Ethyl Pyruvate Improves Pulmonary Function in Mice with Bleomycin-induced Lung Injury as Monitored with Hyperpolarized 129Xe MR Imaging.

PURPOSE: High Mobility Group Box1 (HMGB1), which is one of the damage-associated molecular pattern molecules relating to various inflammatory diseases, has gained interest as a therapeutic target because of its involvement in wound healing processes. In the present study, we investigated HMGB1 as a potential therapeutic target in a model of lung fibrosis using a preclinical hyperpolarized 129Xe (HPXe) MRI system.
METHODS: Lung injury was induced by intra-peritoneal injection of bleomycin (BLM) in 19 mice. Three weeks post-injection (when fibrosis was confirmed histologically), administration of ethyl pyruvate (EP) and alogliptin (ALG), which are down- and up-regulators of HMGB1, respectively, was commenced in six and seven of the 19 mice, respectively, and continued for a further 3 weeks. A separate sham-instilled group was formed of five mice, which were administered with saline for 6 weeks. Over the second 3-week period, the effects of disease progression and pharmacological therapy in the four groups of mice were monitored by HPXe MRI metrics of fractional ventilation and gas-exchange function.
RESULTS: Gas-exchange function in BLM mice was significantly reduced after 3 weeks of BLM challenge compared to sham-instilled mice (P < 0.05). Ethyl pyruvate was found to improve HPXe MRI metrics of both ventilation and gas exchange, and repair tissue damage (assessed histologically), to a similar level as sham-instilled mice (P < 0.05), whilst ALG treatment caused no significant improvement of pulmonary function.
CONCLUSION: This study demonstrates the down-regulator of HMGB1, EP, as a potential therapeutic agent for pulmonary fibrosis, as assessed by a non-invasive HPXe MRI protocol.

Introduction

In recent years, hyperpolarized (HP) noble gas MRI has been developed as a powerful tool for functional imaging of lung.[1,2] In particular, hyperpolarized 129Xe (HPXe) MRI can be applied to a wide range of disease phenotypes since it affords information about gas exchange in addition to ventilation function. Sensitivity to gas exchange arises from the fact that HPXe dissolves in alveolar tissue and blood and exhibits a distinct chemical shift from that of gaseous-phase HPXe in the lung airspaces.

We have developed a home-built continuous-flow type Xe polarizer for production of HPXe for preclinical applications, which allows the acquisition of functional images of the lung from spontaneously breathing mice using a preclinical MRI system.[3] Previously, we used this system to evaluate the efficacy of the drug ethyl pyruvate (EP) for treating chronic obstructive pulmonary disease (COPD).[4] In that study, we suggested that High Mobility Group Box1 (HMGB1), which is one of the damage-associated molecular patterns (DAMPs), is involved in COPD disease progression.

The HMGB1 is known to play a crucial role in various inflammatory lung diseases, including COPD.[5,6] When HMGB1 released from necrotic or apoptotic cells binds to its receptors, which are receptor for advanced glycation end (RAGE) products, and toll-like receptors (TLRs) 2 and 4, it activates the transcription factor nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, as shown in Fig. 1.[7-9] Somewhat paradoxically, a moderate expression of HMGB1 has been shown to be linked to wound healing processes through the activation of extracellular signal-regulated kinase (ERK) 1/2 involved in the MAPK pathway, while its overexpression exacerbates inflammation and pathology through NF-κB activation.[10] Ethyl pyruvate has previously been reported to dose-dependently activate ERK 1/2 through inhibition of HMGB1.[11-14] Combining these facts with our previous observations of EP treatment efficacy for a murine COPD model stated above, we hypothesized that EP could show therapeutic efficacy for other lung diseases via wound healing effects by ERK 1/2 activation, following anti-inflammatory responses by NF-κB down-regulation.

Fig. 1

Schematic representation of the extra-cellular production of High Mobility Group Box1, its subsequent binding with receptors receptor for advanced glycation end and toll-like receptors 2/4, and the corresponding activation of cellular signaling pathways. ERK, signal-regulated kinase; HMGB1, High Mobility Group Box1; MAPK, mitogen-activated protein kinase; NF-κB, factor-kappa B; RAGE, receptor for advanced glycation end; TLR, toll-like receptor.

