Investigation of antifibrotic properties of lithium as a potential treatment agent for idiopathic pulmonary fibrosis and comparing its antifibrotic activity with standard of care drugs for IPF

Investigation of antifibrotic properties of lithium as a potential treatment agent for idiopathic pulmonary fibrosis and comparing its antifibrotic activity with standard of care drugs for IPF | 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 Investigation of antifibrotic properties of lithium as a potential treatment agent for idiopathic pulmonary fibrosis and comparing its antifibrotic activity with standard of care drugs for IPF Pavel Idelevich, Kenneth Reed This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4777803/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background In this study, we examined in a proprietary in vitro system the antifibrotic properties of lithium and its modulating effect on telomere maintenance and telomerase activity. Lithium effects were compared with FDA-approved drugs for the treatment of Idiopathic pulmonary fibrosis. Methods Lithium in three different concentrations, Nintedanib and Pirfenidone were tested in InMatrico IPF assay (Xylyx Bio) using a decellularized extracellular matrix obtained from a human lung with Idiopathic pulmonary fibrosis added with primary lung fibroblasts. After 72 hours of drug treatment samples were collected for gene expression analysis and protein secretion analysis. Results Lithium statistically significantly decreased the expression of two out of three tested pro-fibrotic genes. Nintedanib statistically significantly downregulated the expression of one out of three pro-fibrotic genes. Pirfenidone did not decrease the expression of pro-fibrotic genes tested in this study. Lithium and FDA-approved drugs upregulated the expression of genes related to telomere maintenance. lithium and Nintedanib exhibit a statistically significant upregulating effect on gene related to telomerase activity. Pirfenidone did not show such activity. Lithium in a dose-dependent, statistically significant manner suppresses profibrotic protein secretion. Nintedanib demonstrated a similar activity. Pirfenidone treatment failed to do so. Conclusions Lithium in the present study demonstrated the ability to modulate major factors related to lung fibrosis development and progression favorably, better or comparable to the “gold standard” drugs. Idiopathic pulmonary fibrosis lithium Nintedanib Pirfenidone telomere TGF-beta1 Figures Figure 1 Figure 2 Background Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal fibrotic lung disease of unknown cause. IPF may present anytime during middle-to-late adulthood but most commonly arises in the sixth and seventh decades. Overall, the prognosis for IPF is poor, with a mean survival of about 2.5-5 years[ 1 ]. The pathogenesis and etiology of IPF are not fully understood. Genetic and epigenetic changes, disturbance in protein synthesis, profibrotic growth factors, and epithelial-to-mesenchymal transition are thought to play a role in the pathophysiology of IPF. The level of active transforming growth factor-beta (TGF-beta) is increased in the lungs of patients with IPF. Possible profibrotic processes associated with TGF-beta activation include inhibition of aortic endothelial cell (AEC) proliferation, differentiation of fibroblast to myofibroblasts[ 2 ], and activation of programming that promotes the mesenchymal transition of epithelial cells[ 3 ]. Several profibrotic genes involved in fibrosis formation include Alpha smooth muscle actin 2 (ACTA2)[ 4 ], Collagen type 1 alpha 1 (COL1A1)[ 5 ], and Fibronectin 1 (FN1)[ 4 ]. Accelerated cellular senescence is hypothesized to play a significant role in the pathogenesis of IPF. Premature shortened telomere length is associated with IPF[ 6 ]. We performed a laboratory investigation of our leading anti-fibrotic compound (Lispiro) compared to FDA-approved IPF drugs. We checked levels of TGF-beta secretion, profibrotic gene expression profiles, and telomerase markers. The investigation was performed in a proprietary in vitro system (Xylyx Bio, Inc,). Primary human lung fibroblasts were cultured in a biopsy-derived lung matrix from a healthy person and a patient with IPF. In this system, the Lispiro compound demonstrated superiority in anti-fibrotic activity over both pirfenidone and nintedanib. Material and methods Drug testing assay setup Drug testing was performed using the InMatrico® IPF Assay (Xylyx Bio), a cell-based assay that uses engineered human lung fibrotic lesions comprised of human primary lung fibroblasts and decellularized extracellular matrix (dECM) scaffolds derived from human IPF lungs. Human lung tissues were procured and processed as previously described[ 7 , 8 ]. Briefly, human donor lungs declined for transplantation and consented for research use were procured under protocols approved by the Institutional Review Board at the State University of New York (SUNY) Downstate Medical Center. IPF lung donor diagnoses were confirmed by a pulmonary pathologist. Normal lung donors used as controls, had no history, diagnosis, or evidence of smoking, aspiration pneumonia, asthma, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, interstitial lung disease, or lung cancer. Lungs were procured in standard fashion, flushed with cold organ preservation solution, transported on ice, and made available without identifiers. Lung histopathology was performed by a lung pathologist using a standard pulmonary fibrosis scoring rubric[ 9 ]. Only regions from the right middle and right lower lobes of IPF lungs with fibrosis score ≥ 2 were used. Lung tissues were decellularized using a proprietary combination of chemicals, enzymes, and surfactants to obtain IPF and normal lung extracellular matrices. Lung dECM scaffolds (diameter: 7 mm, thickness: 1 mm) were placed in a 48-well plate. Primary Human Pulmonary Fibroblasts (PromoCell) were expanded according to the manufacturer’s instructions on collagen-coated flasks in Fibroblast Growth Medium (PromoCell) supplemented with 2% fetal calf serum with 5% CO2 and 100% humidity at 37°C and used at passage 5–8. Before use, fibroblast growth media were changed to low-serum media supplemented with 0.2% fetal calf serum for 24 hours. A cell suspension of 10 µL containing 2 × 10 5 human primary lung fibroblasts was added to each well, and assay plates were incubated at 37°C. After 1 hour, low-serum media supplemented with 0.2% fetal calf serum was added (500 µL/well). After 24 hours, drug treatments were added. Experimental drug candidate lithium carbonate (MW: 73.891 g/mol) was added at 1.5 mM, 5 mM, and 10 mM. IPF standard-of-care drugs were added: pirfenidone (Sigma, P2116) at 