HMGB1 Box A gene therapy to alleviate bleomycin-induced pulmonary fibrosis in rats

HMGB1 Box A gene therapy to alleviate bleomycin-induced pulmonary fibrosis in rats | 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 HMGB1 Box A gene therapy to alleviate bleomycin-induced pulmonary fibrosis in rats Rathasapa Patarat, Suchanart Chuaybudda, Sakawdaurn Yasom, Apiwat Mutirangura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5266547/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2025 Read the published version in BMC Pulmonary Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Pulmonary fibrosis is characterized by the destruction of normal lung tissue and then replacement by abnormal fibrous tissue, leading to an overall decrease in gas exchange function. The effective treatment for pulmonary fibrosis remains unknown. The upstream pathogenesis of pulmonary fibrosis may involve cellular senescence of the lung tissue. Previously, a new gene therapy technology using Box A of the HMGB1 plasmid (Box A) was used to reverse cellular senescence and cure liver fibrosis in aged rats. Methods: Here, we show that Box A is a promising medicine for the treatment of lung fibrosis. In a bleomycin-induced pulmonary fibrosis rat model, Student's t ‐test and one-way ANOVA was used for comparisons among groups of samples. Results: Box A effectively lowered fibrous tissue deposits (from 18.74±0.62 to 3.45±1.19%) and senescent cells (from 3.74±0.40% to 0.89±0.18%) to levels comparable to those of the negative control group. Moreover, after eight weeks, Box A also increased the production of the surfactant protein C (from 3.60±1.68% to 6.82±0.65%). Conclusions Our results demonstrate that Box A is a promising therapeutic approach for pulmonary fibrosis and other senescence-promoted fibrotic lesions. Idiopathic Pulmonary fibrosis DNA damage DNA stability DNA protection Box A of HMGB1 Youth-DNA gap senescence rejuvenation gene therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Pulmonary fibrosis is a chronic lung disease characterized by defective pulmonary structural remodeling [ 1 ]. This remodeling includes excessive irregular fibrous tissue deposition in the extracellular matrix, increased numbers of senescent cells, and loss of normal gas exchange function. [ 2 ]. Pulmonary fibrosis affects approximately 5 million patients worldwide, and its incidence has sharply increased in recent years due to the COVID-19 pandemic [ 3 ]. A meta-analysis revealed that patients who survive severe COVID-19 infection are at risk of developing pulmonary fibrosis disease [ 4 ]. Moreover, severe pulmonary fibrosis disease cases have a mortality rate of 50% annually, with a median survival rate of 3 years after diagnosis. Currently, scientists are able to identify many pathways responsible for the development of pulmonary fibrosis [ 5 ]. Cellular senescence has been identified as a common cause of pulmonary fibrosis [ 6 – 8 ]. The accumulation of senescent cells of various cell types in the tissue leads to dysfunction of multiple normal tissue functions and results in excessive production of fibrous tissue, impaired fibrous tissue clearance, decreased proliferation of alveolar cells and/or endothelial cells, and prolonged aggregation of inflammatory cells. Ultimately, pulmonary fibrosis can develop [ 9 , 10 ]. Currently, the pharmacological targets for pulmonary fibrosis include epigenetic alterations, antifibrogenic agents, fibrolysis agents, and senolytic agents [ 11 – 13 ]. However, effective treatments for pulmonary fibrosis are lacking. For the last decade, our group has studied a new gene therapy for aging, the Box A portion of the high mobility group Box 1 protein (Box A of HMGB1 or Box A). Box A provides DNA protection and increases genomic stability by relieving DNA torsional stress due to DNA double helix denaturation during replication and transcription. Our group has already utilized Box A to treat liver fibrosis in an animal model. We found that administering Box A significantly reduced liver fibrosis to a level comparable to that of the control group [ 14 ]. Therefore, Box A could be a novel therapeutic agent for treating pulmonary fibrosis. In this study, we investigated the ability of Box A to prevent bleomycin-induced pulmonary fibrosis in a rat model. These findings demonstrate the potential of Box A for treating pulmonary fibrosis and can be developed into a new treatment for other senescence-induced fibrotic diseases. Methods 2.1 Plasmid construction and preparation In this study, we used the full-length human Box A sequence of HMGB1 and a scrambled sequence for the plasmid control (PC). The plasmids were subsequently transformed into Escherichia coli (DH5α) (Invitrogen), specifically, NEB® 5-alpha competent E. coli (New England BioLabs). For all plasmid selection, transformed cells were grown on LB agar supplemented with ampicillin or chloramphenicol. The selected colony was then cultured in LB broth supplemented with 100 µg/ml ampicillin and incubated on an incubator shaker at 37°C for 16 hrs. The plasmids were then extracted via a GeneJET Plasmid Maxiprep Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Sequence fidelity was confirmed by Sanger sequencing[ 14 ]. 2.2 Nanoparticle construction and preparation To deliver the plasmids into the rat model, each type of plasmid was coated with Ca-P (Calcium Phosphate) nanoparticle mixture before administration[ 14 ]. Our team prepared the solution at the maximal effective concentration. The highest effective plasmid to Ca‐P nanoparticle solution ratio for transfection was 5 µg of plasmid in 100 µl of Ca‐P nanoparticle solution. The Ca‐P nanoparticle solution was composed of a mixture of 0.5 M calcium chloride (CaCl 2 ) solution (Merck Millipore), 0.01 M sodium carbonate (Na 2 CO 3 ) solution (Merck Millipore), and 0.01 M sodium dihydrogen phosphate monohydrate (NaH 2 PO 4 ·H 2 O) solution (Merck Millipore). The final molar ratio of the CO 3 2− /PO 4 3− nanoparticle solution was 31:1. To mix the solutions together, we followed these steps. First, the plasmid DNA‐calcium complex was prepared by mixing 16 µl of CaCl 2 solution and 5 µg of plasmid DNA, with the final volume adjusted to 50 µl via sterile dH 2 O, and the mixture was maintained at room temperature. Second, the plasmid DNA‐calcium complex was added to 50 µl of a mixture of Na 2 CO 3 and NaH 2 PO 4 ·H 2 O solution (16 µl) and sterile dH 2 O (34 µl). The final solution contained CaPO 4 /plasmid DNA nanoparticles as intended. For the rat model, each plasmid type was calculated on the basis of the rat body weight (100 µg of plasmid DNA per kg of rat body weight). After the amount of solution needed was calculated, the plasmid DNA was coated with Ca‐P nanoparticle solution as described above. Finally, the plasmid DNA‐Ca‐P nanoparticle mixture was freshly prepared before intraperitoneal administration. 