Ethyl Pyruvate Promotes Wound Healing in Elastase-Induced Lung Injury in Mice as Assessed by Hyperpolarized 129Xe Magnetic Resonance Imaging

Ethyl Pyruvate Promotes Wound Healing in Elastase-Induced Lung Injury in Mice as Assessed by Hyperpolarized 129Xe Magnetic Resonance Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ethyl Pyruvate Promotes Wound Healing in Elastase-Induced Lung Injury in Mice as Assessed by Hyperpolarized 129Xe Magnetic Resonance Imaging Atsuomi Kimura, Akihiro Shimokawa, Neil J. Stewart, Rie Hosoi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6772464/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Wound healing process in lung injury involves activation of the mitogen-activated protein kinase (MAPK) pathway. In this study, we investigated the role of the MAPK pathway in wound healing in a murine model of emphysema using hyperpolarized 129 Xe (HP 129 Xe) magnetic resonance imaging (MRI). Procedures Porcine pancreatic elastase was administered intratracheally to 25 mice to induce lung injury. Temporal changes in pulmonary gas exchange function were monitored using HP 129 Xe MRI, which revealed a significant decline in function one day after elastase administration. Treatments with ethyl pyruvate (EP) and nicorandil (Nic), which upregulate and downregulate the MAPK pathway, respectively, were initiated in 12 and 7 of the 25 mice, respectively, and continued for 20 days. Over the 21-day period, HP 129 Xe MRI was performed to monitor the disease progression and treatment efficacy through changes in the metrics of gas exchange and fractional ventilation. Results HP 129 Xe MRI showed that EP significantly improved gas exchange function 14 days after elastase administration, whereas Nic did not show any improvement. Ventilatory function also improved in the EP group, but not in the Nic group, 14 days after elastase administration. Histological analysis showed that EP repaired tissue damage to a level similar to that observed in healthy mice, whereas Nic did not. Conclusions In the present study, we provided some insight into the role of the MAPK pathway in wound healing in elastase-induced lung injury, as assessed using the HP 129 Xe MRI protocol. hyperpolarized 129Xe magnetic resonance imaging elastase-induced lung injury mitogen-activated protein kinase ethyl pyruvate nicorandil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hyperpolarized 129 Xe (HP 129 Xe) magnetic resonance imaging (MRI) is an established tool for clinically imaging pulmonary function [ 1 – 3 ]. This method has been applied for the functional assessment of various lung diseases in humans [ 4 ]. We developed a unique continuous-flow type HP 129 Xe polarizer and applied it to murine pulmonary functional imaging under spontaneous breathing, which allowed us to evaluate pulmonary function under natural conditions [ 5 , 6 ]. Recently, we have utilized our HP 129 Xe MRI preclinical evaluation system to identify therapeutic drugs for several lung diseases [ 7 – 10 ]. Ethyl pyruvate (EP) was shown to be effective in repairing lung tissue and improving pulmonary function damaged by diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lung cancer [ 8 – 10 ]. A hallmark of lung diseases is the involvement of high mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule, in disease progression [ 11 – 14 ]. Upon lung injury, HMGB1 is released from macrophages and cells undergoing apoptosis or necrosis, which binds to Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), which are highly expressed in alveolar epithelial cells [ 14 , 15 ]. After binding to TLR4, HMGB1 activates the transcription factor nuclear factor kappa B (NF-κB) and induces inflammatory responses [ 16 ]. Furthermore, after binding to RAGE, HMGB1 generates reactive oxygen species (ROS) in the cytoplasm [ 17 ], and these ROS activate the mitogen-activated protein kinase (MAPK) signaling pathway in addition to the NF-κB pathway, exacerbating the pathology [ 18 , 19 ]. In contrast, moderate activation of extracellular signal-regulated kinases (ERK) (1/2), which are involved in the MAPK pathway, has been reported to be paradoxically involved in wound healing in skin disorders and diabetes-induced tissue damage [ 20 – 22 ]. Overexpression of HMGB1 has been shown to aggravate the pathology of lung diseases [ 15 , 23 ], whereas EP has been reported to downregulate HMGB1 production, deactivate NF-κB, and upregulate the expression of ERK(1/2) in a dose-dependent manner [ 24 – 26 ]. EP has also been reported to increase intracellular ROS and activate ERK to inhibit melanogenesis in B16F10 melanoma cells for the treatment of hyperpigmentation disorders [ 27 ]. Here, we hypothesize that EP modulates the expression levels of HMGB1 and intracellular ROS, deactivate NF-κB, and moderately activates the MAPK/ERK pathway to exert coordinated anti-inflammatory and tissue repair effects and improve pulmonary function. In this study, we attempted to confirm this hypothesis by observing the treatment response to EP in elastase-induced acute lung injury using HP 129 Xe MRI. To the best of our knowledge, there have been no reports on the application of EP in the treatment of elastase-induced lung injury. The treatment response of EP was compared with that of nicorandil (Nic), which exhibits its protective effect against lung injury through suppression of ROS production and downregulation of the NF-kB and MAPK pathways [ 28 ]. Materials and Methods Ethics Statement The animal study protocol was approved by the Institutional Animal Care and Use Committee of Division of Health Sciences, Graduate School of Medicine, The University of Osaka (approval no. 24-03-03). Animal Preparation A total of 33 mice, 6-week-old, male ddY mice (Japan SLC Ltd., Hamamatsu, Japan) were divided into four groups: (i) sham-instilled group ( n = 8), (ii) porcine pancreatic elastase (PPE)-treated group ( n = 6), (iii) EP-treated group ( n = 12), and (iv) Nic-treated group ( n = 7). Sham-instilled mice received 40 µL of saline intra-tracheally for five consecutive days per week for 21 days. A saline solution of PPE (40 µL, 300 U/kg body weight; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was intra-tracheally administered to each mouse in the PPE-, EP-, and Nic-treated groups to induce lung injury (the day of PPE administration was set as day 0). One day after PPE administration, EP and Nic treatments were initiated in the EP- and Nic-treated groups, respectively, following a similar protocol as previously reported [ 9 ]. In brief, a 40 µL saline solution of EP (2.6 mg/kg body weight; Tokyo Chemical Industry Ltd, Tokyo, Japan) or Nic (2.4 mg/kg body weight; BIOMOL International, Pennsylvania, USA) was intra-tracheally administered to each mouse in the EP- or Nic-treated group for five consecutive days per week for a 20-day period. The PPE-treated mice were intra-tracheally administered with 40 µL saline solution for five consecutive days per week for a 20-day period. In all cases, prior to instillation, mice were anesthetized with 5% isoflurane (ISOFLU®, Dainippon Sumitomo Pharmaceutical Co. Ltd, Osaka, Japan). The survival rate of the 21-day procedure was 100% in all groups. MRI measurements of sham-instilled mice were conducted on day 0. PPE was administered to each mouse in the EP-, Nic-, and PPE-treated groups on day 0, as described above. One day after PPE administration, MRI measurements were performed for the EP-, Nic-, and PPE-treated groups. During the remaining of the 20-day period, MRI measurements were performed on days 7, 14, and 21 after PPE administration for all groups. A home-built glass mask was attached to the mouth of each mouse to deliver HP 129 Xe and oxygen and remove exhaust gases. For respiratory-gated imaging, a pressure sensor (AD Instruments Ltd., Dunedin, New Zealand) was positioned on the abdomen of each mouse. During MRI measurements, mice were anesthetized with 2% isoflurane, and their body temperature was maintained using warm water circulating through a rubber tube placed on the abdomen. MRI Measurements All MRI measurements were performed using Agilent Unity INOVA 400 WB spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA) with a 9.4 T vertical magnet (Oxford Instruments Plc., Oxford, UK) and a Highland L-500 Gradient Amp system (Highland Technology, Inc. California, USA). A self-shielded imaging probe with a Litz coil switchable to 129 Xe and 1 H frequencies, 32-mm in diameter and 15-mm in length (Clear Bore DSI-1117; Doty Scientific, Inc., Columbia, SC, USA) was used. High-purity xenon gas (> 99.995%) with 129 Xe in its natural abundance, 26.4%, mixed with nitrogen (Japan Air Liquide, Tokyo, Japan) was used to produce HP 129 Xe for MRI measurements using a home-built continuous-flow type apparatus [ 5 – 10 ]. A gas mixture