* & Eung-Gook Kim *
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Emphysema is a chronic obstructive pulmonary disease (COPD) characterized by disruption of the alveolar wall, resulting in enlargement of the air space, and airway inflammation and remodeling accompanied by limitation of expiratory airflow (1-3). Several hypotheses regarding the pathogenic mechanism have been proposed including an imbalance between proteolysis and anti-proteolysis, oxidative stress and anti-oxidative stress, and inflammation (4-6). Although many therapeutic approaches targeting these processes have been attempted, they have so far been unsuccessful. Currently, conservative treatment with bronchodilators and steroids is the preferred treatment (7). Considering the current status of unmet clinical need in the treatment of emphysema, conduct of additional intensive study on cellular and molecular mechanisms of COPD pathogenesis is required.
The senescence hypothesis has recently emerged as a new pathogenic mechanism of COPD (8, 9). Senescence is a cellular state in which cell cycle is arrested at G1/S phase accompanied by characteristic changes including senescence-associated secretory phenotype (SASP) are detected (10). There are two approaches to targeting senescence; development of senolytics capable of killing senescent cells such as dasatanib and quercetin, and senomorphics, which attenuate disease progression by reducing of SASP mediators (11-13). Many components of SASP overlap with those of inflammation (10). Thus, it seems reasonable to assume that senomorphic drugs can be used for treatment of inflammatory diseases such as emphysema.
In our previous study, regorafenib, a multi-receptor tyrosine kinase inhibitor drug, was identified as a potential senomorphic agent capable of exerting a therapeutic effect in porcine pancreatic elastase (PPE)-induced emphysema in mice (14). A key element of the experimental setup for that purpose was to administer regorafenib after the development of emphysematous phenotypes. The PPE model shows active inflammation in the early phase, 3-7 days after instillation of PPE. To further explore its preventive effect on development of emphysema, in the current study, we attempted to examine the anti-inflammatory function of regorafenib. PPE-induced enlargement of air space and deterioration of obstructive lung functions was prevented by earlier administration of regorafenib. In addition, we were able to determine its anti-inflammatory role, reducing the levels of key inflammatory mediators, IL-1β, IL-6, and CXCL1/KC.
Regorafenib, a well-known inhibitor of multi-receptor tyrosine kinases, is approved for therapy of metastatic colorectal cancer, and is also used as the second-line treatment in patients with hepatocellular carcinoma (15, 16). The findings of our previous study demonstrated a therapeutic effect of regorafenib in a PPE-induced animal model of emphysema (which might be in part due to the attenuation of senescence) (14). In the present study, we asked whether regorafenib has a preventive effect on the development of PPE-induced emphysema in mice. To determine whether regorafenib functions as we considered, mice were prepared as three groups and treated with saline (control), PPE, and PPE plus regorafenib (5 mg/kg), respectively. Regorafenib was administered orally for one day before and seven days after instillation of PPE, as shown in Fig. 1A. Two weeks after the termination of regorafenib instillation, measurement of mean chord length (Lm) was performed to assess changes in the volume of alveolar space. Treatment with PPE alone resulted in increased Lm ∼5 times compared with saline-treated control mice (Fig. 1B; quantified in C). Of note, PPE-induced increase in Lm was blocked by ∼50% after treatment with regorafenib (Fig. 1B, C). A lung function test was performed using the flexiVent system in order to further assess the changes in lung function (Fig. 1D and Supplementary Fig. 1). Treatment of PPE induced alterations in the parameters that reflect emphysematous changes; a significant increase in Cst (static compliance), A (total lung capacity), and K (form of deflating PV-loop) (Fig. 1D). These parameters were significantly restored by treatment with regorafenib (Fig. 1D). These results suggest the potential for prevention of PPE-induced emphysema in mice with administration of regorafenib in the earlier phase.