2.3 Animal disease models and therapeutic diagram All animal procedures were reviewed and approved by the Animal Care and Use Committee, Medical Faculty, Chulalongkorn University, Thailand, Approval No. 032/2564. Forty-eight male Wistar rats (6–8 weeks of age) were purchased from Nomura Siam International, Bangkok, Thailand. After 1 week of acclimatization, all the animals were housed in a temperature-controlled chamber (25 ± 0.5°C) with a 12:12‐hour light/dark cycle with a standard diet, and sterilized water was provided ad libitum . All the rats were monitored daily and weighed weekly until they reached the desired weight and age (300–350 grams and 8–10 weeks of age). The rats of the desired age and weights were randomly assigned to subgroups by a laboratory technician who was blinded to the characteristics of the rats. Forty-eight male Wistar rats were used in this study and were randomly assigned to six subgroups. (Fig. 1 ) 6-week Vehicle Control group ; Eight rats were given intraperitoneal normal saline(NSS) beginning on Day 0, thrice per week, and given for three weeks. At Day 21 or after the animal had received 9 doses of normal saline, normal saline administration was stopped. Fourteen days later, PC-Calcium Phosphate (100 µg/kg) was administered intraperitoneally(i.p.) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77. 6-week Disease Model group : Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning on Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of bleomycin, bleomycin administration was stopped. Fourteen days later, we administered intraperitoneal PC-Calcium Phosphate (100 µg/kg) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77. 6-week Treatment Group : Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning on the Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of bleomycin, bleomycin administration was stopped. Fourteen days later, we administered intraperitoneal Box A-Calcium Phosphate (100 µg/kg) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77. 8-week Vehicle Control group : Eight rats were given intraperitoneal normal saline beginning on Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of normal saline, normal saline administration was stopped. Fourteen days later, intraperitoneal PC-Calcium Phosphate (100 µg/kg) was administered once every week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91. 8-week Disease Model group : Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning Day 0 thrice per week for three weeks. Bleomycin was stopped on Day 21 or after 9 doses. Fourteen days later, intraperitoneal PC-Calcium Phosphate (100 µg/kg) was administered once every week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91. 8-week Treatment Group : Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning Day 0 thrice per week for three weeks. Bleomycin was stopped on Day 21 or after 9 doses. Fourteen days later, intraperitoneal Box A-Calcium Phosphate (100 µg/kg) was administered once per week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91. 2.3 Detection of blood chemistry, and complete blood count. Blood samples from all the rats were collected at the beginning of the acclimatization period, before bleomycin/normal saline was administered, before plasmid/treatment was administered, and just after euthanization. The blood samples were collected and shipped to the Pathology Laboratory Department, Small Animal Hospital, Faculty of Veterinary Medicine, Chulalongkorn University, for analysis of the complete blood count and glucose, creatinine, total protein, albumin, globulin, alanine transaminase, alkaline phosphatase, C-reactive protein, and blood urea nitrogen (BUN) levels. All of the blood samples for the complete blood count were measured via a ProCyte Dx analyzer (IDEXX, USA), and all of the blood samples for blood chemistry were measured via a Catalyst One Chemistry analyzer (IDEXX, USA). 2.4 SA-β‐Gal staining After euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For the SA-β‐Gal(senescence associated – beta – galactosidase) staining method, lungs were fixed in 4% paraformaldehyde (PFA) before being embedded in optimal cutting temperature (OCT) compound (Sakura, Tissue‐Tek) and cryosectioned at a thickness of 10 µm. After rehydration of the lung sections in PBS for 10 minutes, the sections were subjected to SA‐β‐gal staining via a Cell Signaling Kit (9860, Beverly, MA, USA) with 15‐min of fixation followed by incubation at 37°C in the staining solution for at least 12 hr. Images of the sections were captured via a Leica DM1000 inverted microscope with a color camera. SA‐β‐gal in lung sections was quantified. The images were analyzed via densitometry. ImageJ software (open source) was used to analyze the area with SA‐β‐Gal staining. 2.5 Histopathological analysis After euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For Masson’s trichrome staining, lungs were fixed in 10% neutral buffered formalin for less than 48 hours, processed into paraffin blocks, and cut into 5-µm sections. The slides were subjected to Masson’s trichrome staining according to standard procedures for histopathological analysis. The Masson trichrome-stained slides were captured via a Leica DM1000 inverted microscope with a color camera. The images were evaluated for fibrous tissue accumulation and degree of fibrosis in the lung according to the standard histopathological analysis of pulmonary fibrosis. ImageJ software (open source) was used to analyze the densitometry of the fibrotic tissue. 2.6 Immunohistochemistry (IHC) After euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For IHC staining, lungs were fixed in 10% neutral buffered formalin for less than 48 hours, processed into paraffin blocks, and cut into 5-µm sections. The immunohistochemistry (IHC) procedure was as follows: deparaffinize at 65°C for 15 min, rehydrate in descending alcohol series, rinse with hydrogen peroxide, rest for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 100°C for 20 min, rinse with PBS, and then block with 2% FBS. Then, the sections were incubated with either (1:50) rabbit anti-SFTPC antibody (ABC99, Abcam®) or (1:500) rabbit anti-DYKDDDDK tag (FLAG) antibody (#14793, Cell Signaling) at room temperature overnight. After that, the slides were rinsed with PBS and incubated with an HRP-linked anti-rabbit IgG antibody (7074 V, Cell Signaling®) at 30°C for 1 hour. Then, the slides were washed with PBS, followed by incubation with ABC solution at 30°C for 30 minutes. The slides were then washed in PBS, incubated with DAB substrate (Merck®) at room temperature for 10 minutes, and then rinsed with tap water. Moreover, on slides with an anti-DYKDDDDK tag, hematoxylin was used for counterstaining. However, none of the anti-SFTPC slides were a counterstained. Finally, the sections were captured via a Leica DM1000 inverted microscope with a color camera. For quantification of immunohistochemical staining in lung sections, the images were analyzed via densitometry. ImageJ software (open source) was used to analyze the area with positive staining. 