Pulmonary fibrosis is characterized by progressive and irreversible scar formation and alveolar septal wall thickening, which lead to severe respiratory dysfunction and ultimately death. Steroids and immunosuppressants have been found to be ineffective for improving prognosis.[15] At present, two novel drugs, nintedanib and pirfenidone, which have shown efficacy for slowing the decline of lung function, are still in a regime of conditional recommendation against use.[16] As such, preclinical studies have been actively pursued in order to develop novel therapeutic drugs using animal models of fibrosis, such as bleomycin (BLM)-challenge for mice.[17,18] In recent studies, the transforming growth factor-β1 (TGF-β1) involved in the Smad signaling pathway has been widely investigated as a potential therapeutic target.[19-20] However, to the best of our knowledge, there exist only one study reporting on the involvement of HMGB1 and the effect of EP in lung fibrosis progression, in which wound healing was not investigated.[11]

In the present study, we monitored the response of pulmonary function to treatment with EP in a BLM mouse model of fibrosis by HPXe MRI, following the non-invasive preclinical MRI protocol presented in Ref. 4. Additionally, in order to further investigate the relationship between HMGB1 and ERK 1/2 expression in disease and treatment processes, the efficacy of a Dipeptidyl Peptidase-4 (DPP-4) inhibitor, alogliptin (ALG), was also monitored because ALG is known to down-regulates ERK 1/2 expression probably through the inhibition of endogenous HMGB1 protease, DPP4.[21,22] The results were compared to support the involvement of ERK 1/2 expression in wound healing processes.

Methods

Animal preparation

All experimental and animal care procedures conformed to the guidelines of Osaka University.

A total of 24 male, 6-week-old, ddY mice weighing 30–35 g were purchased from Japan SLC, Inc. (Shizuoka, Japan). Mice were divided into four groups: 1) sham-instilled group (N = 5), 2) BLM-challenged group (N = 6), 3) EP-treated group (N = 6), and 4) ALG-treated group (N = 7). To induce fibrotic lung injury, a 75 μL saline solution of BLM (1.2 U/kg body weight; Sigma-Aldrich, St Louis, MO, USA) was intratracheally administrated to each mouse of the BLM-challenged, EP- and ALG-treated groups, while the sham-instilled mice were intratracheally administered with a 75 μL saline solution.[23] Three weeks after BLM-challenge, EP and ALG administration was commenced in the EP- and ALG-treated group, respectively, following a similar protocol to that in Ref. 4. Briefly, a 40 μL solution of EP (2.6 mg/kg body weight; Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) or ALG (200 mg/kg body weight; Takeda Pharmaceutical Co., Ltd, Osaka, Japan) in saline was administered to each mouse in the EP- or ALG-treated group every weekday for an additional 3 weeks. Therefore, in total, 6 weeks were required to prepare the EP- and ALG-treated groups. The sham-instilled mice were intratracheally administered with 40 μL saline solution every weekday for the additional 3 week period, in order to ensure the same instillation process for all mice. In all cases, mice were anesthetized with 2% isoflurane (ISOFLU; Sumitomo Dainippon Pharma Co. Ltd, Osaka, Japan) prior to instillation. The survival rates of the whole 6-week procedure were 100%, 100%, 86%, and 100% for BLM-challenged, EP-treated, ALG-treated and sham-instilled groups, respectively.

Magnetic resonance imaging

The MRI measurements of all groups were performed at 3, 4, 5, and 6 weeks after commencement of saline or BLM challenge (i.e., for EP- and ALG-treated groups; 0, 1, 2, and 3 weeks from commencement of EP or ALG treatment). Immediately before all MR measurements, mice were anesthetized with 2% isoflurane, and non-invasive respiratory-gated imaging was performed without tracheal intubation.

All MRI measurements were performed on an Agilent Unity INOVA 400WB high-resolution NMR system equipped with VNMR 6.1C software (Agilent Technologies, Inc., Santa. Clara, CA, USA). To acquire respiratory-gated images from spontaneously breathing mice, the protocol described previously was used.[4] A respiratory sensor was employed to synchronize the acquisition of HPXe lung images with inspiratory or expiratory phases.

A home-built continuous-flow type 129Xe polarizer was used to produce HPXe.[24] A gas mixture of HPXe and N2 (Xe in natural abundance, 129Xe fraction 0.26: 70%, N2: 30%) was continuously delivered to the mouse in the MRI scanner at a flow rate of 50 mL/min. Mice spontaneously breathed the polarized gas after it was mixed with O2 (flow rate 9 ml/min). Hyperpolarized 129Xe images were acquired using a balanced steady-state free precession sequence modified by adding four 180°-pulses or two 90°-pulses in order to extract information about pulmonary gas-exchange or ventilation function, as described previously.[4,25] Acquisition parameters are replicated here for completeness: 1000-μs Gaussian-shaped radio-frequency (RF) pulse of flip angle α = 40°; acquisition bandwidth, 88 kHz; TR/TE = 3.6 ms/1.8 ms; echo train length, 8; number of shots, 4; number of averages, 8; coronal slice thickness, 20 mm; matrix, 64 × 32 with a FOV of 80 × 25 mm2.