containing HP 129 Xe and N 2 (70% HP 129 Xe and 30% N 2 ) was continuously supplied to mice placed in NMR probe at a rate of 50 ml/min through a mask attached to their head. In the mask, O 2 (Japan Air Liquide, Tokyo, Japan) was mixed with Xe/N 2 gas mixture at a rate of 12 ml/min immediately prior to inhalation. The mice spontaneously inhaled a 56:24:20 volume mixture of Xe:N 2 :O 2 gases. Assessment of Pulmonary Function The pulmonary function of gas exchange metric f D (%), the rate of HP 129 Xe magnetization diffusing from the gas phase (alveolar air space) to the dissolved phase (alveolar tissue and blood) within a given exchange time, was assessed from HP 129 Xe MR images acquired using a balanced steady-state free precession (bSSFP) sequence, as described previously [ 7 , 9 ]. A parametric map of f D for each mouse was obtained via pixel-by-pixel analysis using MATLAB (MathWorks, Inc., Natick, MA, USA). The map was then averaged to obtain the whole lung f D value. The measurements were repeated three times, and the obtained f D values were averaged. Similarly, the fractional ventilation r a , the alveolar volume fraction of gas turned over in a single breath, was also assessed [ 7 , 9 ]. A parametric map of r a and whole lung r a value were derived using the same process as for f D , and these values were compared between the groups. Acquisition parameters of HP 129 Xe images were as follows: 1000-µs Gaussian-shaped radiofrequency (RF) pulse of flip angle θ = 40º; 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 field of view of 80 × 25 mm 2 . Acquisition was commenced after confirming a steady state signal by monitoring 129 Xe MR spectra obtained by the application of an 8º hard RF pulse with an interval of 2 s. Histology After completion of MRI experiments, mice were euthanized with a lethal dose of carbon dioxide gas. Lungs were extracted, fixed in 10% formalin, and processed for histological examination by staining with hematoxylin and eosin (H&E), as previously described [ 7 ]. Four coronal slides taken close to the center of the lungs per mouse were captured using a digital microscope (Celestron LCD Microscope PRO ; Celestron, LLC., Torrance, CA, USA). The captured images were used to evaluate the mean linear intercept (MLI) and mean bronchial wall thickness ( h bw_histology ). The MLI and h bw_histology values were determined from five regions of the lung (right upper lobe, right middle lobe, right lower lobe, and the upper and lower lobes of the left lung). Each set of five values was averaged over the four slides, and the resulting five averaged regional MLI and h bw _histology values were averaged to yield a single mean MLI and h bw _histology value for each mouse of the sham-instilled, PPE- and EP-, and Nic-treated groups. Statistical Analysis Statistical analyses were performed using Student’s t -test or one-way ANOVA with Tukey–Kramer post-hoc analysis to identify significant differences between groups. All data are presented as mean ± standard error and/or box-and-whisker plots, and differences in signal intensities were considered significant at P < 0.05. Results Temporal Changes in Pulmonary Gas Exchange Function Temporal changes in representative f D parametric maps and f D values for the sham-instilled, PPE-, EP-, and Nic-treated groups are illustrated in Fig. 1 a and 1 b, respectively. Notably, EP and Nic treatments were initiated one day after PPE administration (set as day 0). When compared with the f D of the sham-instilled group on day 0 ( f D _sham−instilled = 6.8 ± 0.7%), significant decrease in the f D values of the PPE- ( f D _PPE−treated = 5.1 ± 0.4%), EP- ( f D _EP−treated = 4.7 ± 1.0%), and Nic-treated ( f D _Nic−treated = 4.8 ± 0.8%) groups was observed on day 1 after PPE administration ( P < 0.01). Significant decrease in the f D value of the PPE-treated group compared with that of the sham-instilled group was consistent for 21 days after PPE administration. Similarly, decrease in the f D values of both EP- and Nic-treated groups was observed until day 7 after PPE administration. However, the f D value of the EP-treated group recovered to a similar level as that of the sham-instilled group on day 14 after PPE administration ( f D _sham−instilled = 6.9 ± 0.6% and f D _EP−treated = 6.6 ± 0.9%), but was significantly higher ​​than that of the PPE and Nic groups ( f D _PPE−treated = 4.0 ± 1.2% and f D _Nic−treated = 4.9 ± 1.0%, P < 0.05). This recovery continued until day 21. However, the f D value of the Nic group did not recover over the remaining period. Temporal Changes in Pulmonary Ventilation Function Temporal changes in the representative r a parametric maps and r a values of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig. 2 a and 2 b, respectively. In contrast to the results of f D measurements, the r a values of the PPE- ( r a _PPE−treated = 0.20 ± 0.06), EP- ( r a _EP−treated = 0.20 ± 0.05), and Nic-treated ( r a _Nic−treated = 0.19 ± 0.07) groups were not decreased on day 1 after PPE administration compared with the r a value of the sham-instilled group on day 0 ( r a _sham−instilled = 0.27 ± 0.02). This tendency persisted until day 7. On day 14, the r a values of the PPE- ( r a _PPE−treated = 0.18 ± 0.06) and Nic-treated ( r a _Nic−treated = 0.18 ± 0.03) groups were significantly decreased compared with that of the sham-instilled group ( r a _sham−instilled = 0.25 ± 0.02, P < 0.05). However, the r a value of the EP-treated group ( r a _EP−treated = 0.23 ± 0.03) was similar to that of the sham-instilled group. This tendency was observed until day 21. Histograms of Pulmonary Gas Exchange Function Figure 3 shows the comparative results of representative histograms of f D values obtained from f D maps of a sham-instilled mouse on day 0, a PPE-treated mouse on day 1, and a PPE-treated mouse on day 21. Histology Representative histological images obtained from mice of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig. 4 a. The whole lung mean MLI values obtained from the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig. 4 b. As illustrated in Fig. 4 a, alveolar enlargement was observed in the PPE- and Nic-treated groups. The mean MLI of the PPE-treated group ( MLI PPE−treated = 46.3 ± 9.4 µm) was significantly higher than that of the sham-instilled and EP-treated groups ( MLI Sham−instilled = 38.0 ± 2.0 µm and MLI EP−treated = 37.8 ± 5.6 µm, P < 0.05). The MLI of the Nic-treated group ( MLI Nic−treated = 42.8 ± 2.7 µm) was not significantly different from that of the sham-instilled, PPE-, and EP-treated groups. Representative histological images and whole lung mean h bW_histology values obtained from mice of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig. 5 . There were no significant differences in h bW_histology values between the groups. Mechanisms of Therapeutic Effect of EP and Nic Figure 6 depicts mechanisms of therapeutic effect of EP (Fig. 6 a) and Nic (Fig. 6 b) against elastase-induced lung injury proposed in this study. Discussion In the present study, we successfully monitored the temporal changes in pulmonary function, f D and r a , induced by PPE administration using HP 129 Xe MRI (Figs. 1 and 2 ). One day after PPE administration, the f D values of the PPE-, EP-, and Nic-treated groups were significantly lower than that of the sham-instilled group on day 0 (Fig. 1 ). Emphysema was previously reported to develop seven days after PPE administration [ 29 ]. Therefore, our data suggest that the f D metric can detect changes in pulmonary function due to pathology before emphysema development. This observation is similar to that of our previous study using a mouse model of lung cancer, in which inflammation-induced alveolar septal wall thickening reduced f D values prior to the onset of lung cancer [ 10 ]. On the other hand, one day after PPE administration, no significant changes were observed in the r a values of the PPE-, EP-, and Nic-treated groups compared with that of the sham-instilled group on day 0 (Fig. 2 ). In our previous study, we reported that inflammation-induced bronchial wall thickening reduced r a values [ 7 ]. In the present study, bronchial wall thickening was not observed in any histological images of the PPE-, EP-, or Nic-treated groups compared with those of the sham-instilled group (Fig. 5 ), suggesting no changes in r a values were observed one 1 day after PPE administration. Considering the above observations, although no alveolar septal wall thickening was observed histologically 21 days after PPE administration (Fig. 4 ), we assumed that alveolar septal wall thickening might occur without bronchial wall thickening due to inflammation on day 1 after PPE administration, before the onset of emphysema. Previously, using a mouse model of elastase-induced lung injury, we reported temporal