2 mM and nintedanib (Selleckchem, S1010) at 1 µM. DMSO was used as vehicle and negative control. After 72 hours of drug treatment, samples were collected for analysis. Gene expression readout Gene expression analysis was performed as previously described[ 7 ]. Briefly, after 72 hours of drug treatment, engineered human lung fibrotic lesions were collected and stored in 300 µL RNAlater Stabilization Solution (Invitrogen) at − 80ºC. RNA extraction was performed using a RNeasy Fibrous Tissue Mini Kit (QIAGEN). Samples were homogenized in lysis buffer containing guanidine-isothiocyanate using a PowerLyzer homogenizer (QIAGEN) and treated with proteinaseK for 10 min at 55ºC. Lysates were washed with ethanol through silica membrane columns and incubated with DNase I for 10 minutes. RNA was eluted into microcentrifuge tubes using RNase-free water. TaqMan Real-Time PCR assays were performed to quantify relative expressions of ACTA2, COL1A1, FN1, DKC1, and TERC. GAPDH was used as a reference gene. All samples were analyzed as biological replicates in triplicate. Protein secretion readout Protein secretion analysis was performed as previously described[ 7 ]. Briefly, after 72 hours of drug treatment, engineered human lung fibrotic lesion supernatants were collected, centrifuged at 3,000 rpm for 5 min at 4°C, and stored at − 80°C. Enzyme-linked immunosorbent assays (ELISA) were performed to quantify the secretion of human transforming growth factor beta 1 (R&D Systems, DB100B) according to the manufacturer’s instructions. All samples were analyzed as biological replicates in triplicate. Statistical analyses To assess the significance of the differences between samples for gene expression or protein secretion, two-sided Welch t-tests were conducted (independent samples) with the function T.TEST from the Excel software (MS Office Suite). For the gene expression, the distributions of ΔCt (targeted gene vs. GAPDH endogenous control) were considered for the control (no drug) and drug-treated samples, over N = 4 biological replicates (control) and N = 3 biological replicates (drug-treated); each biological replicate was averaged over three technical replicates with the same sample. For the protein secretion experiments, the protein concentration distributions were compared, with N = 3 biological replicates per sample. A significance level of 0.05 was considered for the p-values. Results Modulation of gene expression by Lithium Carbonate The effect of the presence of Lithium Carbonate on human normal lung fibroblasts cultured onto ECM with IPF was monitored by RT-qPCR to assess the impact of Lithium Carbonate on the gene expression. The collected RNA amounts were found similar between samples, suggesting similar growth conditions without a global gene expression bias (See Supplementary Fig. 1, Additional File 1). Especially, the genes ACTA2 (Alpha smooth muscle actin 2), COL1A1 (Collagen type I alpha 1), FN1 (Fibronectin 1), DKC1 (Dyskerin pseudouridine synthase 1) and TERC (Human Telomerase Reverse Transcriptase) were quantified in the absence (control) or presence of Lithium Carbonate (10 mM, 5 mM, and 1.5 mM), Nintedanib (1 µM), or Pirfenidone (2 mM). The relative expression of these genes was expressed as the fold change expression compared to the control sample (absence of drug), as illustrated in the Fig. 1 . In Fig. 1 (a), the expression of the gene ACTA2 is decreased in the presence of Lithium Carbonate at concentrations of 5 mM and 1.5 mM (p-values < 0.05). No significant difference is observed between these two conditions either (See Supplementary Table 1, Additional File 1), meaning that Lithium Carbonate seems to have an inhibitory effect on the expression of the gene ACTA2 at least for concentrations higher than 1.5 mM, with no notable amplified effect at higher concentrations. The distribution for Lithium Carbonate at 10 mM does not exhibit any significant effect on the ACTA2 gene expression, which seems contradictory with the effect observed for 5 mM and 1.5 mM. Given the error bars associated with this distribution, it seems reasonable that this apparent contradictory observation could result from an insufficient statistical power on this measurement, as it is counter-intuitive that a higher concentration of the drug would inhibit its effect. The distribution for the sample with Nintedanib treatment shows a decrease in ACTA2 gene expression close to the two-fold reduction in the Lithium Carbonate samples at 5 mM and 1.5 mM, but this decrease is not statistically significant at the level 5%. The Pirfenidone sample does not seem to be impacted by the drug treatment. The Fig. 1 (b) presents the expression level for the gene COL1A1. None of the drug-treated samples seem to be impacted by the treatment at a significant level; it is especially clear for the Lithium Carbonate samples with good accuracy of the measurements (as represented by the error bars) and associated p-values (See Supplementary Table 1, Additional File 1). The Nintedanib and Pirfenidone samples exhibit an apparent up-regulation of the COL1A1 gene, but these changes do not appear significant at the level 5%, with noisy measurements for the Pirfenidone sample. In Fig. 1 (c), the expression of the gene FN1 appears to be down-regulated similarly (See Supplementary Table 1, Additional File 1) for the drug-treated samples with Lithium Carbonate (5 mM and 1.5 mM) and the Nintedanib, with a two-fold decrease, while the Pirfenidone sample does not display a significant difference compared to the control sample. Similarly to the gene ACTA2, the drug-treated sample with Lithium Carbonate 10 mM shows a gene expression level similar to the control sample, which is counter-intuitive given the significant effect observed for lower Lithium Carbonate concentration treatments. A low statistical power for the distribution of the sample Lithium Carbonate 10 mM cannot be excluded. The DKC1 gene expression is displayed in the Fig. 1 (d), in which all drug-treated samples except Lithium Carbonate 10 mM are observed significantly up-regulated with a 2–4 fold increase for the Lithium Carbonate 5 mM and 1.5 mM and a 3–8 fold increase for the Nintedanib and Pirfenidone samples. No statistically significant differences are highlighted between the up-regulated samples (See Supplementary Table 1, Additional File 1), but the distributions for the Lithium Carbonate 5 mM and 1.5 mM appear more homogeneous than the other drug-treated samples (from error bars in Fig. 1 (d)). Finally, the Fig. 1 (e) shows the expression level of the gene TERC, which appears to be up-regulated for all the drug-treated samples except the Lithium Carbonate 1.5 mM and Pirfenidone. In detail, these upregulations