Pulmonary functional evaluation by HPXe MRI

Metrics of fractional ventilation, r and xenon gas exchange rate, f, of HPXe were evaluated as reported previously.[4] Only brief details are reproduced here for completeness.

First, r the alveolar volume fraction of gas turned over in a single breath, was evaluated. After gas-phase HPXe magnetization in the lung was destroyed by a saturation pre-pulse, a set of inspiratory images was acquired after n breathing cycles. Value n was incremented from 1 to 10 in steps of one, and then to 12, 15, and 20; in total, 13 images were acquired. From these images, a r map was obtained by pixel-by-pixel linear least squares fitting of the signal intensity as a function of n, using in-house MATLAB (The MathWorks, Inc., Natick, MA, USA) routines.

The xenon polarization transfer contrast (XTC) method was used to evaluate the parameter f, the rate of HPXe magnetization diffusing from the gas-phase to dissolved-phase within a given exchange time.[26] The XTC measurement was performed at the end-expiratory phase, and an f map was derived by pixel-by-pixel analysis using in-house MATLAB routines as for r.

Finally, r or f maps were reprocessed with a 2 × 2 median filter, and r or f values were averaged over the whole of the lungs for each mouse.

Histology

After HPXe MR measurements were completed, mice were killed with a lethal dose of carbon monoxide gas. Lungs were extracted, immersed in 10% formalin at 25 cmH2O and processed for histology by staining with hematoxylin and eosin (H&E). Slides were evaluated to assess morphological changes in the lungs after 6 weeks. Coronal H&E-stained lung images were obtained from each mouse. Five regional images (right upper, middle and lower lobes, and upper and lower regions of the left lung, each of dimensions 175 μm ×131.25 μm) were then captured from each coronal slide using a digital microscope (Celestron LCD Microscope PRO ; Celestron, LLC., Torrance, CA, USA). Septal wall thickness, h (μm), values were calculated in the five regions from four separate slices and averaged for each mouse. All digital images were processed using ImageJ (National Institutes of Health, Bethesda, MD, USA).

In order to monitor morphological changes induced 3 weeks after BLM administration (i.e., the beginning of MRI measurements), an additional histological analysis group (HA_3w, N = 5 mice) was prepared 3 weeks post-BLM-challenge using the same protocol as described above.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) with Tukey–Kramer post-hoc analysis to identify differences between groups. All data are presented as mean ± standard deviation (SD) or box-and-whisker plots, and differences are considered significant at the P < 0.05 level.

Results

Figure 2 displays representative, Fig. 2a f and Fig. 2br maps obtained from the sham-instilled, BLM-challenged, EP-treated, and ALG-treated groups. Figure 3 shows longitudinal changes in the group-mean values of f and r, respectively, at 3–6 weeks post-BLM or saline challenge. The administration of EP and ALG was commenced at week 3, when a significant decrease of mean f value for BLM-challenged (4.8 ± 1.0), EP-treated (f = 4.8 ± 0.8) and ALG-treated (f = 4.8 ± 1.4) groups compared to that of the sham-instilled group (f=7.4 ± 1.1) was observed (P < 0.05). The mean f value of the EP-treated group gradually increased over the course of the treatment and was significantly larger (f = 6.8 ± 0.8) than that of the BLM-challenged group (f = 4.3 ± 0.9) at week 6 (P < 0.05), while the BLM-challenged group showed relatively little change in f during the whole observation period. On the other hand, the mean f values of the ALG-treated group at weeks 5 and 6 were still significantly smaller than those of the sham-instilled group.

Fig. 2

Representative parametric maps of (a) fD and (b) ra derived from longitudinal studies of mice in each of the four groups, from top to bottom: sham-instilled; bleomycin (BLM)-challenged; ethyl pyruvate (EP)-treated; alogliptin (ALG)-treated. In all cases, the horizontal direction shows the time course from the initial intra-tracheal injection of BLM or saline.

Fig. 3

Box plots of the temporal change of mean (a) fD and (b) ra values for all mice, separated by group, as a function of time post-intratracheal injection of bleomycin or saline. Significant differences between groups are indicated by solid lines (*: P < 0.05). ALG, alogliptin; BLM, bleomycin; EP, ethyl pyruvate; IT, intra tracheal.