Inflammation has a major function in the development of emphysema. Instillation of PPE induces inflammation in the early phase as a key pathogenic event. A recent study reported on discovery of a regulatory role of regorafenib in LPS-induced neuroinflammation (17). Therefore, we attempted to determine whether regorafenib might exert a protective effect through modulation of PPE-induced inflammatory response. As shown in the scheme (Fig. 2A), regorafenib was administered for two days, and mice were sacrificed on the third day after injection of PPE, and analysis of the bronchoalveolar lavage (BAL) fluid was performed (Fig. 2B-G, and Supplementary Table 1). Cytology was performed for measurement of total and differential cell numbers in BAL fluid. In the PPE group, the total cell count increased to ∼2 × 105/ml compared to 1.2 × 104/ml in the saline group (Fig. 2B, C). In the PPE plus regorafenib group, PPE-induced increase in total cell counts was blocked by treatment with increasing concentrations of regorafenib in a dose-dependent manner (Fig. 2C). The regorafenib effect was extended in a similar manner to differential cell counts; PPE-induced increase in the numbers of recruited macrophages, neutrophils, eosinophils, and lymphocytes in BAL fluid was significantly blocked by regorafenib in a dose-dependent manner (Fig. 2D-G). Taken together, our findings indicated that the inflammatory response is affected by regorafenib at the initial phase in acute PPE-induced events, which may explain the preventive effect in PPE-induced emphysema in mice.
We next examined the expression of inflammatory mediators in the early PPE-induced injury. For this purpose, three groups of mice were prepared as shown in Fig. 2A. BAL fluid was collected and incubated on murine inflammation antibody array blots, and measurement of the optical density was performed to assess the expression levels of each inflammatory mediator (Fig. 3A, B). A number of mediators showed a positive response in the PPE-treated group, but IL-1β, IL-6, CXCL1/KC, and TIMP-1 were reduced on their expression patterns in PPE plus regorafenib group (Fig. 3A, B). To confirm these results, enzyme-linked immunosorbent assay (ELISA) was performed with BAL fluids from three groups of mice. A significant increase in their expression was observed in the PPE-treated group compared with the saline group (Fig. 3C-F). Consistent with the array results, data from ELISA confirmed the reducing effect on the expression of all of these inflammatory mediators (Fig. 3C-F). However, the expression levels of IL-6, CXCL1/KC, and TIMP-1 but not IL-1β were notable. Next, we performed immunohistochemistry (IHC) in PPE-treated tissues for analysis of the expression of pro-inflammatory cytokines (Fig. 4). For this purpose, three groups of mice were prepared as shown in Fig. 4A. As consistently shown in Fig. 3, increased expression of IL-6, CXCL1/KC, and TIMP-1 was observed in the PPE-treated group and decreased expression was observed in the PPE plus regorafenib group (Fig. 4B-E). These regorafenib-responsive mediators might explain the preventive effect of regorafenib in PPE-induced emphysema in mice.
The findings of the current study demonstrated a preventive role of regorafenib in development of PPE-induced emphysema in mice. Inflammation is a key pathogenic event in emphysema; therefore, we focused on its anti-inflammatory activity. In agreement with this idea, both the recruitment of inflammatory cells and the levels of inflammatory mediators were reduced by treatment with regorafenib (Fig. 2-4). Considering the clinically unmet need in the treatment of emphysema, earlier management using an anti-inflammatory approach may aid in slowing the progression of this incurable disease.
Lung macrophages have an essential function in development of emphysema and its progression through regulation of inflammation and immune responses (18). In particular, because of their strategic localization, alveolar macrophages (AMs) are continuously exposed to external pathogens and irritants and they respond by producing proinflammatory mediators such as TNF-α, IL-1β, IL-6, and CXCL1/KC (19-21). The results of our analysis for BAL fluid from PPE-treated mice confirmed a marked increase of AMs (Fig. 2D). In agreement, the levels of their inflammatory mediators also increased (Fig. 2, 3). IL-6 is a major inflammatory cytokine, thus, closely linked with the pathogenesis of emphysema and promotes emphysema (22). CXCL1/KC is released from macrophages (23), and recruits neutrophils which in turn attract monocytes via secretion of monocyte chemotactic protein 1 (24). Thus, CXCL1/KC plays a critical role in the recruitment of neutrophils and monocytes in the inflammatory site. TIMP-1 is a critical regulator of extracellular matrix degradation that contributes to the airway remodeling during the development of emphysema (25, 26). The number of AMs and the expression of all of these mediators except TNF-α were downregulated by treatment with regorafenib in the lung parenchyme as well as BAL fluid (Fig. 3, 4). Notably, TNF-α did not show a response to regorafenib, even though TNF-α is also controlled by NF-κB transcription factors like IL-1β, IL-6, and CXCL1/KC (27). As described above, PPE-induced inflammation can be enhanced by a co-operative network between inflammatory cells and cytokines. The anti-inflammatory activity of regorafenib is believed to exert its preventive effect against development of emphysema.