changes in alveolar septal wall thickness in vivo (Figure E5 of Ref. [ 29 ]). In this study, significant thickening of the alveolar septal walls was observed in vivo on day 1 after PPE administration, prior to the development of emphysema on day 7. In addition, no alveolar septal wall thickening was observed in vivo on day 7 after PPE administration. This study supports these assumptions. Notably, in addition to inflammation, a reduction in alveolar tissue volume was reported one day after PPE administration [ 29 ]. This finding suggests that, in the present study, tissue destruction to some extent might have already started on day 1 after PPE administration, which is supported by shifts in f D values caused by PPE administration (Fig. 3 ). When compared with the f D values of a sham-instilled mouse on day 0, the f D values of a PPE-treated mouse on day 1 showed a wider distribution and tended to shift to lower values, similar to that of a PPE-treated mouse on day 21. Therefore, to observe therapeutic effects, it is rational to initiate EP and Nic treatment one day after PPE administration as a significant decrease in f D value was observed at this time point in the present study. The decrease in f D value of the PPE-treated group was consistent from day 1 to day 21 after PPE administration (Fig. 1 ). As mentioned above, emphysema develops seven days after PPE administration [ 29 ]. Therefore, the decrease in f D value on day 7 after PPE administration was due to the early onset of emphysematous lesions. In contrast, the r a value of the PPE-treated group was significantly lower than that of the sham-instilled group on day 14 after PPE administration (Fig. 2 ), implying that emphysematous lesions fully developed at this time point. In contrast, the r a value of the EP-treated group was higher than that of the PPE- and Nic-treated groups on day 14 after PPE administration and recovered to a level similar to that of the sham-instilled group, similar to that of f D value (Figs. 1 and 2 ). The recovery of both f D and r a continued until day 21 after PPE administration, and the MLI of the EP-treated group was significantly lower than that of the PPE-treated group (Fig. 4 ). Therefore, these data suggest that by starting EP treatment one day after PPE administration, decline in pulmonary function associated with the onset of emphysematous lesions could be improved, as measured by HP 129 Xe MRI. However, in the Nic-treated group, the decline in the r a and f D values did not improve (Figs. 1 and 2 ). Moreover, the MLI of the Nic-treated group did not improve compared with that of the PPE-treated group (Fig. 4 ), supporting the results of the pulmonary functional assessments. Regulation of the MAPK/ERK pathway has been reported to promote tissue repair during cutaneous wound healing [ 20 ]. The MAPK/ERK pathway has been reported to be activated during wound healing in a murine diabetic wound model [ 21 ]. Regulation of HMGB1 expression has been reported to be involved in wound healing process via the MAPK/ERK pathway [ 22 ]. Based on these reports, we hypothesize that EP regulates the expression levels of HMGB1 and the MAPK/ERK pathway to exert anti-inflammatory and tissue repair effects. Our previous study using a mouse model of bleomycin-induced lung injury supported this hypothesis, in which we observed lung structural and functional changes caused by the upregulation and downregulation of HMGB1 expression [ 9 ]. However, in this study, we were unable to directly elucidate the involvement of intracellular ROS and MAPK/ERK pathway in the recovery of injured tissue. In the present study, we investigated the involvement of intracellular ROS and MAPK/ERK pathway in elastase-induced emphysema in mice by evaluating and comparing the pharmacological effects of EP and Nic. Intracellular ROS production has been reported to contribute to elastase-induced lung injury [ 30 ]. Further, HMGB1 and NF-kB have also been reported to be involved in elastase-induced lung injury [ 31 , 32 ]. Furthermore, EP activated the ROS-MAPK/ERK pathway to inhibit melanogenesis in B16F10 melanoma cells [ 27 ]. Therefore, we hypothesized that EP moderately upregulates the expression levels of HMGB1 and activates the ROS-MAPK/ERK pathway to exert anti-inflammatory and tissue repair effects (Fig. 6 a). In contrast, Nic suppressed ROS production and showed its protective effect through downregulation of the NF-kB and MAPK pathways (Fig. 6 b) [ 28 ]. In the present study, Nic treatment did not improve pulmonary function, indicating the role of the MAPK/ERK pathway in wound healing process. In other words, non-activation of the MAPK/ERK pathway by Nic treatment seemed to impair tissue repair and lead to the development of emphysema and pulmonary functional decline. Although Nic has been reported to show protective effects against acute lung injury [ 28 ], collapse-induced lung injury [ 33 ], pulmonary fibrosis [ 34 , 35 ], pulmonary artery endothelial damage in pulmonary hypertension [ 36 ], and other lung disorders [ 37 ], its therapeutic effects have not been reported. In the present study, inflammation was detected one day after PPE administration, and Nic treatment was started at this stage to suppress the inflammation through inhibition of intracellular ROS production and NF-kB activation [ 28 ]. However, at this stage, in addition to inflammation, there appeared to be some extent of tissue destruction, as mentioned previously. We assumed that Nic did not show tissue repair effects via the MAPK/ERK pathway and was unable to exert a therapeutic effect considering the above reason. In contrast, EP downregulated HMGB1 expression, increased ROS production, and moderately upregulated the MAPK/ERK pathway (Fig. 6 a) [ 24 – 27 ], resulting in tissue repair and suppression in the onset of emphysema, leading to improvement in pulmonary function. Conclusions In the present study, we successfully monitored temporal changes in pulmonary function of gas exchange and ventilation caused by the development of emphysema using a preclinical HP 129 Xe MRI protocol. The treatment responses of EP and Nic against the development of emphysema were monitored and compared. EP treatment effectively repaired tissue damage and improved pulmonary function in elastase-induced lung injury, whereas Nic showed no therapeutic effects. The present results suggest therapeutic efficacy of EP in wound healing process via activation of the MAPK/ERK pathway. Declarations Acknowledgments: The authors are grateful to all members of the Department of Medical Physics and Engineering, Area of Medical Imaging Technology and Science, Division of Health Sciences, Graduate School of Medicine, The University of Osaka, for their valuable comments and helpful discussions. The authors would like to thank Editage (www.editage.jp) for English language editing. Author Contributions: Atsuomi Kimura designed the study. Material preparation, data collection, and analysis were conducted by all authors. Atsuomi Kimura wrote the first draft of the manuscript, and all authors have read and approved the final manuscript. Funding: This work was supported in part by the Grant-in-Aids of Scientific Research (No. 20H04516) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. Data Availability: The datasets generated in this study are available from the corresponding author upon reasonable request. Ethical Approval The animal study protocol was approved by the Institutional Animal Care and Use Committee of Division of Health Sciences, Graduate School of Medicine, The University of Osaka (approval no. 24-03-03). Conflicts of Interest The authors declare that they have no conflict of interest. References MacLeod JL, Khan HM, Franklin A, Myc L, Shim YM (2025) Hyperpolarized xenon-129 MRI: Narrative review of clinical studies, testing, and implementation of advanced pulmonary in vivo imaging and its diagnostic applications. 