The mean r value of the EP-treated group was (r = 0.24 ± 0.02) at week 6, a significant improvement compared with week 3 (r = 0.20 ± 0.02) (P < 0.05) and larger than that of the BLM-challenged and ALG-treated groups (r = 0.16 ± 0.03, r = 0.15±0.05, respectively) (P < 0.05). The mean r value of the ALG-treated group remained low compared with the sham-instilled group, i.e., at a similar level to that of the BLM-challenged group, at weeks 4–6.

Figure 4a depicts group-mean values of the septal wall thickness, h, and Fig. 4b representative histological results for all groups including the histological analysis group (HA_3w). The mean h value of the HA_3w group (mice killed after 3 weeks of BLM-challenge) was hHA_=10.8±1.7 μm, significantly larger than that of the sham-instilled mice at the same time-point (P<0.05). Similarly, terminal (6-week) h values of BLM and ALG-treated groups (h_BLM-challenged = 10.8 ± 1.9 μm and h_ALG-treated = 11.6 ± 1.6 μm) were larger than that of the sham-instilled group (h_sham-instilled = 6.4 ± 0.7 μm, P < 0.01), while the mean h of the EP-treated group was significantly smaller than that of both the BLM and ALG-treated groups (h_EP-treated = 7.6 ± 1.0 μm, P < 0.05) and comparable to that of the sham-instilled group (P > 0.05).

Fig. 4

(a) Box plots of the mean septal wall thickness (h) obtained from mice in each of the five groups, from left to right: sham-instilled; histological analysis (HA_3w), bleomycin -challenged; ethyl pyruvate-treated; alogliptin-treated. (b) Representative examples of hematoxylin and eosin stained histology slides obtained from each group. ALG, alogliptin; BLM, bleomycin; EP, ethyl pyruvate.

Figure 5 shows the correlations between MRI metrics of lung function and the histological h value, evaluated at week 6. A significant, positive correlation was found between r and f (P < 0.001), while negative correlations were found between h and f, and h and r (P < 0.001).

Fig. 5

Correlations between hyperpolarized 129Xe (HPXe) MRI-derived parameters of pulmonary function (ra and fD), and histology-derived septal wall thickness (h) obtained from the sham-instilled (□), bleomycin-challenged (▲), ethyl pyruvate-treated (○), and alogliptin-treated (△) groups after the 6-week experimental protocol. Pearson’s r values and P values of statistical significance are noted in each plot.

Discussion

In the present study, the temporal changes of pulmonary ventilation and gas-exchange function in mice treated with EP and ALG as therapeutic agents for BLM-induced lung injury were monitored by HPXe MRI for 3 weeks, treatment commencing 3 weeks post-BLM-challenge. According to Moeller et al,[17] the efficacy of anti-fibrotic agents should be evaluated at least 7 days after BLM-challenge and in the phase of established fibrosis rather than the phase of inflammation. In our measurements, week 3 was determined as the point where sufficient symptoms of fibrosis were observed; a significant decrease of the mean f values for the BLM-challenged, EP-treated and ALG-treated groups compared to that of the sham-instilled group. This choice was supported by histological measurements of significantly elevated h in the HA_3w group. Therefore, the administration of EP was commenced from this time-point. However, we note one report in which EP treatment was investigated for 2 weeks after only 3 days of BLM-challenge,[11] thus, the choice of timing for commencement of fibrosis treatment is still a matter of debate.

At week 4, both f and r values tended to be slightly lower than those at week 3; however, after week 4, both metrics remained approximately constant for the BLM-challenged group (i.e., the absolute values of the metrics did not significantly degrade for the remainder of the observation period) (f = 4.8 ± 1.0 vs. f = 4.3 ± 0.9, r = 0.21 ± 0.04 vs. r = 0.16 ± 0.03). However, it should be recalled that a typical feature of BLM-induced lung injury is partial reversibility of pathology over time[15]; initially, fibrosis occurs following an acute inflammation stage with the expression of signals such as TGF-β1, but subsequently, the inflammatory reactions gradually disappear and the pathology proceeds with fibrotic lung remodeling.[27,28] Babin et al. reported that inflammation in a rat model was markedly attenuated after 21 days of BLM-challenge.[29] Therefore, our observations suggest the acute inflammatory reaction attenuated over time, but since fibrosis was already established around weeks 3–4, f and r did not change significantly (i.e., implying that fibrosis predominantly determines the pulmonary functional status and is irreversible, as expected).