represent around a two-fold increase for Lithium Carbonate 10 mM and 5 mM and a 1.5-fold increase for Nintedanib. Modulation of the human TGF-β1 protein secretion by Lithium Carbonate After the study of the expression of various genes implied in the IFP ECM, the secretion level of the human TGF-β1 protein was measured by ELISA quantification for human lung fibroblasts cultured onto either normal or IPF ECM substrates. The Fig. 2 summarizes these measurements, with the statistical differences between distributions assessed by two-sided Welch t-test p-values (See Supplementary Table 2, Additional File 1). For fibroblasts cultured onto normal ECM, the secretion level of the human TGF-β1 protein does not appear significantly perturbed for drug-treated samples Lithium Carbonate 1.5 mM, Nintedanib, and Pirfenidone, with protein concentration around 500–600 pg/mL. The sample Lithium Carbonate 5 mM is not statistically significant from the control (no drug treatment) at the level 5% (p-value = 0.052) but the small error bars for this distribution and the similar level of protein secretion compared to the Lithium Carbonate 10 mM seems to indicate a downregulation of the human TGF-β1 protein secretion starting for concentrations around 5–10 mM in normal ECM. For fibroblasts cultured onto IPF ECM, all drug-treated samples except Pirfenidone exhibit a significant downregulation of the human TGF-β1 protein secretion with protein concentration around 400 pg/mL for Lithium Carbonate 10 mM and 5 mM and Nintedanib and 500 pg/mL for Lithium Carbonate 1.5 mM (around 650 pg/mL for control sample). The protein secretion levels for untreated samples (control) between fibroblasts cultured onto either normal ECM or IPF ECM are not statistically different. The Pirfenidone treatment does not seem to regulate the human TGF-β1 protein secretion for either normal or IPF ECM conditions. The Nintedanib treatment is observed to downregulate the human TGF-β1 protein secretion only for IPF ECM conditions, but its effects for normal ECM remain quite unclear due to the overlap between error bars of its distribution and the control one. The Lithium Carbonate samples present a continuous downregulation of the human TGF-β1 protein for increasing concentrations of Lithium Carbonate, with higher downregulation for higher doses. This downregulation does not appear to depend on the physiological state of the ECM, since no significant differences are observed for Lithium Carbonate-treated samples between their normal ECM and IPF distributions, for each Lithium Carbonate concentration (See Supplementary Table 2, Additional File 1). The Table 1 summarizes the main experimental observations on the modulation of gene expression and protein secretion of normal lung fibroblasts cultured onto normal or IPF ECM, by Lithium Carbonate treatment. While the treatment with Lithium Carbonate 10 mM shows unclear patterns, concentrations between 5 mM and 1.5 mM seem to lead to the higher modulations observed in the frame of this study, with higher effects observed compared to Nintedanib 1 µM treatment. Table 1 Summary of the modulation of gene expression and protein secretion Treatment LC 10 mM LC 5 mM LC 1.5 mM Nintedanib (1 µM) Pirfenidone (2 mM) ACTA 2 gene n.s. Downregulated Downregulated n.s. n.s. COL1A1 gene n.s. n.s. n.s. n.s. n.s. FN1 gene n.s. Downregulated Downregulated Downregulated n.s. DKC1 gene n.s. Upregulated Upregulated Upregulated Upregulated TERC gene Upregulated Upregulated Upregulated Upregulated Upregulated hTGF-β1 protein Downregulated Downregulated Downregulated Downregulated n.s. Discussion In this study, we investigated the effect of lithium carbonate on the expression of pro-fibrotic genes, pro-fibrotic protein secretion, and genes associated with telomere maintenance and telomerase activity. We tested the effect of lithium in comparison with Nintedanib and Pirfenidone, drugs in use in clinical practice for the treatment of IPF. The goal of this study is to explore the therapeutic potential of lithium for the treatment of IPF. Currently FDA-approved drugs for IPF have limited therapeutic potential and do not change dramatically the short life expectancy for patients with this disease[ 10 ]. Unfortunately for these patients, there remains a great unmet need for a more effective treatment with a reduced side effect profile. IPF pathogenesis is known to be related to pro-fibrotic pathways activation as well as premature telomere shortening[ 6 , 11 – 13 ]. We studied the expressions of ACTA2, COL1A1, and FN1, known as fibrosis-associated genes [ 4 , 14 ]. We also studied the expression of DKC1, a gene involved in telomere maintenance[ 15 ], and TERC, a telomerase-associated gene[ 16 ]. We checked secretion levels of TGF beta1, one of the major factors driving fibrosis in multiple tissue types[ 17 ]. Lithium at concentrations of 5mM and 1.5mM for fibrosis-associated genes statistically significantly decreased the expression of ACTA2. Neither Nintedanib nor Pirfenidone have statistically significant effects on ACTA2 expression. Lithium does not have statistically significant impact on COL1A1 expression levels. Interestingly, Nintedanib and Pirfenidone up-regulate the COL1A1 gene. In the case of Nintedanib, this up-regulation reached statistical significance. FN1 is downregulated by Lithium at concentrations of 5mM and 1.5mM and by Nintedanib, but not by Pirfenidone. Only lithium at concentrations of 5mM and 1.5mM downregulated FN1 to show statistical significance. As a result of this part of the study, we can conclude that Lithium shows a more favorable inhibition profile of fibrosis-associated genes than both Nintedanib and Pirfenidone. On the DKC1 and TERC genes, lithium treatment in 5mM and 1.5mM concentrations as well as Nintedanib and Pirfenidone induced statistically significant upregulation of DKC1: 10M and 5mM concentrations of lithium and Nintedanib upregulated statistically significant expression of TERC. Pirfenidone failed to do so. Based on these data, we conclude that lithium has an upregulating effect on the expression of telomere-associated genes, similar to the effect of Nintedanib and superior to Pirfenidone. Finally, all three concentrations of lithium induce a statistically significant reduction in TGF-beta1 secretion in a dose-dependent manner. Lithium in contrast to Nintedanib suppresses TGF-beta1 secretion in both normal and IPF extracellular matrix (ECM). Pirfenidone in this study does not show TGF-beta1 secretion modulation activity. We conclude that lithium is similar to Nintedanib in its ability to downregulate TGF-beta1 secretion in IPF ECM but potentially, taking into account lithium activity in normal ECM, lithium may have both therapeutic and