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Sci Rep 5:14043 Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted Editorial decision: Major revisions 06 Oct, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers invited by journal 16 Jun, 2025 Editor assigned by journal 29 May, 2025 First submitted to journal 28 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6772464","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472206087,"identity":"da3e78c5-0af2-4ffa-b1b9-9abe47a576ee","order_by":0,"name":"Atsuomi Kimura","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5702-276X","institution":"Osaka University: Osaka Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Atsuomi","middleName":"","lastName":"Kimura","suffix":""},{"id":472206088,"identity":"5779f351-4847-4b76-ad07-ff15bb9f4a20","order_by":1,"name":"Akihiro Shimokawa","email":"","orcid":"","institution":"Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Shimokawa","suffix":""},{"id":472206089,"identity":"c422c18f-88cf-4eea-bd2a-caae022c7df4","order_by":2,"name":"Neil J. Stewart","email":"","orcid":"","institution":"The University of Sheffield","correspondingAuthor":false,"prefix":"","firstName":"Neil","middleName":"J.","lastName":"Stewart","suffix":""},{"id":472206090,"identity":"90279472-9ca9-466b-a129-d77de66e8b83","order_by":3,"name":"Rie Hosoi","email":"","orcid":"","institution":"Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Rie","middleName":"","lastName":"Hosoi","suffix":""},{"id":472206091,"identity":"f99cda29-675d-47f3-8115-dc006ab91b22","order_by":4,"name":"Hirohiko Imai","email":"","orcid":"","institution":"Gifu University School of Medicine Graduate School of Medicine: Gifu Daigaku Igakubu Daigakuin Igakukei Kenkyuka","correspondingAuthor":false,"prefix":"","firstName":"Hirohiko","middleName":"","lastName":"Imai","suffix":""},{"id":472206092,"identity":"2fd2c5fe-9bb8-4e37-938b-6479c280eaf3","order_by":5,"name":"Hideaki Fujiwara","email":"","orcid":"","institution":"Osaka Daigaku Daigakuin Igakukei Kenkyuka Igakubu","correspondingAuthor":false,"prefix":"","firstName":"Hideaki","middleName":"","lastName":"Fujiwara","suffix":""}],"badges":[],"createdAt":"2025-05-29 04:04:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6772464/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6772464/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11307-025-02073-6","type":"published","date":"2025-12-10T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85177021,"identity":"818e095c-6918-4589-8263-53cbb331d783","added_by":"auto","created_at":"2025-06-23 06:39:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":826236,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal changes in representative\u003cem\u003e f\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e parametric maps of mice of each of the four groups, from top to bottom: sham-instilled; PPE-treated; EP-treated; Nic-treated (a). Box plots of temporal changes in the mean \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values for all mice, separated by groups (b). The time course from the initial intra-tracheal injection of saline or PPE is shown horizontally. Significant differences between groups are indicated by solid lines (*: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **:\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Of note, EP and Nic treatments were initiated one day after PPE administration on day 0. The first \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e map of sham-instilled mouse was obtained from day 0, whereas the first maps of PPE-, EP-, and Nic-treated mice were obtained from day 1\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/d1839d4365095ae67c709b93.png"},{"id":85177026,"identity":"4c798f61-a0e1-4fbd-9c9b-96ab5d5061e1","added_by":"auto","created_at":"2025-06-23 06:39:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":827636,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal changes in representative\u003cem\u003e r\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e parametric maps of mice of each of the four groups, from top to bottom: sham-instilled; PPE-treated; EP-treated; Nic-treated (a). Box plots of temporal changes in the mean \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values for all mice, separated by groups (b)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/2dfa949bd5934a41e0665844.png"},{"id":85177019,"identity":"516b6610-ed14-4d5c-995f-e81269fcc38c","added_by":"auto","created_at":"2025-06-23 06:39:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":604065,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative histograms of \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values obtained from sham-instilled mice (a), PPE-treated mice on day 1 (b), and PPE-treated mice on day 21 (c). The global mean \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e = 7.2% (a), 5.1% (b), and 4.9% (c). After PPE injection, \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values tended to shift to lower values without a decrease in the total area of the histogram, suggesting regional transfer from higher \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e sites to lower \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e sites as a result of the pathological process\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/1fcf48ecf49c257b1b1bb7da.png"},{"id":85177030,"identity":"bc673079-f5ed-4e4b-a121-e24a2efb7121","added_by":"auto","created_at":"2025-06-23 06:39:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2933032,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative H\u0026amp;E-stained histology slides obtained from five lung regions of a mouse from each of the four groups, from top to bottom: sham-instilled; PPE-treated; EP-treated; Nic-treated (a). RU, right upper lobe; RM, right middle lobe; RL, right lower lobe; LU, upper region of the left lobe; LL, lower region of the left lobe. Box plots of mean MLI values obtained from mice of each of the four groups (b)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/2ea53b06dbae907f064e6baf.png"},{"id":85178545,"identity":"348269a2-a904-468a-b1ce-8ed8e05ff21c","added_by":"auto","created_at":"2025-06-23 06:55:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1483550,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative H\u0026amp;E-stained histology slides obtained from the four groups, from left to right: sham-instilled; PPE-treated, EP-treated; Nic-treated (a). Box plots showing the mean bronchial wall thickness (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebw_histology\u003c/em\u003e\u003c/sub\u003e) values obtained from mice of each of the four groups\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/a7a30e55bea26b6bc880b6f8.png"},{"id":85177028,"identity":"34b0ea3c-2e54-45b2-a5d1-2e4061e8e9ad","added_by":"auto","created_at":"2025-06-23 06:39:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":604395,"visible":true,"origin":"","legend":"\u003cp\u003eProposed mechanisms of therapeutic effect of EP against elastase-induced lung injury. a) EP downregulates HMGB1 expression and moderately activates intracellular ROS and MAPK/ERK pathway, thereby initiating wound healing process. b) Nic, which inhibits intracellular ROS and deactivates the MAPK/ERK pathway, does not exhibit a positive therapeutic effect\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/ed6dd538e0587617e5d747bb.png"},{"id":98244153,"identity":"df4786af-2c2d-42fa-86a2-e17ec16e2bc8","added_by":"auto","created_at":"2025-12-15 16:13:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7821118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6772464/v1/ec56bdb4-33e8-42b1-9a62-a7c674af15fe.pdf"}],"financialInterests":"","formattedTitle":"Ethyl Pyruvate Promotes Wound Healing in Elastase-Induced Lung Injury in Mice as Assessed by Hyperpolarized 129Xe Magnetic Resonance Imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHyperpolarized \u003csup\u003e129\u003c/sup\u003eXe (HP \u003csup\u003e129\u003c/sup\u003eXe) magnetic resonance imaging (MRI) is an established tool for clinically imaging pulmonary function [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This method has been applied for the functional assessment of various lung diseases in humans [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We developed a unique continuous-flow type HP \u003csup\u003e129\u003c/sup\u003eXe polarizer and applied it to murine pulmonary functional imaging under spontaneous breathing, which allowed us to evaluate pulmonary function under natural conditions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recently, we have utilized our HP \u003csup\u003e129\u003c/sup\u003eXe MRI preclinical evaluation system to identify therapeutic drugs for several lung diseases [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Ethyl pyruvate (EP) was shown to be effective in repairing lung tissue and improving pulmonary function damaged by diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and lung cancer [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA hallmark of lung diseases is the involvement of high mobility group box 1 (HMGB1), a damage-associated molecular pattern molecule, in disease progression [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Upon lung injury, HMGB1 is released from macrophages and cells undergoing apoptosis or necrosis, which binds to Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE), which are highly expressed in alveolar epithelial cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. After binding to TLR4, HMGB1 activates the transcription factor nuclear factor kappa B (NF-κB) and induces inflammatory