Regarding the possible mechanism underlying suppression of inflammatory cytokine production by regorafenib, NF-κB transcription factor can be considered a major target. NF-κB enhances inflammatory processes via upregulation of proinflammatory mediators (27). It has been demonstrated that regorafenib effectively alleviated these processes in cancer through inhibition of NF-κB activation (16). Analogously, by targeting NF-κB, regorafenib may reduce the inflammatory processes in emphysema. Indeed, the findings of the current study demonstrated a reducing effect of regorafenib on recruitment of inflammatory cells and secretion of proinflammatory mediators in BAL fluid (Fig. 2, 3). YAP has recently been reported as an upstream regulator of NF-κB activation that contributes to resolution of lung inflammation (28). I-κB, an inhibitory subunit of NF-κB, was identified by the authors as a transcriptional target of YAP. Thus, YAP has the capacity to suppress the activity of NF-κB via upregulation of I-κB. Given that, it is tempting to speculate that regorafenib may inhibit activation of NF-κB through suppression of YAP activity or downregulation of its levels.
Regarding the pathogenic mechanisms of emphysema, our study did not examine the effect of regorafenib on proteolysis and oxidative stress. Of note, PPE-induced upregulation of TIMP-1, which neutralizes matrix metalloprotease (MMPs), was inhibited by regorafenib. PPE-induced upregulation of TIMP-1 may be a compensatory event in response to excessive production/activation of MMPs (29). The molecular mechanism underlying downregulation of TIMP-1 by regorafenib remains to be determined. A close association between inflammation and oxidative stress has been reported (30), thus, we can speculate on the potential of regorafenib in reducing oxidative stress.
The PPE-induced emphysema model has several disadvantages; it does not allow for complete simulation of human emphysema, where lesions occur mainly within the centrilobular area and show progression over a long-term period (31). Thus, the findings of this study should be cautiously extrapolated to human emphysema. However, this model has some advantages with regard to acute development and widespread panlobular lesions (32). In particular, this model provides a tool for use in analysis of inflammatory signaling, upstream and downstream mediators (33). Regarding future study, examination of the regorafenib effect in a model of cigarette smoke exposure that features slow development of the phenotypes of human emphysema would be meaningful.
In summary, our findings demonstrate that development of emphysema can be attenuated by treatment with regorafenib by reducing the inflammatory process and restoring lung function. Thus, oral administration of regorafenib may represent a novel approach in prevention of emphysema. Along with the findings of our previous study demonstrating the senomorphic effect of regorafenib, the current findings suggest its potential application to inflammatory diseases including pulmonary emphysema.
All chemicals were of analytical grade. Porcine pancreatic elastase (PPE) was purchased from Elastin Products Company, Incorporated (Owensville, MO, USA), Regorafenib was purchased from Selleck Chemicals (Houston, TX, USA). ZoletilⓇ50 was purchased from Virbac (Carros Cedex, France), and Rompun was purchased from Elanco (Buenos Aires, Argentina). Hematoxylin and Eosin Y solution, biotinylated secondary antibody, red blood cell lysis buffer, and 3,3’-Diaminobenzidine (DAB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hanks’ Balanced Salt Solution and Proteinase K were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Mice (C57BL/6) were purchased from Daehanbiolink (Eumseong, Korea). ELISA kits for mouse IL-1β (#MLB00C), IL-6 (#M6000B), CXCL1/KC (#MKC00B-1), and TIMP-1 (#MTM 100) were purchased from R&D Systems (Minneapolis, MN, USA). Anti-IL-6 antibody (ab208113) from abcam (Cambridge, UK), CXCL1 antibody (12335-1-AP) from Proteintech (Rosemont, IL, USA), and TIMP-1 antibody (#AF980) from R&D Systems were purchased respectively. Mouse inflammation antibody array (Cat # ab133999) was purchased from abcam. NovaUltraTM Hema-Diff Stain Kit was obtained from IHC WORLD (Woodstock, MD, USA).
