Curcumin modulates airway remodelling‐contributing genes—the significance of transcription factors

Abstract Bronchial epithelial cells and fibroblasts play an essential role in airway remodelling, due to their protective and secretory functions. There are many studies proving that infection caused by human rhinovirus may contribute to the process of airway remodelling. The beneficial properties of curcumin, the basic ingredient of turmeric, have been proved in many studies. Therefore, the aim of this study was the evaluation of curcumin immunomodulatory properties in development of airway remodelling. Fibroblasts (WI‐38 and HFL1) and epithelial cells (NHBE) were incubated with curcumin. Additionally, remodelling conditions were induced with rhinovirus (HRV). Airway remodelling genes were determined by qPCR and immunoblotting. Moreover, NF‐κB, c‐Myc and STAT3 were silenced to analyse the pathways involved in airway remodelling. Curcumin reduced the expression of the genes analysed, especially MMP‐9, TGF‐β and collagen I. Moreover, curcumin inhibited the HRV‐induced expression of MMP‐9, TGF‐β, collagen I and LTC4S (p < 0.05). NF‐κB, c‐Myc and STAT3 changed their course of expression. Concluding, our study shows that curcumin significantly downregulated gene expression related to the remodelling process, which is dependent on NF‐κB and, partially, on c‐Myc and STAT3. The results suggest that the remodelling process may be limited and possibly prevented, however this issue requires further research.

remodelling. Repeated HRV-induced respiratory illnesses during infancy and early childhood are strongly associated with an increased risk of subsequent asthma. 4 Recurrent human rhinovirus infections are a potential stimulus for remodelling, which leads to the hypothesis that HRV infections may play a central role in the start of the airway remodelling leading to asthma. Recent studies have identified a number of potential pathways that could causally link recurrent HRV infection of the lower airway with airway remodelling and asthma development. Recent studies have identified a number of potential pathways that could causally link recurrent HRV infection of the lower airway with airway remodelling and asthma development. Leigh et al. 5 presented a hypothesis that early childhood HRV infections might play a role in the development of the airway remodelling which manifests even before the confirmed diagnosis of asthma. Rhinovirus has been detected in subepithelial layers and cells, including fibroblasts in asthmatic airways, probably because of a disrupted and inflamed epithelium. 6 It was recently found that fibroblasts from asthmatic patients enhance the replication of rhinovirus and induce a subsequent vigorous proinflammatory response with IL-6 and IL-8 production. 7 There is also a confirmation that HRV infection of the airway epithelium both in vitro and in vivo results in robust upregulation of IL-11 production. 8 This suggests that HRV infection of airway epithelial cells is capable of upregulating a number of mediators implicated in the airway remodelling process.
Furthermore, such rhinovirus replication was augmented in TGFβ-treated fibroblasts from asthmatic patients. 9 The cytopathic effects of viral infection on epithelial cells predispose to an acute inflammatory response and could enhance airway remodelling. 10 Rhinovirus infection also induces cell cytotoxicity and thereby reduces the cell proliferation rate resulting in an impaired repair process of the bronchial epithelial cells. 11 Skevaki et al. 12 demonstrated that RV-specific basal FGF release is time-and dose-dependent and results from both transcriptional upregulation and release from epithelial cells upon induction of cytotoxicity.
Curcumin is a polyphenolic compound derived from roots of popular Indian turmeric plant and used as a spice, but also in food colouring. The beneficial aspects of curcumin intake have been shown previously in many studies, [13][14][15] and curcumin is very popular food ingredient, recommended by WHO at a dosage up to 3 mg per kg of body weight per day. 16 However, its poor solubility and rapid biotransformation to inactive metabolites limit the utility of formulated curcumin; therefore, products that provide >100-fold better absorption than unformulated curcumin are considered highly bioavailable. 17 Immunomodulatory properties of curcumin have been reported recently, 18 and benefits of curcumin in chronic phase of asthma have been investigated due to the complexity and involvement of many factors in the disease. Curcumin, having many beneficial properties, can be used as a potential therapeutic drug for the treatment of asthma. In Chauhan's study, 19 intranasal curcumin effectively attenuated structural changes in asthmatic mice airways which occur either as a result of chronic allergic airway inflammations, injury or the repair process which leads to airway remodelling.
It effectively suppressed recruitment of inflammatory cells to the lungs compared to dexamethasone. Moreover, the study also proved the effect of intranasal curcumin on extracellular matrix depositions and substantial reduction in the replacement of epithelial cells by goblet cells.
Curcumin also affects the level of metalloproteinases (MMPs) in extracellular sites by modulating the inducers, such as growth factors and cytokines (IL-1α, IL-1β, TNFα, TNFβ, HGF etc.), occurring primarily at the transcriptional level and initiated by the binding of the stimulating factor to its cell surface receptor. 20 There are only a few studies concerning the action of curcumin in asthma, but based on the literature, it might be concluded that curcumin is worth further studies in this disease as an anti-inflammatory agent.
The aim of the study was to evaluate the curcumin effect on the airway remodelling-related gene expression in the context of rhinovirus infections, HRV-2 and HRV-16, as the factor which induces changes leading to airway remodelling. We also analysed the action of curcumin under conditions of the knockout of transcription factors-NF-κB, c-myc and STAT3. All the experiments (n = 6) were performed after reaching 80-90% confluence (passage three to nine) by the cells. The viability of the cells was assessed by adding 10 µl of Presto Blue (BD Pharmingen, Franklin Lakes, NJ, USA), and the absorbance was measured at 570 nm.