2.7 Statistics The data were analyzed for their distribution before the appropriate analysis tools were selected. Student's t -test was used for comparisons between two sets of samples, and one‐way ANOVA was used for comparisons among multiple groups of samples. Statistical analyses were performed with GraphPad Prism V9.5 for Windows (GraphPad Software, Inc.). Results 3.1 Effectiveness of Box A in reducing senescence in bleomycin-induced pulmonary fibrosis After our treatment of rats with bleomycin-induced pulmonary fibrosis with Box A protein following our experimental protocol (Fig. 1 ) , we sacrificed the rats, harvested the lung tissue for senescence-associated beta-galactosidase staining, and analyzed the area of staining. We calculated the area of senescence for each group (Fig. 2 ) . The groups that received NSS + plasmid control, bleomycin + plasmid control, or bleomycin + Box A for 6 weeks exhibited senescence areas encompassing 0.36 ± 0.04%, 3.8 ± 0.19%, and 1.04 ± 0.21% (mean ± SD) of the total tissue area, respectively. The groups that received NSS + plasmid control, bleomycin + plasmid control, or bleomycin + Box A for 8 weeks exhibited senescence areas encompassing 0.35 ± 0.05%, 3.74 ± 0.40%, and 0.89 ± 0.18% (mean ± SD) of the total tissue area, respectively. The differences between the groups treated with bleomycin + plasmid control for 6 weeks and with bleomycin + Box A for 6 weeks were statistically significant (p value < 0.0001). The differences between the groups treated with bleomycin + plasmid control for 8 weeks and with bleomycin + Box A for 8 weeks were also statistically significant (p value < 0.0001). We also collected blood samples from the rats after Box A was administered to treat the induced pulmonary fibrosis (Supplemental Fig. 1). Moreover, to confirm that the Box A-producing plasmid had reached the lung tissue, entered the cells and produced Box A as intended, we tagged Box A with a DYKDDDDK tag attached to the end of the Box A protein. The results of immunohistostaining are shown in Supplemental Fig. 2. The results clearly revealed that the plasmid had entered the target cells and produced Box A protein. 3.2 Effectiveness of Box A in reducing fibrosis in bleomycin-induced pulmonary fibrosis After our treatment of bleomycin-induced pulmonary fibrosis in the rats with Box A protein following our experimental protocol, we sacrificed the rats, harvested the lung tissue for histostaining via Masson-Trichrome staining, and analyzed the area of fibrous tissue deposits. We subsequently calculated the area of fibrosis for each group (Fig. 3 ) . The fibrotic tissue areas of the rats in the NSS + plasmid control, bleomycin + plasmid control, and bleomycin + Box A groups after 6 weeks were 3.25 ± 0.97%, 18.60 ± 1.49%, and 3.58 ± 1.11% (mean ± SD) of the total area, respectively. For the groups that received NSS + plasmid control, bleomycin + plasmid control, or bleomycin + Box A for 8 weeks, the fibrotic areas were 3.05 ± 0.52%, 18.74 ± 1.66%, and 4.11 ± 1.36% (mean ± SD), respectively. The differences between the groups treated with bleomycin + plasmid control for 6 weeks and with bleomycin + Box A for 6 weeks were statistically significant (p value < 0.0001). The differences between the groups treated with bleomycin + plasmid control for 8 weeks and with bleomycin + Box A for 8 weeks were statistically significant (p value < 0.0001). 3.3 Effectiveness of Box A on surfactant protein C production in bleomycin-induced pulmonary fibrosis After our treatment of bleomycin-induced pulmonary fibrosis in the rats with Box A protein following our experimental protocol, we sacrificed the rats and harvested the lung tissue for anti-sftPC (anti-surfactant protein C) immunohistostaining and analyzed the area of staining. We calculated the area of surfactant protein C for each group as follows. The groups that received NSS + plasmid control, bleomycin + plasmid control, or bleomycin + plasmid control for 6 weeks exhibited surfactant protein C areas representing 13.71 ± 2.64%, 3.44 ± 0.96%, and 2.52 ± 1.38% (mean ± SD) of the total tissue area, respectively. The groups that received NSS + plasmid control, bleomycin + plasmid control, or bleomycin + plasmid control for 8 weeks exhibited surfactant protein C areas representing 13.01 ± 1.55%, 3.60 ± 1.68%, and 6.82 ± 0.65% (mean ± SD) of the total tissue area, respectively. The differences between the groups treated with bleomycin + plasmid control for 6 weeks and with bleomycin + Box A for 6 weeks were not statistically significant (p value = 0.2606). The differences between the groups treated with bleomycin + plasmid control for 8 weeks and with bleomycin + Box A for 8 weeks were statistically significant (p value = 0.0039). Discussion Our research demonstrated that administering the Box A-producing plasmid to rats with bleomycin-induced pulmonary fibrosis significantly reduced the number of senescent cells and fibrotic deposit area in the rat lung tissue within 6 to 8 weeks compared with those in the control group. The results also revealed a significant improvement in surfactant protein production, although this was seen at only the 8-week time point and did not completely return to baseline levels . Regarding how Box A helps reduce senescence, our team has recently published research findings about the mechanism involved[ 13 – 17 ]. To summarize, the administered plasmid produces a Box A portion of the HMGB1 protein, and these Box A proteins then spread throughout the cells. Some Box A is transferred into the cell nucleus, where it promotes DNA stabilization, DNA stress relief, DNA gap production, chromatin structural changes, and DNA protection. Once senescent cells or presenescent cells have received sufficient Box A and achieved DNA stabilization, these cells reduce the DDR and consequently emerge from the senescent state and return to normal. The possibility of Box A clearing fibrosis may be due to a reduction in the number of senescent cells. After Box A reversed the senescent phenotype, these cells began to function normally. These rejuvenated cells include fibrocytes, fibroblasts, and tissue-specific macrophages. These cells are involved in the creation, retention, and destruction of fibrotic tissue[ 18 – 20 ]. Typically, when these cells function normally, the fibroblasts and fibrocytes create and rearrange the fibrous tissue during the tissue healing process. After the tissue is healed, the normal fibroblasts and fibrocytes stop producing more fibrous tissue, and the fibrous material degrades by itself or is destroyed by tissue-specific macrophages. With respect to the ability of Box A to promote surfactant protein production, the rejuvenated cells include not only fibroblasts and macrophages inside the tissue. Box A also affects all cells, including epithelial cells and tissue progenitor cells[ 21 – 22 ]. When senescent epithelial cells are rejuvenated, their protein-producing function returns to normal. Therefore, epithelial cells can produce normal functioning surfactant protein again[ 23 – 24 ]. Additionally, as progenitor cells return to normal, they enter the cell cycle and differentiate to replace destroyed epithelial cells in the tissue[ 25 ]. Because cell division and cell differentiation require time, surfactant protein production is a slower process than rejuvenation and fibrosis clearance. Conclusions As pulmonary fibrosis continues to be an incurable disease, finding or developing novel and effective treatments is essential. An examination of the effect of Box A in treating bleomycin-induced pulmonary fibrosis in a rat model revealed that Box A could treat pulmonary fibrosis by reducing the number of senescent cells in the diseased tissue, with a significant direct correlation with the fibrotic tissue area and an inverse correlation with surfactant protein production. Some genetic mutations, such as surfactant protein mutations, can also lead to pulmonary fibrosis. The process of developing fibrosis includes chronic inflammation resulting from the production of a faulty surfactant protein, which leads to the accumulation of oxidative stress, ultimately leading to cellular senescence. The ability of Box A to help rejuvenate senescent cells and help cure pulmonary fibrosis should be an example for future research and the development of new treatments for other diseases involving cellular senescence. Abbreviations anti-sftPC anti - surfactant protein C Ble Bleomycin Box A Box A portion of the high mobility group Box 1 protein BUN blood urea nitrogen Ca-P Calcium Phosphate IHC immunohistochemistry i.p. intraperitoneally NSS normal saline solution OCT optimal cutting temperature PC plasmid control SA-β‐Gal senescence associated – beta – galactosidase Declarations Ethics approval and consent to participate All the animal procedures were reviewed and approved by the Animal Care and Use Committee, Medical Faculty, Chulalongkorn University, Thailand, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines (Approval No. 032/2564). Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding This work was supported by the Applied Research of Rejuvenating DNA by Genomic Stability Molecule (REDGEM) for the treatment of age-associated disease. National Research Council of Thailand (NRCT), the National Science and Technology Development Agency, Thailand [Research Chair Grant, P-19-50189] Authors' contributions RP and AM conceived the study and designed the analysis; RP and SC designed the methodology and investigation; RP analyzed and wrote the original draft of the paper; AM supplied grant support, reviewed and edited the article; RP, SC, and SY handled the animals; AM and SY are involved in supervision and visualization; and all authors read and approved the final manuscript. 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The cellular kinetics of lung alveolar epithelial cells and its relationship with lung tissue repair after acute lung injury. Respir Res. 2016;17(1):164. 10.1186/s12931-016-0480-y . Additional Declarations No competing interests reported. Supplementary Files supplement.docx Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2025 Read the published version in BMC Pulmonary Medicine → Version 1 posted Editorial decision: Revision requested 28 Oct, 2024 Editor assigned by journal 28 Oct, 2024 Submission checks completed at journal 25 Oct, 2024 First submitted to journal 15 Oct, 2024 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. 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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-5266547","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371192980,"identity":"c52ead6f-1c58-4de9-ad11-c3c62110d0cf","order_by":0,"name":"Rathasapa Patarat","email":"","orcid":"","institution":"Chulalongkorn University, King Chulalongkorn Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rathasapa","middleName":"","lastName":"Patarat","suffix":""},{"id":371192981,"identity":"988071ac-629a-45c8-9220-cc046327240d","order_by":1,"name":"Suchanart Chuaybudda","email":"","orcid":"","institution":"Chulalongkorn University, King Chulalongkorn Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Suchanart","middleName":"","lastName":"Chuaybudda","suffix":""},{"id":371192982,"identity":"abda0955-a7a1-4815-9a48-d5d51dd7bf49","order_by":2,"name":"Sakawdaurn Yasom","email":"","orcid":"","institution":"Chulalongkorn University, King Chulalongkorn Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sakawdaurn","middleName":"","lastName":"Yasom","suffix":""},{"id":371192983,"identity":"afc7fe4b-484a-4706-9e23-49b3f10aa264","order_by":3,"name":"Apiwat Mutirangura","email":"data:image/png;base64,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","orcid":"","institution":"Chulalongkorn University, King Chulalongkorn Memorial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Apiwat","middleName":"","lastName":"Mutirangura","suffix":""}],"badges":[],"createdAt":"2024-10-15 07:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5266547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5266547/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12890-025-03522-2","type":"published","date":"2025-01-31T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68341353,"identity":"4eacdf3c-0258-4154-8e97-ea4d4f3ec568","added_by":"auto","created_at":"2024-11-06 08:55:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100055,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagrams of bleomycin-induced pulmonary fibrosis treatment in a rat model. The rats were given either bleomycin (15 mg/kg/dose, i.p.) or normal saline at 3 doses/week for 3 weeks, allowed to rest for 2 weeks, and then given either Box A plasmid-calcium-phosphate nanoparticles (Box A) (100 μg/kg/week, i.p.) or plasmid control-calcium-phosphate nanoparticles (PCs) (100 μg/kg/week, i.p.), and the injections were continued for either 6 or 8 weeks.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/89d0b96ada47baf875da8abb.png"},{"id":68342891,"identity":"c1099588-9505-4da2-8c61-0fcec90991fe","added_by":"auto","created_at":"2024-11-06 09:11:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":458709,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Box A on cellular senescence in bleomycin-induced pulmonary fibrosis rats (n=6). (A) SA-β-gal staining of rat lung sections. (B) Quantitative analysis of images of SA-β-gal-stained rat lungs. The values represent the means±SDs. *\u003cem\u003ep\u003c/em\u003e ≤ 0.05, **\u003cem\u003ep\u003c/em\u003e ≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e ≤ 0.001 \u003cem\u003et\u003c/em\u003e‐test\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/e00a93ee65bbe4d18130619d.png"},{"id":68340227,"identity":"9ac390bb-1cc0-4b96-83b0-020e731af108","added_by":"auto","created_at":"2024-11-06 08:47:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":529313,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Box A on fibrotic tissue deposits in bleomycin-induced pulmonary fibrosis rats (n=6). Masson-Trichrome staining of rat lung sections (A). Quantitative analysis of pictures of Masson-Trichrome-stained rat lungs (B). The values represent the means ± SDs. *\u003cem\u003ep\u003c/em\u003e ≤ 0.05, **\u003cem\u003ep\u003c/em\u003e ≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e ≤ 0.001 \u003cem\u003et\u003c/em\u003e‐test\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/dff17c23558d60b8f424ad1f.png"},{"id":68341766,"identity":"62fb0651-0348-474a-96d8-f67805fe93c0","added_by":"auto","created_at":"2024-11-06 09:03:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":432285,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Box A on surfactant protein C production in bleomycin-induced pulmonary fibrosis rats (n=6). (A) Immunohistostaining with an anti-sftpc antibody in rat lung sections. (B) Quantitative analysis of the images of immunohistostaining in the rat lung. The values represent the means±SDs. *\u003cem\u003ep\u003c/em\u003e ≤ 0.05, **\u003cem\u003ep\u003c/em\u003e ≤ 0.01, ***\u003cem\u003ep\u003c/em\u003e ≤ 0.001 \u003cem\u003et\u003c/em\u003e‐test\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/12fff5c7c073c48ddd8b6fd2.png"},{"id":75351483,"identity":"0783636d-1fb2-42b5-b99f-04f48b0fb230","added_by":"auto","created_at":"2025-02-03 16:11:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2280232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/cdf4dce3-9b01-4495-89df-3bfa1433f3af.pdf"},{"id":68340231,"identity":"39f362a4-b3be-428c-80ce-8acb1467f954","added_by":"auto","created_at":"2024-11-06 08:47:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":427752,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-5266547/v1/374216969c9bea0c000547e6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"HMGB1 Box A gene therapy to alleviate bleomycin-induced pulmonary fibrosis in rats","fulltext":[{"header":"Background","content":"\u003cp\u003ePulmonary fibrosis is a chronic lung disease characterized by defective pulmonary structural remodeling [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This remodeling includes excessive irregular fibrous tissue deposition in the extracellular matrix, increased numbers of senescent cells, and loss of normal gas exchange function. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pulmonary fibrosis affects approximately 5\u0026nbsp;million patients worldwide, and its incidence has sharply increased in recent years due to the COVID-19 pandemic [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A meta-analysis revealed that patients who survive severe COVID-19 infection are at risk of developing pulmonary fibrosis disease [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, severe pulmonary fibrosis disease cases have a mortality rate of 50% annually, with a median survival rate of 3 years after diagnosis.\u003c/p\u003e \u003cp\u003eCurrently, scientists are able to identify many pathways responsible for the development of pulmonary fibrosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cellular senescence has been identified as a common cause of pulmonary fibrosis [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The accumulation of senescent cells of various cell types in the tissue leads to dysfunction of multiple normal tissue functions and results in excessive production of fibrous tissue, impaired fibrous tissue clearance, decreased proliferation of alveolar cells and/or endothelial cells, and prolonged aggregation of inflammatory cells. Ultimately, pulmonary fibrosis can develop [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Currently, the pharmacological targets for pulmonary fibrosis include epigenetic alterations, antifibrogenic agents, fibrolysis agents, and senolytic agents [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, effective treatments for pulmonary fibrosis are lacking.\u003c/p\u003e \u003cp\u003eFor the last decade, our group has studied a new gene therapy for aging, the Box A portion of the high mobility group Box 1 protein (Box A of HMGB1 or Box A). Box A provides DNA protection and increases genomic stability by relieving DNA torsional stress due to DNA double helix denaturation during replication and transcription. Our group has already utilized Box A to treat liver fibrosis in an animal model. We found that administering Box A significantly reduced liver fibrosis to a level comparable to that of the control group [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, Box A could be a novel therapeutic agent for treating pulmonary fibrosis. In this study, we investigated the ability of Box A to prevent bleomycin-induced pulmonary fibrosis in a rat model. These findings demonstrate the potential of Box A for treating pulmonary fibrosis and can be developed into a new treatment for other senescence-induced fibrotic diseases.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003e2.1\u003c/b\u003e \u003cb\u003ePlasmid construction and preparation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we used the full-length human Box A sequence of HMGB1 and a scrambled sequence for the plasmid control (PC). The plasmids were subsequently transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e (DH5α) (Invitrogen), specifically, NEB\u0026reg; 5-alpha competent \u003cem\u003eE. coli\u003c/em\u003e (New England BioLabs). For all plasmid selection, transformed cells were grown on LB agar supplemented with ampicillin or chloramphenicol. The selected colony was then cultured in LB broth supplemented with 100 \u0026micro;g/ml ampicillin and incubated on an incubator shaker at 37\u0026deg;C for 16 hrs. The plasmids were then extracted via a GeneJET Plasmid Maxiprep Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Sequence fidelity was confirmed by Sanger sequencing[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2\u003c/b\u003e \u003cb\u003eNanoparticle construction and preparation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo deliver the plasmids into the rat model, each type of plasmid was coated with Ca-P (Calcium Phosphate) nanoparticle mixture before administration[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our team prepared the solution at the maximal effective concentration. The highest effective plasmid to Ca‐P nanoparticle solution ratio for transfection was 5 \u0026micro;g of plasmid in 100 \u0026micro;l of Ca‐P nanoparticle solution. The Ca‐P nanoparticle solution was composed of a mixture of 0.5 M calcium chloride (CaCl\u003csub\u003e2\u003c/sub\u003e) solution (Merck Millipore), 0.01 M sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) solution (Merck Millipore), and 0.01 M sodium dihydrogen phosphate monohydrate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO) solution (Merck Millipore). The final molar ratio of the CO\u003csub\u003e3\u003c/sub\u003e \u003csup\u003e2\u0026minus;\u003c/sup\u003e/PO\u003csub\u003e4\u003c/sub\u003e \u003csup\u003e3\u0026minus;\u003c/sup\u003e nanoparticle solution was 31:1. To mix the solutions together, we followed these steps. First, the plasmid DNA‐calcium complex was prepared by mixing 16 \u0026micro;l of CaCl\u003csub\u003e2\u003c/sub\u003e solution and 5 \u0026micro;g of plasmid DNA, with the final volume adjusted to 50 \u0026micro;l via sterile dH\u003csub\u003e2\u003c/sub\u003eO, and the mixture was maintained at room temperature. Second, the plasmid DNA‐calcium complex was added to 50 \u0026micro;l of a mixture of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e and NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO solution (16 \u0026micro;l) and sterile dH\u003csub\u003e2\u003c/sub\u003eO (34 \u0026micro;l). The final solution contained CaPO\u003csub\u003e4\u003c/sub\u003e/plasmid DNA nanoparticles as intended. For the rat model, each plasmid type was calculated on the basis of the rat body weight (100 \u0026micro;g of plasmid DNA per kg of rat body weight). After the amount of solution needed was calculated, the plasmid DNA was coated with Ca‐P nanoparticle solution as described above. Finally, the plasmid DNA‐Ca‐P nanoparticle mixture was freshly prepared before intraperitoneal administration.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3\u003c/b\u003e \u003cb\u003eAnimal disease models and therapeutic diagram\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e All animal procedures were reviewed and approved by the Animal Care and Use Committee, Medical Faculty, Chulalongkorn University, Thailand, Approval No. 032/2564. Forty-eight male Wistar rats (6\u0026ndash;8 weeks of age) were purchased from Nomura Siam International, Bangkok, Thailand. After 1 week of acclimatization, all the animals were housed in a temperature-controlled chamber (25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C) with a 12:12‐hour light/dark cycle with a standard diet, and sterilized water was provided \u003cem\u003ead libitum\u003c/em\u003e. All the rats were monitored daily and weighed weekly until they reached the desired weight and age (300\u0026ndash;350 grams and 8\u0026ndash;10 weeks of age). The rats of the desired age and weights were randomly assigned to subgroups by a laboratory technician who was blinded to the characteristics of the rats. Forty-eight male Wistar rats were used in this study and were randomly assigned to six subgroups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e6-week Vehicle Control group\u003c/b\u003e; Eight rats were given intraperitoneal normal saline(NSS) beginning on Day 0, thrice per week, and given for three weeks. At Day 21 or after the animal had received 9 doses of normal saline, normal saline administration was stopped. Fourteen days later, PC-Calcium Phosphate (100 \u0026micro;g/kg) was administered intraperitoneally(i.p.) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e6-week Disease Model group\u003c/b\u003e: Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning on Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of bleomycin, bleomycin administration was stopped. Fourteen days later, we administered intraperitoneal PC-Calcium Phosphate (100 \u0026micro;g/kg) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e6-week Treatment Group\u003c/b\u003e: Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning on the Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of bleomycin, bleomycin administration was stopped. Fourteen days later, we administered intraperitoneal Box A-Calcium Phosphate (100 \u0026micro;g/kg) once every week for 6 weeks (Day 35, 42, 49, 56, 63, 70). All rats were euthanized on Day 77.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e8-week Vehicle Control group\u003c/b\u003e: Eight rats were given intraperitoneal normal saline beginning on Day 0 thrice per week for three weeks. On Day 21 or after 9 doses of normal saline, normal saline administration was stopped. Fourteen days later, intraperitoneal PC-Calcium Phosphate (100 \u0026micro;g/kg) was administered once every week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e8-week Disease Model group\u003c/b\u003e: Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning Day 0 thrice per week for three weeks. Bleomycin was stopped on Day 21 or after 9 doses. Fourteen days later, intraperitoneal PC-Calcium Phosphate (100 \u0026micro;g/kg) was administered once every week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003e8-week Treatment Group\u003c/b\u003e: Eight rats were given intraperitoneal Bleomycin (15 mg/kg) beginning Day 0 thrice per week for three weeks. Bleomycin was stopped on Day 21 or after 9 doses. Fourteen days later, intraperitoneal Box A-Calcium Phosphate (100 \u0026micro;g/kg) was administered once per week for 8 weeks (Day 35, 42, 49, 56, 63, 70, 77, 84). All rats were euthanized on Day 91.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 Detection of blood chemistry, and complete blood count.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBlood samples from all the rats were collected at the beginning of the acclimatization period, before bleomycin/normal saline was administered, before plasmid/treatment was administered, and just after euthanization. The blood samples were collected and shipped to the Pathology Laboratory Department, Small Animal Hospital, Faculty of Veterinary Medicine, Chulalongkorn University, for analysis of the complete blood count and glucose, creatinine, total protein, albumin, globulin, alanine transaminase, alkaline phosphatase, C-reactive protein, and blood urea nitrogen (BUN) levels. All of the blood samples for the complete blood count were measured via a ProCyte Dx analyzer (IDEXX, USA), and all of the blood samples for blood chemistry were measured via a Catalyst One Chemistry analyzer (IDEXX, USA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.4 SA-β‐Gal staining\u003c/h2\u003e \u003cp\u003eAfter euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For the SA-β‐Gal(senescence associated \u0026ndash; beta \u0026ndash; galactosidase) staining method, lungs were fixed in 4% paraformaldehyde (PFA) before being embedded in optimal cutting temperature (OCT) compound (Sakura, Tissue‐Tek) and cryosectioned at a thickness of 10 \u0026micro;m. After rehydration of the lung sections in PBS for 10 minutes, the sections were subjected to SA‐β‐gal staining via a Cell Signaling Kit (9860, Beverly, MA, USA) with 15‐min of fixation followed by incubation at 37\u0026deg;C in the staining solution for at least 12 hr. Images of the sections were captured via a Leica DM1000 inverted microscope with a color camera. SA‐β‐gal in lung sections was quantified. The images were analyzed via densitometry. ImageJ software (open source) was used to analyze the area with SA‐β‐Gal staining.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.5 Histopathological analysis\u003c/h3\u003e\n\u003cp\u003eAfter euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For Masson\u0026rsquo;s trichrome staining, lungs were fixed in 10% neutral buffered formalin for less than 48 hours, processed into paraffin blocks, and cut into 5-\u0026micro;m sections. The slides were subjected to Masson\u0026rsquo;s trichrome staining according to standard procedures for histopathological analysis. The Masson trichrome-stained slides were captured via a Leica DM1000 inverted microscope with a color camera. The images were evaluated for fibrous tissue accumulation and degree of fibrosis in the lung according to the standard histopathological analysis of pulmonary fibrosis. ImageJ software (open source) was used to analyze the densitometry of the fibrotic tissue.\u003c/p\u003e\n\u003ch3\u003e2.6 Immunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eAfter euthanization, the rat tissues were immediately dissected and fixed in fresh fixative buffer. For IHC staining, lungs were fixed in 10% neutral buffered formalin for less than 48 hours, processed into paraffin blocks, and cut into 5-\u0026micro;m sections. The immunohistochemistry (IHC) procedure was as follows: deparaffinize at 65\u0026deg;C for 15 min, rehydrate in descending alcohol series, rinse with hydrogen peroxide, rest for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 100\u0026deg;C for 20 min, rinse with PBS, and then block with 2% FBS. Then, the sections were incubated with either (1:50) rabbit anti-SFTPC antibody (ABC99, Abcam\u0026reg;) or (1:500) rabbit anti-DYKDDDDK tag (FLAG) antibody (#14793, Cell Signaling) at room temperature overnight. After that, the slides were rinsed with PBS and incubated with an HRP-linked anti-rabbit IgG antibody (7074 V, Cell Signaling\u0026reg;) at 30\u0026deg;C for 1 hour. Then, the slides were washed with PBS, followed by incubation with ABC solution at 30\u0026deg;C for 30 minutes. The slides were then washed in PBS, incubated with DAB substrate (Merck\u0026reg;) at room temperature for 10 minutes, and then rinsed with tap water. Moreover, on slides with an anti-DYKDDDDK tag, hematoxylin was used for counterstaining. However, none of the anti-SFTPC slides were a counterstained. Finally, the sections were captured via a Leica DM1000 inverted microscope with a color camera. For quantification of immunohistochemical staining in lung sections, the images were analyzed via densitometry. ImageJ software (open source) was used to analyze the area with positive staining.\u003c/p\u003e\n\u003ch3\u003e2.7 Statistics\u003c/h3\u003e\n\u003cp\u003eThe data were analyzed for their distribution before the appropriate analysis tools were selected. Student's \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two sets of samples, and one‐way ANOVA was used for comparisons among multiple groups of samples. Statistical analyses were performed with GraphPad Prism V9.5 for Windows (GraphPad Software, Inc.).