responses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, after binding to RAGE, HMGB1 generates reactive oxygen species (ROS) in the cytoplasm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and these ROS activate the mitogen-activated protein kinase (MAPK) signaling pathway in addition to the NF-κB pathway, exacerbating the pathology [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, moderate activation of extracellular signal-regulated kinases (ERK) (1/2), which are involved in the MAPK pathway, has been reported to be paradoxically involved in wound healing in skin disorders and diabetes-induced tissue damage [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Overexpression of HMGB1 has been shown to aggravate the pathology of lung diseases [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], whereas EP has been reported to downregulate HMGB1 production, deactivate NF-κB, and upregulate the expression of ERK(1/2) in a dose-dependent manner [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. EP has also been reported to increase intracellular ROS and activate ERK to inhibit melanogenesis in B16F10 melanoma cells for the treatment of hyperpigmentation disorders [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we hypothesize that EP modulates the expression levels of HMGB1 and intracellular ROS, deactivate NF-κB, and moderately activates the MAPK/ERK pathway to exert coordinated anti-inflammatory and tissue repair effects and improve pulmonary function. In this study, we attempted to confirm this hypothesis by observing the treatment response to EP in elastase-induced acute lung injury using HP \u003csup\u003e129\u003c/sup\u003eXe MRI. To the best of our knowledge, there have been no reports on the application of EP in the treatment of elastase-induced lung injury. The treatment response of EP was compared with that of nicorandil (Nic), which exhibits its protective effect against lung injury through suppression of ROS production and downregulation of the NF-kB and MAPK pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThe animal study protocol was approved by the Institutional Animal Care and Use Committee of Division of Health Sciences, Graduate School of Medicine, The University of Osaka (approval no. 24-03-03).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal Preparation\u003c/h3\u003e\n\u003cp\u003eA total of 33 mice, 6-week-old, male ddY mice (Japan SLC Ltd., Hamamatsu, Japan) were divided into four groups: (i) sham-instilled group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), (ii) porcine pancreatic elastase (PPE)-treated group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6), (iii) EP-treated group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;12), and (iv) Nic-treated group (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;7). Sham-instilled mice received 40 \u0026micro;L of saline intra-tracheally for five consecutive days per week for 21 days. A saline solution of PPE (40 \u0026micro;L, 300 U/kg body weight; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was intra-tracheally administered to each mouse in the PPE-, EP-, and Nic-treated groups to induce lung injury (the day of PPE administration was set as day 0). One day after PPE administration, EP and Nic treatments were initiated in the EP- and Nic-treated groups, respectively, following a similar protocol as previously reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In brief, a 40 \u0026micro;L saline solution of EP (2.6 mg/kg body weight; Tokyo Chemical Industry Ltd, Tokyo, Japan) or Nic (2.4 mg/kg body weight; BIOMOL International, Pennsylvania, USA) was intra-tracheally administered to each mouse in the EP- or Nic-treated group for five consecutive days per week for a 20-day period. The PPE-treated mice were intra-tracheally administered with 40 \u0026micro;L saline solution for five consecutive days per week for a 20-day period. In all cases, prior to instillation, mice were anesthetized with 5% isoflurane (ISOFLU\u0026reg;, Dainippon Sumitomo Pharmaceutical Co. Ltd, Osaka, Japan). The survival rate of the 21-day procedure was 100% in all groups.\u003c/p\u003e \u003cp\u003eMRI measurements of sham-instilled mice were conducted on day 0. PPE was administered to each mouse in the EP-, Nic-, and PPE-treated groups on day 0, as described above. One day after PPE administration, MRI measurements were performed for the EP-, Nic-, and PPE-treated groups. During the remaining of the 20-day period, MRI measurements were performed on days 7, 14, and 21 after PPE administration for all groups. A home-built glass mask was attached to the mouth of each mouse to deliver HP \u003csup\u003e129\u003c/sup\u003eXe and oxygen and remove exhaust gases. For respiratory-gated imaging, a pressure sensor (AD Instruments Ltd., Dunedin, New Zealand) was positioned on the abdomen of each mouse. During MRI measurements, mice were anesthetized with 2% isoflurane, and their body temperature was maintained using warm water circulating through a rubber tube placed on the abdomen.\u003c/p\u003e\n\u003ch3\u003eMRI Measurements\u003c/h3\u003e\n\u003cp\u003eAll MRI measurements were performed using Agilent Unity INOVA 400 WB spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA) with a 9.4 T vertical magnet (Oxford Instruments Plc., Oxford, UK) and a Highland L-500 Gradient Amp system (Highland Technology, Inc. California, USA). A self-shielded imaging probe with a Litz coil switchable to \u003csup\u003e129\u003c/sup\u003eXe and \u003csup\u003e1\u003c/sup\u003eH frequencies, 32-mm in diameter and 15-mm in length (Clear Bore DSI-1117; Doty Scientific, Inc., Columbia, SC, USA) was used.\u003c/p\u003e \u003cp\u003eHigh-purity xenon gas (\u0026gt;\u0026thinsp;99.995%) with \u003csup\u003e129\u003c/sup\u003eXe in its natural abundance, 26.4%, mixed with nitrogen (Japan Air Liquide, Tokyo, Japan) was used to produce HP \u003csup\u003e129\u003c/sup\u003eXe for MRI measurements using a home-built continuous-flow type apparatus [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A gas mixture containing HP \u003csup\u003e129\u003c/sup\u003eXe and N\u003csub\u003e2\u003c/sub\u003e (70% HP \u003csup\u003e129\u003c/sup\u003eXe and 30% N\u003csub\u003e2\u003c/sub\u003e) was continuously supplied to mice placed in NMR probe at a rate of 50 ml/min through a mask attached to their head. In the mask, O\u003csub\u003e2\u003c/sub\u003e (Japan Air Liquide, Tokyo, Japan) was mixed with Xe/N\u003csub\u003e2\u003c/sub\u003e gas mixture at a rate of 12 ml/min immediately prior to inhalation. The mice spontaneously inhaled a 56:24:20 volume mixture of Xe:N\u003csub\u003e2\u003c/sub\u003e:O\u003csub\u003e2\u003c/sub\u003e gases.\u003c/p\u003e\n\u003ch3\u003eAssessment of Pulmonary Function\u003c/h3\u003e\n\u003cp\u003eThe pulmonary function of gas exchange metric \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e (%), the rate of HP \u003csup\u003e129\u003c/sup\u003eXe magnetization diffusing from the gas phase (alveolar air space) to the dissolved phase (alveolar tissue and blood) within a given exchange time, was assessed from HP \u003csup\u003e129\u003c/sup\u003eXe MR images acquired using a balanced steady-state free precession (bSSFP) sequence, as described previously [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A parametric map of \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e for each mouse was obtained via pixel-by-pixel analysis using MATLAB (MathWorks, Inc., Natick, MA, USA). The map was then averaged to obtain the whole lung \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value. The measurements were repeated three times, and the obtained \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values were averaged.\u003c/p\u003e \u003cp\u003eSimilarly, the fractional ventilation \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, the alveolar volume fraction of gas turned over in a single breath, was also assessed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A parametric map of \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e and whole lung \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value were derived using the same process as for \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e, and these values were compared between the groups.\u003c/p\u003e \u003cp\u003eAcquisition parameters of HP \u003csup\u003e129\u003c/sup\u003eXe images were as follows: 1000-\u0026micro;s Gaussian-shaped radiofrequency (RF) pulse of flip angle θ\u0026thinsp;=\u0026thinsp;40\u0026ordm;; acquisition bandwidth, 88 kHz; TR/TE\u0026thinsp;=\u0026thinsp;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 \u0026times; 32 with a field of view of 80 \u0026times; 25 mm\u003csup\u003e2\u003c/sup\u003e. Acquisition was commenced after confirming a steady state signal by monitoring \u003csup\u003e129\u003c/sup\u003eXe MR spectra obtained by the application of an 8\u0026ordm; hard RF pulse with an interval of 2 s.