All experiments using mice were submitted to the Chungbuk National University Animal Experiment Ethics Committee and conducted according to the approved protocol (CBNUA-1639-21-02). Mice (eight weeks old) were randomly divided into three or five groups in each experiment set, and each group was composed of 3-8 animals per experiment. PPE (0.2 U/kg) was administered in mice anesthetized with Zoletil plus Rompun (Zoletil:Rompun:Saline = 1:1:8) using intranasal instillation. In the PPE plus regorafenib group, regorafenib was administered by oral gavage at 5 mg/kg once a day for the indicated times. Following collection of the lungs, the lung function test, BAL fluid analysis, inflammation antibody array, ELISA, and immunohistochemistry were performed.
The lung tissues were prepared as paraffin sections, followed by staining with hematoxylin and eosin (H&E). Five random fields per mouse were photographed under a microscope using a digital camera (× 200 magnification), and an analysis was performed using ImageJ software. Estimation of the mean chord length (Lm) of the airspace was based on the average size of alveoli.
The lung function test was performed using the flexiVent FX system (SCIREQ Inc., Montreal Qc, Canada). Following administration of anesthesia, mice were connected to the flexiVent system through a catheter inserted in the airway. Measurement of various parameters of perturbation was performed using the flexiVent system according to the manufacturer’s instructions. Calculation and quantification of each parameter was based on an average of three measurements per mouse.
An incision was made in the neck skin of the anesthetized mouse using surgical forceps and scissors, and the airway was cut to approximately 1/3. A catheter was placed through the clipped prayer, tied, and secured using surgical sutures. One ml of Hanks’ Balanced Salt Solution (HBSS) was added to a 1 ml syringe, connected to the catheter, injected through the respiratory tract, and then recollected three times to obtain BAL fluid. The BAL fluid was collected and centrifuged for 15 min at 1,500 rpm at 4°C. The supernatant was then rapidly frozen at −80°C in order to quantify the expression of cytokines. For analysis of immune cells, BAL cell pellets were lysed with red blood cell lysis buffer, neutralized with HBSS, and centrifuged at 1,500 rpm for 15 min at 4°C. BAL cells were then prepared by cytocentrifuging using CellSpin (Hanil Scientific Inc., Korea). In brief, BAL cells were centrifuged at 600 rpm for 15 min in Cellspin, and slides were stained using the NovaUltraTM Hema-Diff Stain Kit according to the manufacturer’s instructions. Five different areas from each stained slide were photographed under a microscope, and counting and analysis of stained cells was performed using ImageJ software.
For analysis of cytokines, mouse inflammation antibody array was performed according to the manufacturers’ protocols. In brief, antibody array membranes were blocked using 10% fetal bovine serum (FBS) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, followed by incubation with BAL fluid at 4°C overnight. The membranes were washed with TBS-T, followed by incubation with paired horseradish peroxidase conjugated secondary antibody. The ECL system was used for detection of immuno-reactive spots using the ChemiDoc imaging systems (Bio-Rad Laboratories, Inc. CA, USA). ImageJ software was used for quantification of immunoreactive dot intensities.
ELISA was performed according to the manufacturers’ protocols. In brief, 50 μl BAL fluid (in triplicate) were incubated on 96-well plates for 2 h at 20°C, followed by aspiration and washing, and then added to the secondary antibody on each well for 2 h at 20°C. Finally, the substrate solution was added to wells and incubated for 30 min, and the reaction was terminated with stop solution. Optical density was determined using a microplate reader set to 450 nm. Analysis and graphing of the detected data was performed using GraphPad Prism 8.0 software.