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e3.1 Effectiveness of Box A in reducing senescence in bleomycin-induced pulmonary fibrosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter our treatment of rats with bleomycin-induced pulmonary fibrosis with Box A protein following our experimental protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, we sacrificed the rats, harvested the lung tissue for senescence-associated beta-galactosidase staining, and analyzed the area of staining. We calculated the area of senescence for each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The groups that received NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, or bleomycin\u0026thinsp;+\u0026thinsp;Box A for 6 weeks exhibited senescence areas encompassing 0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04%, 3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19%, and 1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of the total tissue area, respectively. The groups that received NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, or bleomycin\u0026thinsp;+\u0026thinsp;Box A for 8 weeks exhibited senescence areas encompassing 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%, 3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40%, and 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of the total tissue area, respectively. The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 6 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 6 weeks were statistically significant (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 8 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 8 weeks were also statistically significant (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also collected blood samples from the rats after Box A was administered to treat the induced pulmonary fibrosis (Supplemental Fig.\u0026nbsp;1). Moreover, to confirm that the Box A-producing plasmid had reached the lung tissue, entered the cells and produced Box A as intended, we tagged Box A with a DYKDDDDK tag attached to the end of the Box A protein. The results of immunohistostaining are shown in Supplemental Fig.\u0026nbsp;2. The results clearly revealed that the plasmid had entered the target cells and produced Box A protein.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Effectiveness of Box A in reducing fibrosis in bleomycin-induced pulmonary fibrosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter our treatment of bleomycin-induced pulmonary fibrosis in the rats with Box A protein following our experimental protocol, we sacrificed the rats, harvested the lung tissue for histostaining via Masson-Trichrome staining, and analyzed the area of fibrous tissue deposits. We subsequently calculated the area of fibrosis for each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The fibrotic tissue areas of the rats in the NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, and bleomycin\u0026thinsp;+\u0026thinsp;Box A groups after 6 weeks were 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97%, 18.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49%, and 3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of the total area, respectively. For the groups that received NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, or bleomycin\u0026thinsp;+\u0026thinsp;Box A for 8 weeks, the fibrotic areas were 3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52%, 18.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66%, and 4.11\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), respectively. The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 6 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 6 weeks were statistically significant (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 8 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 8 weeks were statistically significant (p value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effectiveness of Box A on surfactant protein C production in bleomycin-induced pulmonary fibrosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter our treatment of bleomycin-induced pulmonary fibrosis in the rats with Box A protein following our experimental protocol, we sacrificed the rats and harvested the lung tissue for anti-sftPC (anti-surfactant protein C) immunohistostaining and analyzed the area of staining. We calculated the area of surfactant protein C for each group as follows. The groups that received NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, or bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 6 weeks exhibited surfactant protein C areas representing 13.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64%, 3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96%, and 2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of the total tissue area, respectively. The groups that received NSS\u0026thinsp;+\u0026thinsp;plasmid control, bleomycin\u0026thinsp;+\u0026thinsp;plasmid control, or bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 8 weeks exhibited surfactant protein C areas representing 13.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.55%, 3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68%, and 6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65% (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of the total tissue area, respectively. The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 6 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 6 weeks were not statistically significant (p value\u0026thinsp;=\u0026thinsp;0.2606). The differences between the groups treated with bleomycin\u0026thinsp;+\u0026thinsp;plasmid control for 8 weeks and with bleomycin\u0026thinsp;+\u0026thinsp;Box A for 8 weeks were statistically significant (p value\u0026thinsp;=\u0026thinsp;0.0039).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur research demonstrated that administering the Box A-producing plasmid to rats with bleomycin-induced pulmonary fibrosis significantly reduced the number of senescent cells and fibrotic deposit area in the rat lung tissue within 6 to 8 weeks compared with those in the control group. The results also revealed a significant improvement in surfactant protein production, although this was seen at only the 8-week time point and did not completely return to baseline levels .\u003c/p\u003e \u003cp\u003eRegarding how Box A helps reduce senescence, our team has recently published research findings about the mechanism involved[\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To summarize, the administered plasmid produces a Box A portion of the HMGB1 protein, and these Box A proteins then spread throughout the cells. Some Box A is transferred into the cell nucleus, where it promotes DNA stabilization, DNA stress relief, DNA gap production, chromatin structural changes, and DNA protection. Once senescent cells or presenescent cells have received sufficient Box A and achieved DNA stabilization, these cells reduce the DDR and consequently emerge from the senescent state and return to normal.\u003c/p\u003e \u003cp\u003eThe possibility of Box A clearing fibrosis may be due to a reduction in the number of senescent cells. After Box A reversed the senescent phenotype, these cells began to function normally. These rejuvenated cells include fibrocytes, fibroblasts, and tissue-specific macrophages. These cells are involved in the creation, retention, and destruction of fibrotic tissue[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Typically, when these cells function normally, the fibroblasts and fibrocytes create and rearrange the fibrous tissue during the tissue healing process. After the tissue is healed, the normal fibroblasts and fibrocytes stop producing more fibrous tissue, and the fibrous material degrades by itself or is destroyed by tissue-specific macrophages.