preventive effects for developing lung fibrosis. Conclusions To conclude, lithium showed a favorable ability to influence key factors in the development and progression of lung fibrosis, performing as well as or better than the ‘gold standard’ medications. Further studies are warranted. Abbreviations IPF Idiopathic pulmonary fibrosis TGF-beta transforming growth factor-beta AEC aortic endothelial cell ACTA2 Alpha smooth muscle actin 2 COL1A1 Collagen type 1 alpha 1 FN1 Fibronectin 1 FDA Food and Drugs Administration dECM decellularized extracellular matrix SUNY State University of New York MW Molar weight DMSO Dimethyl sulfoxide ELISA Enzyme-linked immunosorbent assays GAPDH glyceraldehyde-3-phosphate dehydrogenase ECM Extracellular matrix RT-qPCR Reverse transcription quantitative polymerase chain reaction DKC1 Dyskerin pseudouridine synthase 1 TERC Telomerase RNA component Declarations Ethics approval Human donor lungs declined for transplantation and consented for research use were procured under protocols approved by the Institutional Review Board at the State University of New York (SUNY) Downstate Medical Center. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests Lispiro owns a patent for using lithium for the treatment of IPF. Funding The study was funded by Lispiro LLC. Authors’ contributions Dr. Pavel Idelevich and Dr. Kenneth M Reed equally contributed to the designing and execution of the study, as well as to the drafting and editing of this manuscript. Acknowledgments We are deeply thankful to Xylyx Bio for their help in the execution of the study. References Bois RM. du. An earlier and more confident diagnosis of idiopathic pulmonary fibrosis. European Respiratory Review. 2012;21(124):141–6. Scotton CJ, Chambers RC. Molecular Targets in Pulmonary Fibrosis: The Myofibroblast in Focus. Chest. 2007;132(4):1311–21. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proceedings of the National Academy of Sciences. 2006;103(35):13180–5. Peyser R, MacDonnell S, Gao Y, Cheng L, Kim Y, Kaplan T, et al. Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing. Am J Respir Cell Mol Biol. 2019;61(1):74–85. Tsitoura E, Trachalaki A, Vasarmidi E, Mastrodemou S, Margaritopoulos GA, Kokosi M et al. Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality. Front Immunol [Internet]. 2021 Nov 17 [cited 2024 Jul 19];12. Duckworth A, Gibbons MA, Allen RJ, Almond H, Beaumont RN, Wood AR, et al. Telomere length and risk of idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease: a mendelian randomisation study. Lancet Respiratory Med. 2021;9(3):285–94. Germanguz I, Aranda E, Xiong JC, Kissel N, Nichols A, Gadee E et al. Fibrotic Human Lung Extracellular Matrix as a Disease- Specific Substrate for Models of Pulmonary Fibrosis. Journal of Respiratory Medicine and Lung Disease [Internet]. 2019 Nov 11 [cited 2024 Jul 19];4(1). O’Neill JD, Anfang R, Anandappa A, Costa J, Javidfar JJ, Wobma HM, et al. Decellularization of Human and Porcine Lung Tissues for Pulmonary Tissue Engineering. Ann Thorac Surg. 2013;96(3):1046–56. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41(4):467–70. Sgalla G, Iovene B, Calvello M, Ori M, Varone F, Richeldi L. Idiopathic pulmonary fibrosis: pathogenesis and management. Respir Res. 2018;19(1):32. Alder JK, Chen JJL, Lancaster L, Danoff S, Su S, chih, Cogan JD et al. Short telomeres are a risk factor for idiopathic pulmonary fibrosis. Proceedings of the National Academy of Sciences. 2008;105(35):13051–6. Cronkhite JT, Xing C, Raghu G, Chin KM, Torres F, Rosenblatt RL, et al. Telomere Shortening in Familial and Sporadic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2008;178(7):729–37. Maharaj S, Shimbori C, Kolb M. Fibrocytes in pulmonary fibrosis: a brief synopsis. Eur Respiratory Rev. 2013;22(130):552–7. Dou F, Liu Q, Lv S, Xu Q, Wang X, Liu S, et al. FN1 and TGFBI are key biomarkers of macrophage immune injury in diabetic kidney disease. Med (Baltim). 2023;102(45):e35794. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature. 1999;402(6761):551–5. Nagpal N, Agarwal S. Telomerase RNA processing: Implications for human health and disease. Stem Cells. 2020;38(12):1532–43. Meng X, ming, Nikolic-Paterson DJ, Lan HY. TGF-β: the master regulator of fibrosis. Nat Rev Nephrol. 2016;12(6):325–38. Additional Declarations Competing interest reported. Lispiro owns a patent for using lithium for the treatment of IPF. Supplementary Files AdditionalFile1.docx DATALISXYL001GeneExpression20230426.xlsx DATALISXYL001ProteinSecretion20230426.xlsx DATALispiroELISATGFbeta20221223.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Aug, 2024 Editor assigned by journal 31 Jul, 2024 Submission checks completed at journal 31 Jul, 2024 First submitted to journal 21 Jul, 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. 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-4777803","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":334633045,"identity":"a72879be-29ef-4500-8939-8a821922c85c","order_by":0,"name":"Pavel Idelevich","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIie3Qu2rDMBSAYZuAvYRmFQSsJyhIGBpCQh6j8zEGaXEhY7eqBDQle8FDX6GlUDrKCDoZuha6uBQya2ygQ5R4tZw1UP2L4KAPXYLA5zvXGjIILuyqQoGSw0A1pwhYErVknh4JnCRBS4JQsEy0E3eTuKYNLGdJFK+p2r1p/nitv+0pi+RSdJPpukgJEJ5Gw5pUm1rfPH8xYkmeXqluQhRjCIjOJGKgQ2lJCQeislcX+djyX0vuJN4eCaclN/3kM3+3P6YhQgNlCQM8LvpPmT78aHsxTuUwV9VGzunTuFgqIO63TEbZvTF/MzyKq5XZSYRxyV+MuV0kzud3fEj3vIdg4d7t8/l8/7M9jQtmV3mrbEAAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Idelevich","suffix":""},{"id":334633046,"identity":"496da544-3e90-4e4e-9807-8c7bf54051f0","order_by":1,"name":"Kenneth Reed","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Reed","suffix":""}],"badges":[],"createdAt":"2024-07-21 17:42:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4777803/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4777803/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63350566,"identity":"78639690-3e2f-4e66-9d5f-a7cb8f5219cc","added_by":"auto","created_at":"2024-08-27 08:17:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":529962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression in matrico\u003c/strong\u003e. Gene expression quantification by RT-qPCR of human normal lung fibroblasts in IPF ECM, expressed as RQ (fold change) compared to control sample (no drug), in presence of Lithium Carbonate (LC; 10 mM, 5 mM, 1.5 mM), Nintedanib (1 µM) or Pirfenidone (2 mM), for genes (a) ACTA2; (b) COL1A1; (c) FN1; (d) DKC1 and (e) TERC. Error bars correspond to the standard deviation (N = 3 biological replicates; each biological replicate is estimated on an average of 3 technical replicates). Significance was assessed by p-values for drugs vs. Control ΔCt distributions from two-tailed Welch t-test (* p \u0026lt; 0.05; ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/258d1533ca7a3dde8011f5ad.jpeg"},{"id":63350571,"identity":"b7503311-2f65-48c6-a793-f62209a804de","added_by":"auto","created_at":"2024-08-27 08:17:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSecretion of human TGF-β1 in matrico.