\u003c/p\u003e\n\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003eAfter completion of MRI experiments, mice were euthanized with a lethal dose of carbon dioxide gas. Lungs were extracted, fixed in 10% formalin, and processed for histological examination by staining with hematoxylin and eosin (H\u0026amp;E), as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Four coronal slides taken close to the center of the lungs per mouse were captured using a digital microscope (Celestron LCD Microscope PRO\u0026thinsp;\u0026lt;\u0026thinsp;CE44345\u0026gt;; Celestron, LLC., Torrance, CA, USA). The captured images were used to evaluate the mean linear intercept (MLI) and mean bronchial wall thickness (\u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebw_histology\u003c/em\u003e\u003c/sub\u003e). The MLI and \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebw_histology\u003c/em\u003e\u003c/sub\u003e values were determined from five regions of the lung (right upper lobe, right middle lobe, right lower lobe, and the upper and lower lobes of the left lung). Each set of five values was averaged over the four slides, and the resulting five averaged regional MLI and \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebw\u003c/em\u003e_histology\u003c/sub\u003e values were averaged to yield a single mean MLI and \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebw\u003c/em\u003e_histology\u003c/sub\u003e value for each mouse of the sham-instilled, PPE- and EP-, and Nic-treated groups.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA with Tukey\u0026ndash;Kramer post-hoc analysis to identify significant differences between groups. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error and/or box-and-whisker plots, and differences in signal intensities were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTemporal Changes in Pulmonary Gas Exchange Function\u003c/h2\u003e \u003cp\u003eTemporal changes in representative \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e parametric maps and \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values for the sham-instilled, PPE-, EP-, and Nic-treated groups are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, respectively. Notably, EP and Nic treatments were initiated one day after PPE administration (set as day 0). When compared with the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e of the sham-instilled group on day 0 (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_sham\u0026minus;instilled\u003c/sub\u003e = 6.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%), significant decrease in the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values of the PPE- (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_PPE\u0026minus;treated\u003c/sub\u003e = 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%), EP- (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_EP\u0026minus;treated\u003c/sub\u003e = 4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%), and Nic-treated (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_Nic\u0026minus;treated\u003c/sub\u003e = 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%) groups was observed on day 1 after PPE administration (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Significant decrease in the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value of the PPE-treated group compared with that of the sham-instilled group was consistent for 21 days after PPE administration. Similarly, decrease in the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values of both EP- and Nic-treated groups was observed until day 7 after PPE administration. However, the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value of the EP-treated group recovered to a similar level as that of the sham-instilled group on day 14 after PPE administration (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_sham\u0026minus;instilled\u003c/sub\u003e = 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% and \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_EP\u0026minus;treated\u003c/sub\u003e = 6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%), but was significantly higher ​​than that of the PPE and Nic groups (\u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_PPE\u0026minus;treated\u003c/sub\u003e = 4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% and \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e_Nic\u0026minus;treated\u003c/sub\u003e = 4.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This recovery continued until day 21. However, the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value of the Nic group did not recover over the remaining period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTemporal Changes in Pulmonary Ventilation Function\u003c/h2\u003e \u003cp\u003eTemporal changes in the representative \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e parametric maps and \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, respectively. In contrast to the results of \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e measurements, the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values of the PPE- (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_PPE\u0026minus;treated\u003c/sub\u003e = 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06), EP- (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_EP\u0026minus;treated\u003c/sub\u003e = 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05), and Nic-treated (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_Nic\u0026minus;treated\u003c/sub\u003e = 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) groups were not decreased on day 1 after PPE administration compared with the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value of the sham-instilled group on day 0 (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_sham\u0026minus;instilled\u003c/sub\u003e = 0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02). This tendency persisted until day 7. On day 14, the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values of the PPE- (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_PPE\u0026minus;treated\u003c/sub\u003e = 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) and Nic-treated (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_Nic\u0026minus;treated\u003c/sub\u003e = 0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) groups were significantly decreased compared with that of the sham-instilled group (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_sham\u0026minus;instilled\u003c/sub\u003e = 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value of the EP-treated group (\u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e_EP\u0026minus;treated\u003c/sub\u003e = 0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) was similar to that of the sham-instilled group. This tendency was observed until day 21.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistograms of Pulmonary Gas Exchange Function\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the comparative results of representative histograms of \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values obtained from \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e maps of a sham-instilled mouse on day 0, a PPE-treated mouse on day 1, and a PPE-treated mouse on day 21.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eRepresentative histological images obtained from mice of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. The whole lung mean MLI values obtained from the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, alveolar enlargement was observed in the PPE- and Nic-treated groups. The mean MLI of the PPE-treated group (\u003cem\u003eMLI\u003c/em\u003e\u003csub\u003e\u003cem\u003ePPE\u0026minus;treated\u003c/em\u003e\u003c/sub\u003e = 46.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 \u0026micro;m) was significantly higher than that of the sham-instilled and EP-treated groups (\u003cem\u003eMLI\u003c/em\u003e\u003csub\u003e\u003cem\u003eSham\u0026minus;instilled\u003c/em\u003e\u003c/sub\u003e = 38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 \u0026micro;m and \u003cem\u003eMLI\u003c/em\u003e\u003csub\u003e\u003cem\u003eEP\u0026minus;treated\u003c/em\u003e\u003c/sub\u003e = 37.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6 \u0026micro;m, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The MLI of the Nic-treated group (\u003cem\u003eMLI\u003c/em\u003e\u003csub\u003e\u003cem\u003eNic\u0026minus;treated\u003c/em\u003e\u003c/sub\u003e = 42.