\u003c/p\u003e \u003cp\u003eWith respect to the ability of Box A to promote surfactant protein production, the rejuvenated cells include not only fibroblasts and macrophages inside the tissue. Box A also affects all cells, including epithelial cells and tissue progenitor cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When senescent epithelial cells are rejuvenated, their protein-producing function returns to normal. Therefore, epithelial cells can produce normal functioning surfactant protein again[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, as progenitor cells return to normal, they enter the cell cycle and differentiate to replace destroyed epithelial cells in the tissue[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Because cell division and cell differentiation require time, surfactant protein production is a slower process than rejuvenation and fibrosis clearance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAs pulmonary fibrosis continues to be an incurable disease, finding or developing novel and effective treatments is essential. An examination of the effect of Box A in treating bleomycin-induced pulmonary fibrosis in a rat model revealed that Box A could treat pulmonary fibrosis by reducing the number of senescent cells in the diseased tissue, with a significant direct correlation with the fibrotic tissue area and an inverse correlation with surfactant protein production. Some genetic mutations, such as surfactant protein mutations, can also lead to pulmonary fibrosis. The process of developing fibrosis includes chronic inflammation resulting from the production of a faulty surfactant protein, which leads to the accumulation of oxidative stress, ultimately leading to cellular senescence. The ability of Box A to help rejuvenate senescent cells and help cure pulmonary fibrosis should be an example for future research and the development of new treatments for other diseases involving cellular senescence.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eanti-sftPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eanti - surfactant protein C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBle\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBleomycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBox A\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBox A portion of the high mobility group Box 1 protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eblood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCa-P\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCalcium Phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ei.p.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintraperitoneally\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enormal saline solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptimal cutting temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eplasmid control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSA-β‐Gal\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esenescence associated \u0026ndash; beta \u0026ndash; galactosidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal procedures were reviewed and approved by the Animal Care and Use Committee, Medical Faculty, Chulalongkorn University, Thailand, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines (Approval No. 032/2564).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Applied Research of Rejuvenating DNA by Genomic Stability Molecule (REDGEM) for the treatment of age-associated disease. National Research Council of Thailand (NRCT), the National Science and Technology Development Agency, Thailand [Research Chair Grant, P-19-50189]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRP and AM conceived the study and designed the analysis; RP and SC designed the methodology and investigation; RP analyzed and wrote the original draft of the paper; AM supplied grant support, reviewed and edited the article; RP, SC, and SY handled the animals; AM and SY are involved in supervision and visualization; and all authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Assistant Professor Amornpun Sereemaspun, MD PhD for providing ethics and methodology guidance, especially pertaining to the animal model, tissue processing and histology staining.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarratt SL, Creamer A, Hayton C, Chaudhuri N. 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Respir Res. 2016;17(1):164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12931-016-0480-y\u003c/span\u003e\u003cspan address=\"10.1186/s12931-016-0480-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Idiopathic Pulmonary fibrosis, DNA damage, DNA stability, DNA protection, Box A of HMGB1, Youth-DNA gap, senescence, rejuvenation, gene therapy","lastPublishedDoi":"10.21203/rs.3.rs-5266547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5266547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003ePulmonary fibrosis is characterized by the destruction of normal lung tissue and then replacement by abnormal fibrous tissue, leading to an overall decrease in gas exchange function. The effective treatment for pulmonary fibrosis remains unknown. The upstream pathogenesis of pulmonary fibrosis may involve cellular senescence of the lung tissue. Previously, a new gene therapy technology using Box A of the HMGB1 plasmid (Box A) was used to reverse cellular senescence and cure liver fibrosis in aged rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eHere, we show that Box A is a promising medicine for the treatment of lung fibrosis. In a bleomycin-induced pulmonary fibrosis rat model, Student's \u003cem\u003et\u003c/em\u003e‐test and one-way ANOVA was used for comparisons among groups of samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Box A effectively lowered fibrous tissue deposits (from 18.74±0.62 to 3.45±1.19%) and senescent cells (from 3.74±0.40% to 0.89±0.18%) to levels comparable to those of the negative control group. Moreover, after eight weeks, Box A also increased the production of the surfactant protein C (from 3.60±1.68% to 6.82±0.65%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e Our results demonstrate that Box A is a promising therapeutic approach for pulmonary fibrosis and other senescence-promoted fibrotic lesions.\u003c/p\u003e","manuscriptTitle":"HMGB1 Box A gene therapy to alleviate bleomycin-induced pulmonary fibrosis in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-06 08:46:59","doi":"10.21203/rs.3.rs-5266547/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-28T10:40:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T05:29:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-25T08:32:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2024-10-15T07:26:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69b4807f-afef-46e8-9f49-9cb33cdb24d5","owner":[],"postedDate":"November 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T16:06:39+00:00","versionOfRecord":{"articleIdentity":"rs-5266547","link":"https://doi.org/10.1186/s12890-025-03522-2","journal":{"identity":"bmc-pulmonary-medicine","isVorOnly":false,"title":"BMC Pulmonary Medicine"},"publishedOn":"2025-01-31 15:58:02","publishedOnDateReadable":"January 31st, 2025"},"versionCreatedAt":"2024-11-06 08:46:59","video":"","vorDoi":"10.1186/s12890-025-03522-2","vorDoiUrl":"https://doi.org/10.1186/s12890-025-03522-2","workflowStages":[]},"version":"v1","identity":"rs-5266547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5266547","identity":"rs-5266547","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}