\u003c/strong\u003e Quantification of the human TGF-β1 protein by ELISA for primary human lung fibroblasts in normal lung ECM (blue) or IPF ECM (orange). Error bars correspond to the standard deviation (N = 3 biological replicates). LC = Lithium Carbonate. Significance was assessed by p-values for drugs vs. Control (Normal ECM or IPF ECM) from two-tailed Welch t-test (* p \u0026lt; 0.05; ** p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/c09e619010978b22e29c59c6.jpeg"},{"id":63351334,"identity":"027c7396-1146-4cff-8e5d-0199364b5030","added_by":"auto","created_at":"2024-08-27 08:25:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1162463,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/e1b818bd-e6c6-4a1e-9c2e-e04b6353e0b6.pdf"},{"id":63351319,"identity":"b0abc0a7-e5d6-4250-8b5d-11d01b66263f","added_by":"auto","created_at":"2024-08-27 08:25:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":206707,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/c865cd2e1cfca783b7e337c7.docx"},{"id":63350567,"identity":"62815362-8b06-4042-89e5-f710a4569a9c","added_by":"auto","created_at":"2024-08-27 08:17:40","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":88088,"visible":true,"origin":"","legend":"","description":"","filename":"DATALISXYL001GeneExpression20230426.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/53b8eb3d0a32403df6b83054.xlsx"},{"id":63350569,"identity":"e3faba40-e46b-422d-8492-8db530e3aaac","added_by":"auto","created_at":"2024-08-27 08:17:40","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":55392,"visible":true,"origin":"","legend":"","description":"","filename":"DATALISXYL001ProteinSecretion20230426.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/d5fee597dff291e1476f368f.xlsx"},{"id":63350570,"identity":"444b9d74-1ab4-457e-be91-9c968bb78b89","added_by":"auto","created_at":"2024-08-27 08:17:40","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":55522,"visible":true,"origin":"","legend":"","description":"","filename":"DATALispiroELISATGFbeta20221223.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4777803/v1/ac79ceacf05737edfa685574.xlsx"}],"financialInterests":"Competing interest reported. Lispiro owns a patent for using lithium for the treatment of IPF.","formattedTitle":"Investigation of antifibrotic properties of lithium as a potential treatment agent for idiopathic pulmonary fibrosis and comparing its antifibrotic activity with standard of care drugs for IPF","fulltext":[{"header":"Background","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fatal fibrotic lung disease of unknown cause. IPF may present anytime during middle-to-late adulthood but most commonly arises in the sixth and seventh decades. Overall, the prognosis for IPF is poor, with a mean survival of about 2.5-5 years[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pathogenesis and etiology of IPF are not fully understood. Genetic and epigenetic changes, disturbance in protein synthesis, profibrotic growth factors, and epithelial-to-mesenchymal transition are thought to play a role in the pathophysiology of IPF.\u003c/p\u003e \u003cp\u003eThe level of active transforming growth factor-beta (TGF-beta) is increased in the lungs of patients with IPF. Possible profibrotic processes associated with TGF-beta activation include inhibition of aortic endothelial cell (AEC) proliferation, differentiation of fibroblast to myofibroblasts[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and activation of programming that promotes the mesenchymal transition of epithelial cells[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral profibrotic genes involved in fibrosis formation include Alpha smooth muscle actin 2 (ACTA2)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], Collagen type 1 alpha 1 (COL1A1)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and Fibronectin 1 (FN1)[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccelerated cellular senescence is hypothesized to play a significant role in the pathogenesis of IPF. Premature shortened telomere length is associated with IPF[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe performed a laboratory investigation of our leading anti-fibrotic compound (Lispiro) compared to FDA-approved IPF drugs. We checked levels of TGF-beta secretion, profibrotic gene expression profiles, and telomerase markers. The investigation was performed in a proprietary in vitro system (Xylyx Bio, Inc,). Primary human lung fibroblasts were cultured in a biopsy-derived lung matrix from a healthy person and a patient with IPF. In this system, the Lispiro compound demonstrated superiority in anti-fibrotic activity over both pirfenidone and nintedanib.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDrug testing assay setup\u003c/h2\u003e \u003cp\u003eDrug testing was performed using the InMatrico\u0026reg; IPF Assay (Xylyx Bio), a cell-based assay that uses engineered human lung fibrotic lesions comprised of human primary lung fibroblasts and decellularized extracellular matrix (dECM) scaffolds derived from human IPF lungs. Human lung tissues were procured and processed as previously described[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Briefly, human donor lungs declined for transplantation and consented for research use were procured under protocols approved by the Institutional Review Board at the State University of New York (SUNY) Downstate Medical Center. IPF lung donor diagnoses were confirmed by a pulmonary pathologist. Normal lung donors used as controls, had no history, diagnosis, or evidence of smoking, aspiration pneumonia, asthma, chronic obstructive pulmonary disease, cystic fibrosis, emphysema, interstitial lung disease, or lung cancer. Lungs were procured in standard fashion, flushed with cold organ preservation solution, transported on ice, and made available without identifiers. Lung histopathology was performed by a lung pathologist using a standard pulmonary fibrosis scoring rubric[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Only regions from the right middle and right lower lobes of IPF lungs with fibrosis score\u0026thinsp;\u0026ge;\u0026thinsp;2 were used. Lung tissues were decellularized using a proprietary combination of chemicals, enzymes, and surfactants to obtain IPF and normal lung extracellular matrices. Lung dECM scaffolds (diameter: 7 mm, thickness: 1 mm) were placed in a 48-well