The observation of a significant correlation of f and r values at week 6 (Fig. 5) suggests the BLM model acted to simultaneously impair both ventilation and gas-exchange function. Previously, we reported that reductions in f and r were indicative of septal wall destruction and bronchial wall thickening for a murine COPD model.[4] In that study, the reduction in f correlated with both septal wall and bronchial wall thicknesses, while r correlated with bronchial wall thickening only. Conversely, in the present study, both metrics were shown to be related to septal wall thickening associated with fibrotic lung remodeling (Fig. 5).

Longitudinal observation of f and r enabled quantification of the treatment efficacy of EP. The BLM-induced low f and r values at week 3 were recovered to a similar level as the sham-instilled group by 3 week’s administration of EP (f = 6.8 ± 0.8, f = 7.4 ± 1.3 and r = 0.24 ± 0.02, r = 0.24 ± 0.03, respectively). Since the administration of EP was commenced after 3 weeks of BLM-challenge (i.e., after a significant impairment of pulmonary function was observed), we believe that the measured action of EP was indeed therapeutic rather than preventative against BLM-induced lung injury.

Ethyl pyruvate is known to down-regulate the HMGB1 level through NF-κB inhibition, and moderately up-regulate ERK 1/2 expression downstream of the HMGB1/RAGE signaling pathway.[14,30,31] Hence, the action of EP provides some insight into the involvement of HMGB1 and ERK 1/2 in fibrosis development via the BLM model as shown in Fig. 6a. According to Ebina et al., gradual increase of HMGB1 expression after the inflammatory response stage exacerbates fibrosis.[32] Ethyl pyruvate administration may ameliorate this fibrotic pathology by down-regulation of HMGB1 and up-regulation of ERK 1/2, the latter which is reportedly involved in tissue repair processes.[10] It is also worth noting that Yupingfeng, a Chinese herbal remedy for respiratory ailments, has been found to inhibit HMGB1, which caused a reduction in TGF-β1 production and subsequently diminished collagen deposition in a BLM model of pulmonary fibrosis.[33]

Fig. 6

Proposed contribution of ethyl pyruvate (EP) to fibrosis therapy in the bleomycin model of lung fibrosis. (a) Ethyl pyruvate down-regulates the HMGB1 expression through NF-κB inhibition and moderately activates ERK 1/2, thereby initiating wound healing process. (b) ALG, which up-regulates the HMGB1 expression and deactivates ERK 1/2, did not show a positive effect for fibrosis therapy. BLM, bleomycin; MAPK, mitogen-activated protein kinase; NF-κB, factor-kappa B; RAGE, receptor for advanced glycation end; TLR, toll-like receptors.

In the present study, the septal wall thickness, h, of the EP-treated group was found to be significantly smaller than that of the BLM-challenged group (Fig. 4). This result, combined with the recovery of mean f and r suggests that the EP therapy directly acted against fibrotic tissue damage via ERK 1/2 activation and led to the recovery of pulmonary function in BLM-challenged mice. In order to further elucidate the involvement of ERK 1/2 in tissue repair processes, ALG was administered to BLM-challenged mice, since it is known to down-regulate ERK 1/2 (i.e., opposite to EP) probably through the inhibition of endogenous HMGB1 protease, DPP-4 (Fig. 6b).[21,22] A positive treatment response with ALG was not observed; the longitudinal changes in mean f and r of the ALG-treated group showed similar tendency to those of the BLM-challenged group, and the h value of the ALG-treated group was comparable to the BLM-challenged group and significantly larger than that of the sham-instilled group. These findings indicate that ALG did not act to repair lung tissue damage induced by BLM by ERK 1/2 deactivation and hence caused no significant changes in pulmonary function during the observation period (Fig. 3). In conclusion, we propose that the observed efficacy of EP for BLM-induced lung injury is mediated by regulation of HMGB1 and ERK 1/2 expression. EP is reported to have low toxicity and to be safe at clinically relevant doses, and has recently been enrolled in preliminary clinical trials.[34] As such, we believe our results are of particular interest to pulmonologists, and scientists and clinicians involved in human pulmonary MRI.

Conclusion

In the present study, the efficacy of EP and ALG treatment for BLM-induced lung fibrosis has been investigated by a preclinical HPXe MRI protocol that can easily be applied to assessment of other diseases and novel drug targets. EP treatment was found to improve ventilation and gas-exchange impairment and repair tissue damage associated with lung fibrosis, whilst ALG caused no significant change in pulmonary structure or function. The present results suggest that the inhibition of NF-κB with EP results in regulation of HMGB1 and ERK1/2 expression, thereby leading to therapeutic efficacy.