| Virus preparation and cell infection
Human rhinovirus (HRV) 16 and HRV-2 were purchased from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK). Ohio HeLa cells were infected until cytopathic effects were observed (multiplicity of infection (MOI) of 1, established on the base of literature [21][22][23]. HRV specimens were exposed to the temperature of 58°C for one hour in order to inactivate the virus particles, which was subsequently confirmed by a lack of HRV replication.
The target fibroblast and epithelial cells were infected by the addition of 50 μL vehicle (medium) or HRV16. The cells were incubated for 24 hours (33°C, 5% CO 2 ). 24
Following this, or before infection, the cells were incubated with curcumin (2 μM) for 24 hours (37°C, 5% CO 2 ). The controls were treated with medium only. We have chosen one HRV serotype from each group in order to evaluate potential differences between them.

| RNA isolation and cDNA synthesis
Total RNA was isolated from the cells by using a Total RNA mini kit (A&A Biotechnology, Gdynia, Poland). The RNA was subsequently purified and stored at −80°C. Reverse transcription using 1 μg of total RNA was performed using a 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 metalloproteinase-9, transforming growth factor β1 (TGF-β1), collagen I, disintegrin and metalloprotein-  The membrane was incubated for one hour at room temperature with 5% nonfat milk dissolved in TBST. Subsequently, the membrane was incubated with primary antibodies (mouse) for 12 hours at 4°C and then with secondary anti-mouse IgG secondary antibodies (goat), conjugated with alkaline phosphatase for 90 minutes at room temperature. All the antibodies were purchased from Santa Cruz Biotechnology, Dallas, USA. The bands on the membrane were developed using a BCIP/NBT alkaline phosphatase substrate (Merck Millipore, Darmstadt, Germany) and after that analysed with Image

| Statistical analyses
The obtained results were analysed with software Statistica software (StatSoft, Tulsa, OK). The Shapiro-Wilk test and Levene's test were, respectively, utilized to check the distribution of data as well as the equality of variances. Significant changes were calculated using the ANOVA test with the appropriate post hoc tests as a multiple comparison procedure. p values <0.05 were considered to be statistically significant. TGFβ is strongly implicated in airway remodelling and is released by eosinophils at the site of allergic inflammation and it also promotes metalloproteinase-9 production, so these genes expression is connected. Curcumin decreased the RXFP1 expression in epithelial cells, with no effect on the expression of other genes analysed ( Figure 1F, p < 0.05, Figure 2F). One of the major sites for RXFP1 expression is bronchial epithelium, and epithelial cells are highly secreting. They can have a big impact on the underlying mesenchymal cells, including fibroblasts. Additionally, the effect of rhinovirus was observed in the expression of α-SMA only in fibroblast cells (p < 0.05, Figure 1H) and in the expression of RXFP1 only in epithelial cells (p < 0.05, Figure 1F).

| The effect of curcumin on the modulation of gene expression induced by two rhinovirus serotypes
Interestingly, the effects of HRV-2 were generally stronger in each cell type ( Figure 1A-H and Figure 2A-H). This serotype belongs to a minor HRV group, and these results show the difference between effects of the serotypes of both viruses.