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7 \u0026micro;m) was not significantly different from that of the sham-instilled, PPE-, and EP-treated groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative histological images and whole lung mean \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebW_histology\u003c/em\u003e\u003c/sub\u003e values obtained from mice of the sham-instilled, PPE-, EP-, and Nic-treated groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. There were no significant differences in \u003cem\u003eh\u003c/em\u003e\u003csub\u003e\u003cem\u003ebW_histology\u003c/em\u003e\u003c/sub\u003e values between the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMechanisms of Therapeutic Effect of EP and Nic\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e depicts mechanisms of therapeutic effect of EP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) and Nic (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) against elastase-induced lung injury proposed in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we successfully monitored the temporal changes in pulmonary function, \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e, induced by PPE administration using HP \u003csup\u003e129\u003c/sup\u003eXe MRI (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). One day after PPE administration, the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values of the PPE-, EP-, and Nic-treated groups were significantly lower than that of the sham-instilled group on day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Emphysema was previously reported to develop seven days after PPE administration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, our data suggest that the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e metric can detect changes in pulmonary function due to pathology before emphysema development. This observation is similar to that of our previous study using a mouse model of lung cancer, in which inflammation-induced alveolar septal wall thickening reduced \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values prior to the onset of lung cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the other hand, one day after PPE administration, no significant changes were observed in the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values of the PPE-, EP-, and Nic-treated groups compared with that of the sham-instilled group on day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In our previous study, we reported that inflammation-induced bronchial wall thickening reduced \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the present study, bronchial wall thickening was not observed in any histological images of the PPE-, EP-, or Nic-treated groups compared with those of the sham-instilled group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting no changes in \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e values were observed one 1 day after PPE administration.\u003c/p\u003e \u003cp\u003eConsidering the above observations, although no alveolar septal wall thickening was observed histologically 21 days after PPE administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we assumed that alveolar septal wall thickening might occur without bronchial wall thickening due to inflammation on day 1 after PPE administration, before the onset of emphysema. Previously, using a mouse model of elastase-induced lung injury, we reported temporal changes in alveolar septal wall thickness \u003cem\u003ein vivo\u003c/em\u003e (Figure E5 of Ref. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]). In this study, significant thickening of the alveolar septal walls was observed \u003cem\u003ein vivo\u003c/em\u003e on day 1 after PPE administration, prior to the development of emphysema on day 7. In addition, no alveolar septal wall thickening was observed \u003cem\u003ein vivo\u003c/em\u003e on day 7 after PPE administration. This study supports these assumptions. Notably, in addition to inflammation, a reduction in alveolar tissue volume was reported one day after PPE administration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This finding suggests that, in the present study, tissue destruction to some extent might have already started on day 1 after PPE administration, which is supported by shifts in \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values caused by PPE administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). When compared with the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values of a sham-instilled mouse on day 0, the \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values of a PPE-treated mouse on day 1 showed a wider distribution and tended to shift to lower values, similar to that of a PPE-treated mouse on day 21. Therefore, to observe therapeutic effects, it is rational to initiate EP and Nic treatment one day after PPE administration as a significant decrease in \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value was observed at this time point in the present study.\u003c/p\u003e \u003cp\u003eThe decrease in \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value of the PPE-treated group was consistent from day 1 to day 21 after PPE administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As mentioned above, emphysema develops seven days after PPE administration [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the decrease in \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value on day 7 after PPE administration was due to the early onset of emphysematous lesions. In contrast, the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value of the PPE-treated group was significantly lower than that of the sham-instilled group on day 14 after PPE administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), implying that emphysematous lesions fully developed at this time point. In contrast, the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e value of the EP-treated group was higher than that of the PPE- and Nic-treated groups on day 14 after PPE administration and recovered to a level similar to that of the sham-instilled group, similar to that of \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e value (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The recovery of both \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e continued until day 21 after PPE administration, and the MLI of the EP-treated group was significantly lower than that of the PPE-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, these data suggest that by starting EP treatment one day after PPE administration, decline in pulmonary function associated with the onset of emphysematous lesions could be improved, as measured by HP \u003csup\u003e129\u003c/sup\u003eXe MRI. However, in the Nic-treated group, the decline in the \u003cem\u003er\u003c/em\u003e\u003csub\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003ef\u003c/em\u003e\u003csub\u003e\u003cem\u003eD\u003c/em\u003e\u003c/sub\u003e values did not improve (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, the MLI of the Nic-treated group did not improve compared with that of the PPE-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), supporting the results of the pulmonary functional assessments.\u003c/p\u003e \u003cp\u003eRegulation of the MAPK/ERK pathway has been reported to promote tissue repair during cutaneous wound healing [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The MAPK/ERK pathway has been reported to be activated during wound healing in a murine diabetic wound model [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Regulation of HMGB1 expression has been reported to be involved in wound healing process via the MAPK/ERK pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Based on these reports, we hypothesize that EP regulates the expression levels of HMGB1 and the MAPK/ERK pathway to exert anti-inflammatory and tissue repair effects. Our previous study using a mouse model of bleomycin-induced lung injury supported this hypothesis, in which we observed lung structural and functional changes caused by the upregulation and downregulation of HMGB1 expression [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, in this study, we were unable to directly elucidate the involvement of intracellular ROS and MAPK/ERK pathway in the recovery of injured tissue.