plate. Primary Human Pulmonary Fibroblasts (PromoCell) were expanded according to the manufacturer\u0026rsquo;s instructions on collagen-coated flasks in Fibroblast Growth Medium (PromoCell) supplemented with 2% fetal calf serum with 5% CO2 and 100% humidity at 37\u0026deg;C and used at passage 5\u0026ndash;8. Before use, fibroblast growth media were changed to low-serum media supplemented with 0.2% fetal calf serum for 24 hours. A cell suspension of 10 \u0026micro;L containing 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e human primary lung fibroblasts was added to each well, and assay plates were incubated at 37\u0026deg;C. After 1 hour, low-serum media supplemented with 0.2% fetal calf serum was added (500 \u0026micro;L/well). After 24 hours, drug treatments were added. Experimental drug candidate lithium carbonate (MW: 73.891 g/mol) was added at 1.5 mM, 5 mM, and 10 mM. IPF standard-of-care drugs were added: pirfenidone (Sigma, P2116) at 2 mM and nintedanib (Selleckchem, S1010) at 1 \u0026micro;M. DMSO was used as vehicle and negative control. After 72 hours of drug treatment, samples were collected for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eGene expression readout\u003c/h2\u003e \u003cp\u003eGene expression analysis was performed as previously described[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, after 72 hours of drug treatment, engineered human lung fibrotic lesions were collected and stored in 300 \u0026micro;L RNAlater Stabilization Solution (Invitrogen) at \u0026minus;\u0026thinsp;80\u0026ordm;C. RNA extraction was performed using a RNeasy Fibrous Tissue Mini Kit (QIAGEN). Samples were homogenized in lysis buffer containing guanidine-isothiocyanate using a PowerLyzer homogenizer (QIAGEN) and treated with proteinaseK for 10 min at 55\u0026ordm;C. Lysates were washed with ethanol through silica membrane columns and incubated with DNase I for 10 minutes. RNA was eluted into microcentrifuge tubes using RNase-free water. TaqMan Real-Time PCR assays were performed to quantify relative expressions of ACTA2, COL1A1, FN1, DKC1, and TERC. GAPDH was used as a reference gene. All samples were analyzed as biological replicates in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eProtein secretion readout\u003c/h2\u003e \u003cp\u003eProtein secretion analysis was performed as previously described[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, after 72 hours of drug treatment, engineered human lung fibrotic lesion supernatants were collected, centrifuged at 3,000 rpm for 5 min at 4\u0026deg;C, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Enzyme-linked immunosorbent assays (ELISA) were performed to quantify the secretion of human transforming growth factor beta 1 (R\u0026amp;D Systems, DB100B) according to the manufacturer\u0026rsquo;s instructions. All samples were analyzed as biological replicates in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eTo assess the significance of the differences between samples for gene expression or protein secretion, two-sided Welch t-tests were conducted (independent samples) with the function T.TEST from the Excel software (MS Office Suite). For the gene expression, the distributions of ΔCt (targeted gene vs. GAPDH endogenous control) were considered for the control (no drug) and drug-treated samples, over N\u0026thinsp;=\u0026thinsp;4 biological replicates (control) and N\u0026thinsp;=\u0026thinsp;3 biological replicates (drug-treated); each biological replicate was averaged over three technical replicates with the same sample. For the protein secretion experiments, the protein concentration distributions were compared, with N\u0026thinsp;=\u0026thinsp;3 biological replicates per sample. A significance level of 0.05 was considered for the p-values.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eModulation of gene expression by Lithium Carbonate\u003c/h2\u003e \u003cp\u003eThe effect of the presence of Lithium Carbonate on human normal lung fibroblasts cultured onto ECM with IPF was monitored by RT-qPCR to assess the impact of Lithium Carbonate on the gene expression. The collected RNA amounts were found similar between samples, suggesting similar growth conditions without a global gene expression bias (See Supplementary Fig.\u0026nbsp;1, Additional File 1). Especially, the genes ACTA2 (Alpha smooth muscle actin 2), COL1A1 (Collagen type I alpha 1), FN1 (Fibronectin 1), DKC1 (Dyskerin pseudouridine synthase 1) and TERC (Human Telomerase Reverse Transcriptase) were quantified in the absence (control) or presence of Lithium Carbonate (10 mM, 5 mM, and 1.5 mM), Nintedanib (1 \u0026micro;M), or Pirfenidone (2 mM). The relative expression of these genes was expressed as the fold change expression compared to the control sample (absence of drug), as illustrated in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), the expression of the gene ACTA2 is decreased in the presence of Lithium Carbonate at concentrations of 5 mM and 1.5 mM (p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant difference is observed between these two conditions either (See Supplementary Table\u0026nbsp;1, Additional File 1), meaning that Lithium Carbonate seems to have an inhibitory effect on the expression of the gene ACTA2 at least for concentrations higher than 1.5 mM, with no notable amplified effect at higher concentrations. The distribution for Lithium Carbonate at 10 mM does not exhibit any significant effect on the ACTA2 gene expression, which seems contradictory with the effect observed for 5 mM and 1.5 mM. Given the error bars associated with this distribution, it seems reasonable that this apparent contradictory observation could result from an insufficient statistical power on this measurement, as it is counter-intuitive that a higher concentration of the drug would inhibit its effect. The distribution for the sample with Nintedanib treatment shows a decrease in ACTA2 gene expression close to the two-fold reduction in the Lithium Carbonate samples at 5 mM and 1.5 mM, but this decrease is not statistically\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003esignificant at the level 5%. The Pirfenidone sample does not seem to be impacted by the drug treatment.\u003c/p\u003e \u003cp\u003eThe Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) presents the expression level for the gene COL1A1. None of the drug-treated samples seem to be impacted by the treatment at a significant level; it is especially clear for the Lithium Carbonate samples with good accuracy of the measurements (as represented by the error bars) and associated p-values (See Supplementary Table\u0026nbsp;1, Additional File 1). The Nintedanib and Pirfenidone samples exhibit an apparent up-regulation of the COL1A1 gene, but these changes do not appear significant at the level 5%, with noisy measurements for the Pirfenidone sample.