulatory proteins, regulatory sRNAs, ribosomal density and ribosome occupancy, protein half‐lives or experimental error/noise. The lack of correlation between mRNA and protein expression might be also a result of transcription–translation time difference [23].

Moreover, transcription and translation do not have a linear relationship. Schwanhausser et al. demonstrated that variation in protein expression levels are mainly determined by regulation of translation [108]. Throughout the genome, the correlation between mRNA and protein expression levels is weak, in many studies balancing around 40%. The incongruity is usually due to various regulation levels between transcript and protein [109, 110, 111]. Our study is limited because we did not analyze protein activity separately, especially in cases of inconsistency between mRNA and protein expression. Moreover, the secondary metabolites contained in the tested L. sibiricus extracts could be additionally analyzed separately in terms of their effect on the evaluated gene expressions. As we cannot exclude these aspects during study design, they should be taken into consideration in critical analysis of results from molecular studies. We are also aware that our presumptions regarding potential clinical impact of the results still remain mainly based on speculations, therefore we clearly state that further research are needed in order to explore this issue in depth.

Although inflammation can be a reason for chronic states, tissue damage and pain requiring pharmacological intervention, it is a response of the body to many aggressors [112]. A knowledge of plants could be a valuable source of information, leading to establishing new therapeutic alternatives for inflammatory diseases [19].

The transgenic root extract of L. sibiricus (AtPAP1 TR) alters the expression of genes related to asthma and their effects. However, the molecular‐level effects of L. sibiricus in regulating the disease pathology remain unknown. Our findings also suggest that L. sibiricus can be used as potential treatment of airway remodeling and inflammation.

In conclusion, AtPAP1 TR extract from transformed L. sibiricus root decreases the expression of MMP‐9, arginase 1 and TGF‐β, thus influencing the remodeling process. We suspect that these properties may be attributable to the higher amount of phenolic acids contained in this extract; however, further investigations are needed for wider analysis and a precise evaluation of its mechanisms and other properties.

Disclosures

The authors declare no conflicts of interest.