Following fixation with formalin, the tissues were embedded in paraffin solution. Tissue sections with a thickness of 4 μm were deparaffinized and an antigen retrieval reaction was performed in 10 μg/ml proteinase K solution or 10 mM sodium citrate buffer (pH 6.0). Following antigen retrieval, slides were incubated in blocking buffer (1% BSA, 0.1% cold fish skin gelatin, 0.5% Triton X-100, 0.05% sodium azide) for 1 h. The slides were then incubated in the primary antibody solution at 4°C overnight, followed by washing three times with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T). For diaminobenzidine-HCl (DAB) staining, slides were treated with 0.3% hydrogen peroxide at room temperature (RT) for 20 min, followed by treatment with a biotin-conjugated secondary antibody for 1 h at RT, and with peroxidase-conjugated streptavidin for 30 min at RT. Finally, detection of the signals was performed using the substrate DAB and an analysis was performed using ImageJ (Plugins, Colour deconvolution) and GraphPad Prism 8.0 software.
Data are expressed as mean ± standard error of mean (SEM). An analysis of representative data from at least three independent experiments was performed. P < 0.05 was considered statistically significant. Assessment of statistical significance was performed using an unpaired Student’s t-test (t-test) or One-way ANOVA, and the results are shown in figure legends and graphs (GraphPad Prism software).
The authors have no conflicting interests.
Fig. 1. Preventive effect of regorafenib on development of PPE-induced emphysema in mice. (A) Experimental scheme. In the PPE + regorafenib (Reg) group, regorafenib (5 mg/kg) was administered orally for one day for sensitization before and consecutive seven days after treatment with PPE via the intranasal route. At three weeks after treatment with PPE, mice were analyzed to measurement of Lm and lung function. (B) Representative H&E stained images. Scale bar indicates 100 μm. (C) Quantification of Lm. N = 6/group. (D) Lung function test. Cst (static compliance), A (total lung capacity) and K (form of deflating the PV-loop) parameters are shown. N = 6/group. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, t-test.
Fig. 2. Effect of regorafenib on PPE-induced inflammatory cells in BAL fluid. (A) Experimental scheme. Regorafenib (5 mg/kg) was sensitized and administered orally in PPE + Reg group for two days after treatment with PPE via the intranasal route. (B) Representative images of stained BAL cells. Scale bars indicate 100 μm. (C-G) Measurement of cell numbers in BAL cells (N = 3 per group). Total number of cells (C), macrophages (D), neutrophils (E), eosinophils (F), and lymphocytes (G) were counted on five areas of stained slides. Error bars indicate the mean ± SEM. **P < 0.01, ****P < 0.0001, One-way ANOVA.
Fig. 3. Effect of regorafenib on PPE-induced in inflammatory mediators in BAL fluid. (A) Representative membrane images of the antibody array. (B) Quantification of relative optical density for IL-1β, IL-6, CXCL1/KC, and TIMP-1 in the array membranes. (C-F) Measurement of IL-1β (C), IL-6 (D), CXCL1/KC (E), and TIMP-1 (F) levels in BAL fluid by ELISA. Error bars indicate the mean ± SEM. N = 8 per group, *P < 0.05, **P < 0.01, ****P < 0.0001, t-test.
Fig. 4. Effect of regorafenib on PPE-induced expression of inflammatory mediators in lung parenchyme. (A) Experimental scheme. Mice were sensitized by treatment with regorafenib (5 mg/kg) for one day in the only PPE + Reg group. After PPE treatment, regorafenib was administered orally to mice for six days. On the seventh day, lung tissues were removed from mice, prepared in paraffin sections and stained with antibodies for inflammatory mediators. (B) Representative images of stained paraffin sections. Red arrows indicate the macrophages in inserted boxes. Scale bars indicate 50 μm. (C-E) Quantification of Fig. 4B. IL-6 (C), CXCL1/KC (D), and TIMP-1 (E). Error bars indicate the mean ± SEM. N = 5 per group, *P < 0.05, **P < 0.01, t-test.
Author ORCID Information
Eun-Young Shin Eung-Gook Kim
Funding Information National Research Foundation of Korea Ministry of Science ICT and Future Planning Chungbuk National University |
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