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c), the expression of the gene FN1 appears to be down-regulated similarly (See Supplementary Table\u0026nbsp;1, Additional File 1) for the drug-treated samples with Lithium Carbonate (5 mM and 1.5 mM) and the Nintedanib, with a two-fold decrease, while the Pirfenidone sample does not display a significant difference compared to the control sample. Similarly to the gene ACTA2, the drug-treated sample with Lithium Carbonate 10 mM shows a gene expression level similar to the control sample, which is counter-intuitive given the significant effect observed for lower Lithium Carbonate concentration treatments. A low statistical power for the distribution of the sample Lithium Carbonate 10 mM cannot be excluded.\u003c/p\u003e \u003cp\u003eThe DKC1 gene expression is displayed in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d), in which all drug-treated samples except Lithium Carbonate 10 mM are observed significantly up-regulated with a 2\u0026ndash;4 fold increase for the Lithium Carbonate 5 mM and 1.5 mM and a 3\u0026ndash;8 fold increase for the Nintedanib and Pirfenidone samples. No statistically significant differences are highlighted between the up-regulated samples (See Supplementary Table\u0026nbsp;1, Additional File 1), but the distributions for the Lithium Carbonate 5 mM and 1.5 mM appear more homogeneous than the other drug-treated samples (from error bars in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(d)).\u003c/p\u003e \u003cp\u003eFinally, the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(e) shows the expression level of the gene TERC, which appears to be up-regulated for all the drug-treated samples except the Lithium Carbonate 1.5 mM and Pirfenidone. In detail, these upregulations represent around a two-fold increase for Lithium Carbonate 10 mM and 5 mM and a 1.5-fold increase for Nintedanib.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eModulation of the human TGF-β1 protein secretion by Lithium Carbonate\u003c/h2\u003e \u003cp\u003eAfter the study of the expression of various genes implied in the IFP ECM, the secretion level of the human TGF-β1 protein was measured by ELISA quantification for human lung fibroblasts cultured onto either normal or IPF ECM substrates. The Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes these measurements, with the statistical differences between distributions assessed by two-sided Welch t-test p-values (See Supplementary Table\u0026nbsp;2, Additional File 1).\u003c/p\u003e \u003cp\u003eFor fibroblasts cultured onto normal ECM, the secretion level of the human TGF-β1 protein does not appear significantly perturbed for drug-treated samples Lithium Carbonate 1.5 mM, Nintedanib, and Pirfenidone, with protein concentration around 500\u0026ndash;600 pg/mL. The sample Lithium Carbonate 5 mM is not statistically significant from the control (no drug treatment) at the level 5% (p-value\u0026thinsp;=\u0026thinsp;0.052) but the small error bars for this distribution and the similar level of protein secretion compared to the Lithium Carbonate 10 mM seems to indicate a downregulation of the human TGF-β1 protein secretion starting for concentrations around 5\u0026ndash;10 mM in normal ECM. For fibroblasts cultured onto IPF ECM, all drug-treated samples except Pirfenidone exhibit a significant downregulation of the human TGF-β1 protein secretion with protein concentration around 400 pg/mL for Lithium Carbonate 10 mM and 5 mM and Nintedanib and 500 pg/mL for Lithium Carbonate 1.5 mM (around 650 pg/mL for control sample).\u003c/p\u003e \u003cp\u003eThe protein secretion levels for untreated samples (control) between fibroblasts cultured onto either normal ECM or IPF ECM are not statistically different. The Pirfenidone treatment does not seem to regulate the human TGF-β1 protein secretion for either normal or IPF ECM conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Nintedanib treatment is observed to downregulate the human TGF-β1 protein secretion only for IPF ECM conditions, but its effects for normal ECM remain quite unclear due to the overlap between error bars of its distribution and the control one. The Lithium Carbonate samples present a continuous downregulation of the human TGF-β1 protein for increasing concentrations of Lithium Carbonate, with higher downregulation for higher doses. This downregulation does not appear to depend on the physiological state of the ECM, since no significant differences are observed for Lithium Carbonate-treated samples between their normal ECM and IPF distributions, for each Lithium Carbonate concentration (See Supplementary Table\u0026nbsp;2, Additional File 1).\u003c/p\u003e \u003cp\u003eThe Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the main experimental observations on the modulation of gene expression and protein secretion of normal lung fibroblasts cultured onto normal or IPF ECM, by Lithium Carbonate treatment. While the treatment with Lithium Carbonate 10 mM shows unclear patterns, concentrations between 5 mM and 1.5 mM seem to lead to the higher modulations observed in the frame of this study, with higher effects observed compared to Nintedanib 1 \u0026micro;M treatment.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the modulation of gene expression and protein secretion\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLC 10 mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC 5 mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLC 1.5 mM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNintedanib\u003c/p\u003e \u003cp\u003e(1 \u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePirfenidone (2 mM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACTA 2 gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOL1A1 gene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFN1\u003c/p\u003e \u003cp\u003egene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDKC1\u003c/p\u003e \u003cp\u003egene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTERC\u003c/p\u003e \u003cp\u003egene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eUpregulated\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehTGF-β1 protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eDownregulated\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the effect of lithium carbonate on the expression of pro-fibrotic genes, pro-fibrotic protein secretion, and genes associated with telomere maintenance and telomerase activity. We tested the effect of lithium in comparison with Nintedanib and Pirfenidone, drugs