| Analysis of the differences in the order of curcumin stimulation in rhinovirus infections
Half of the samples were HRV-induced cells and then stimulated with curcumin, and the other half of the samples were infected with HRV cells after pre-incubation with curcumin. It appeared that curcumin added to HRV-infected cells (no difference whether it was HRV-2 or HRV-16) has exerted a stronger effect than added at first, before HRV infection.
Curcumin significantly decreased the HRV-induced MMP-9 expression in fibroblasts and epithelial cells, in comparison with HRV samples ( Figure 1A, p < 0.05, and Figure 2A); however, in the case of HRV infection, the effect of curcumin pre-incubations was also statistically significant (p < 0.05, Figure 1A). A similar effect was observed in the TGFβ expression; however, curcumin was effective in decreasing the HRV-2 and HRV-16 effect only in fibroblasts (p < 0.05, Figure 1B). For collagen I, ADAM33 and LTC4S, their expressions were decreased in comparison with HRV only, when curcumin was added to HRV-induced fibroblasts, but most importantly, in case of collagen, the result was confirmed on the protein level (Figure 1C, D, G, p < 0.05 and Figure 2C, D and G). Oppositely, the same effect of curcumin but in epithelial cells was observed only in the RXFP1 expression (HRV-2) ( Figure 1F, p < 0.05, and Figure 2F). These results suggest that curcumin decrease HRV-induced, remodelling-involved genes expression.

| NF-κB, silencing influences transcription of the genes contributing to airway remodelling process
In  Figure 3A, B, E and F). However, both serotypes of HRV analysed, significantly increased collagen I and α-SMA expression in fibroblasts (p < 0.05, Figure 3C and H), as well as ADAM33 and LTC4S in fibroblasts and epithelial cells (p < 0.05, Figure 3D and G).
Interestingly, pre-incubation with curcumin effectively inhibited the effects of HRV-2 in the expression of α-SMA and the effects of HRV-16 in the case of ADAM33 expression, which were the only effects of curcumin in this set of experiments (p < 0.05, Figure 3H and D respectively).

| c-Myc knockout affects influences transcription of the genes contributing to airway remodelling process
Recent study has shown that upregulation of transcription factor c-Myc may affect the asthma airway remodelling. 27  although the RQ levels were significant in comparison with the control sample, they were lower without knockout ( Figure 4A, C, D, F, p < 0.05, and H, Figure 5A, C, D, E and G). We did not detect the changed TGFβ expression ( Figure 4B, Figure 5B). What is important, curcumin significantly decreased the MMP-9 expression in fibroblasts and epithelial cells, causing also a decrease in the HRVinduced expression in this gene with no difference in the order of stimulation (p < 0.05, Figure 4A). Similarly, curcumin decreased the HRV-induced collagen expression in fibroblasts and epithelial cells (p < 0.05), with no effect, however, when added before HRV infection (p > 0.05, Figure 4C). We observed an upregulatory effect of HRV-2 an HRV-16 on the ADAM33, RXFP1 and LTC4S expression in fibroblasts and epithelial cells (Figure 4D, F and G, p < 0.05, Figure 5D, E and F), whereas the α-SMA expression was increased only in fibroblasts (p < 0.05, Figure 4H). Similarly to the experiments without siRNA silencing, curcumin decreased the HRV-induced expression of RXFP1in epithelial cells, with no difference in the order of stimulation (p < 0.05, Figure 4F). No changes were observed in YKL-40 mRNA expression ( Figure 4E). Figure, Figure 6D, G and Figure 7D, E) and α-SMA (in fibroblasts, Figures 6H, 7F), which were inhibited by curcumin only in the case of ADAM33 and α-SMA (p < 0.05). We did not observe any effects in YKL-40 and RXFP1 (p > 0.05, Figure 6E, F). In our study, RXFP1 was the only gene whose expression was significant in epithelial but not in fibroblast cell lines ( Figure 1F).

F I G U R E 3 Action of curcumin (Cu) in c-Myc knockout cells under Rhinovirus infection conditions. Rhinovirus 2 (H2 in the
These results, however, seem to be justified by studies conducted by To confirm this supposition, we knocked out NF-κB and analysed curcumin action. As shown in Figure 3, the effect of curcumin alone or in the infectious condition was inhibited after NF-κB knockout. This shows that curcumin acts through this transcription factor on MMP-9, TGFβ and COLI ( Figure 3A-C respectively). Additionally, as a confirmation of this hypothesis, we observed lower levels of expression in HRV samples; HRV-induced levels of collagen I, ADAM33, α-SMA and LTC4S were also lower than in samples without NF-κB knockout. In asthma, biological effects of curcumin are associated with several signalling pathways which lead to a decrease in the inflammatory cytokine production and cell infiltration as well as to the inhibition of airway hyperresponsiveness. 47

| CON CLUS IONS
In conclusion, the study confirms that curcumin presents a promising therapeutic target, but most importantly, shows for the first time, the importance of the biological context while delivering it. The order of administration of curcumin and the rhinovirus infection, which may have an important contribution to the development of the remodelling process, significantly affects curcumin effectiveness. These results, although they need to be extended and continued, confirm the usefulness of curcumin in a potential anti-remodelling therapy and also indicate the pathways of its action that may be useful in understanding the mechanisms limiting the process of airway remodelling in the future.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.