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the involvement of intracellular ROS and MAPK/ERK pathway in elastase-induced emphysema in mice by evaluating and comparing the pharmacological effects of EP and Nic. Intracellular ROS production has been reported to contribute to elastase-induced lung injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Further, HMGB1 and NF-kB have also been reported to be involved in elastase-induced lung injury [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Furthermore, EP activated the ROS-MAPK/ERK pathway to inhibit melanogenesis in B16F10 melanoma cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, we hypothesized that EP moderately upregulates the expression levels of HMGB1 and activates the ROS-MAPK/ERK pathway to exert anti-inflammatory and tissue repair effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In contrast, Nic suppressed ROS production and showed its protective effect through downregulation of the NF-kB and MAPK pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the present study, Nic treatment did not improve pulmonary function, indicating the role of the MAPK/ERK pathway in wound healing process. In other words, non-activation of the MAPK/ERK pathway by Nic treatment seemed to impair tissue repair and lead to the development of emphysema and pulmonary functional decline.\u003c/p\u003e \u003cp\u003eAlthough Nic has been reported to show protective effects against acute lung injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], collapse-induced lung injury [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], pulmonary fibrosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], pulmonary artery endothelial damage in pulmonary hypertension [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and other lung disorders [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], its therapeutic effects have not been reported. In the present study, inflammation was detected one day after PPE administration, and Nic treatment was started at this stage to suppress the inflammation through inhibition of intracellular ROS production and NF-kB activation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, at this stage, in addition to inflammation, there appeared to be some extent of tissue destruction, as mentioned previously. We assumed that Nic did not show tissue repair effects via the MAPK/ERK pathway and was unable to exert a therapeutic effect considering the above reason. In contrast, EP downregulated HMGB1 expression, increased ROS production, and moderately upregulated the MAPK/ERK pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], resulting in tissue repair and suppression in the onset of emphysema, leading to improvement in pulmonary function.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, we successfully monitored temporal changes in pulmonary function of gas exchange and ventilation caused by the development of emphysema using a preclinical HP \u003csup\u003e129\u003c/sup\u003eXe MRI protocol. The treatment responses of EP and Nic against the development of emphysema were monitored and compared. EP treatment effectively repaired tissue damage and improved pulmonary function in elastase-induced lung injury, whereas Nic showed no therapeutic effects. The present results suggest therapeutic efficacy of EP in wound healing process via activation of the MAPK/ERK pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe authors are grateful to all members of the Department of Medical Physics and Engineering, Area of Medical Imaging Technology and Science, Division of Health Sciences, Graduate School of Medicine, The University of Osaka, for their valuable comments and helpful discussions. The authors would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Atsuomi Kimura designed the study. Material preparation, data collection, and analysis were conducted by all authors. Atsuomi Kimura wrote the first draft of the manuscript, and all authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported in part by the Grant-in-Aids of Scientific Research (No. 20H04516) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e The animal study protocol was approved by the Institutional Animal Care and Use Committee of Division of Health Sciences, Graduate School of Medicine, The University of Osaka (approval no. 24-03-03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMacLeod JL, Khan HM, Franklin A, Myc L, Shim YM (2025) Hyperpolarized xenon-129 MRI: Narrative review of clinical studies, testing, and implementation of advanced pulmonary in vivo imaging and its diagnostic applications. 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Int Immunopharmacol 64:289\u0026ndash;297\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Zuo X, Wang Q et al (2013) Nicorandil inhibits hypoxia-induced apoptosis in human pulmonary artery endothelial cells through activation of mitoKATP and regulation of eNOS and the NF-κB pathway. Int J Mol Med 32:187\u0026ndash;194\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed LA, El-Maraghy SA, Rizk SM (2015) Role of the KATP channel in the protective effect of nicorandil on cyclophosphamide-induced lung and testicular toxicity in rats. Sci Rep 5:14043\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hyperpolarized 129Xe magnetic resonance imaging, elastase-induced lung injury, mitogen-activated protein kinase, ethyl pyruvate, nicorandil","lastPublishedDoi":"10.21203/rs.3.rs-6772464/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6772464/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eWound healing process in lung injury involves activation of the mitogen-activated protein kinase (MAPK) pathway. In this study, we investigated the role of the MAPK pathway in wound healing in a murine model of emphysema using hyperpolarized \u003csup\u003e129\u003c/sup\u003eXe (HP \u003csup\u003e129\u003c/sup\u003eXe) magnetic resonance imaging (MRI).\u003c/p\u003e\u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003ePorcine pancreatic elastase was administered intratracheally to 25 mice to induce lung injury. Temporal changes in pulmonary gas exchange function were monitored using HP \u003csup\u003e129\u003c/sup\u003eXe MRI, which revealed a significant decline in function one day after elastase administration. Treatments with ethyl pyruvate (EP) and nicorandil (Nic), which upregulate and downregulate the MAPK pathway, respectively, were initiated in 12 and 7 of the 25 mice, respectively, and continued for 20 days. Over the 21-day period, HP \u003csup\u003e129\u003c/sup\u003eXe MRI was performed to monitor the disease progression and treatment efficacy through changes in the metrics of gas exchange and fractional ventilation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHP \u003csup\u003e129\u003c/sup\u003eXe MRI showed that EP significantly improved gas exchange function 14 days after elastase administration, whereas Nic did not show any improvement. Ventilatory function also improved in the EP group, but not in the Nic group, 14 days after elastase administration. Histological analysis showed that EP repaired tissue damage to a level similar to that observed in healthy mice, whereas Nic did not.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn the present study, we provided some insight into the role of the MAPK pathway in wound healing in elastase-induced lung injury, as assessed using the HP \u003csup\u003e129\u003c/sup\u003eXe MRI protocol.\u003c/p\u003e","manuscriptTitle":"Ethyl Pyruvate Promotes Wound Healing in Elastase-Induced Lung Injury in Mice as Assessed by Hyperpolarized 129Xe Magnetic Resonance Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-23 06:39:52","doi":"10.21203/rs.3.rs-6772464/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-10-06T08:58:26+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-16T23:06:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T22:07:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-29T23:50:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Imaging and Biology","date":"2025-05-29T00:03:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-imaging-and-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mibi","sideBox":"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)","snPcode":"11307","submissionUrl":"https://www.editorialmanager.com/mibi/default2.aspx","title":"Molecular Imaging and Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d1a8f9b5-74aa-4caf-a4c5-3a5f05ba0abf","owner":[],"postedDate":"June 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:06:09+00:00","versionOfRecord":{"articleIdentity":"rs-6772464","link":"https://doi.org/10.1007/s11307-025-02073-6","journal":{"identity":"molecular-imaging-and-biology","isVorOnly":false,"title":"Molecular Imaging and Biology"},"publishedOn":"2025-12-10 15:57:39","publishedOnDateReadable":"December 10th, 2025"},"versionCreatedAt":"2025-06-23 06:39:52","video":"","vorDoi":"10.1007/s11307-025-02073-6","vorDoiUrl":"https://doi.org/10.1007/s11307-025-02073-6","workflowStages":[]},"version":"v1","identity":"rs-6772464","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6772464","identity":"rs-6772464","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}