in use in clinical practice for the treatment of IPF. The goal of this study is to explore the therapeutic potential of lithium for the treatment of IPF. Currently FDA-approved drugs for IPF have limited therapeutic potential and do not change dramatically the short life expectancy for patients with this disease[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Unfortunately for these patients, there remains a great unmet need for a more effective treatment with a reduced side effect profile. IPF pathogenesis is known to be related to pro-fibrotic pathways activation as well as premature telomere shortening[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe studied the expressions of ACTA2, COL1A1, and FN1, known as fibrosis-associated genes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We also studied the expression of DKC1, a gene involved in telomere maintenance[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and TERC, a telomerase-associated gene[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We checked secretion levels of TGF beta1, one of the major factors driving fibrosis in multiple tissue types[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Lithium at concentrations of 5mM and 1.5mM for fibrosis-associated genes statistically significantly decreased the expression of ACTA2. Neither Nintedanib nor Pirfenidone have statistically significant effects on ACTA2 expression. Lithium does not have statistically significant impact on COL1A1 expression levels. Interestingly, Nintedanib and Pirfenidone up-regulate the COL1A1 gene. In the case of Nintedanib, this up-regulation reached statistical significance. FN1 is downregulated by Lithium at concentrations of 5mM and 1.5mM and by Nintedanib, but not by Pirfenidone. Only lithium at concentrations of 5mM and 1.5mM downregulated FN1 to show statistical significance. As a result of this part of the study, we can conclude that Lithium shows a more favorable inhibition profile of fibrosis-associated genes than both Nintedanib and Pirfenidone. On the DKC1 and TERC genes, lithium treatment in 5mM and 1.5mM concentrations as well as Nintedanib and Pirfenidone induced statistically significant upregulation of DKC1: 10M and 5mM concentrations of lithium and Nintedanib upregulated statistically significant expression of TERC. Pirfenidone failed to do so. Based on these data, we conclude that lithium has an upregulating effect on the expression of telomere-associated genes, similar to the effect of Nintedanib and superior to Pirfenidone. Finally, all three concentrations of lithium induce a statistically significant reduction in TGF-beta1 secretion in a dose-dependent manner. Lithium in contrast to Nintedanib suppresses TGF-beta1 secretion in both normal and IPF extracellular matrix (ECM). Pirfenidone in this study does not show TGF-beta1 secretion modulation activity. We conclude that lithium is similar to Nintedanib in its ability to downregulate TGF-beta1 secretion in IPF ECM but potentially, taking into account lithium activity in normal ECM, lithium may have both therapeutic and preventive effects for developing lung fibrosis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo conclude, lithium showed a favorable ability to influence key factors in the development and progression of lung fibrosis, performing as well as or better than the \u0026lsquo;gold standard\u0026rsquo; medications. Further studies are warranted.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIdiopathic pulmonary fibrosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTGF-beta\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransforming growth factor-beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eaortic endothelial cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACTA2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlpha smooth muscle actin 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCOL1A1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollagen type 1 alpha 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFN1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibronectin 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drugs Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003edECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edecellularized extracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSUNY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eState University of New York\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolar weight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assays\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReverse transcription quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDKC1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDyskerin pseudouridine synthase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTERC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTelomerase RNA component\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman donor lungs declined for transplantation and consented for research use were procured under protocols approved by the Institutional Review Board at the State University of New York (SUNY) Downstate Medical Center.\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 analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLispiro owns a patent for using lithium for the treatment of IPF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by Lispiro LLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr. Pavel Idelevich and Dr. Kenneth M Reed equally contributed to the designing and execution of the study, as well as to the drafting and editing of this manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are deeply thankful to Xylyx Bio for their help in the execution of the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBois RM. du. An earlier and more confident diagnosis of idiopathic pulmonary fibrosis. European Respiratory Review. 2012;21(124):141\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScotton CJ, Chambers RC. Molecular Targets in Pulmonary Fibrosis: The Myofibroblast in Focus. Chest. 2007;132(4):1311\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN et al. Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proceedings of the National Academy of Sciences. 2006;103(35):13180\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeyser R, MacDonnell S, Gao Y, Cheng L, Kim Y, Kaplan T, et al. Defining the Activated Fibroblast Population in Lung Fibrosis Using Single-Cell Sequencing. Am J Respir Cell Mol Biol. 2019;61(1):74\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsitoura E, Trachalaki A, Vasarmidi E, Mastrodemou S, Margaritopoulos GA, Kokosi M et al. Collagen 1a1 Expression by Airway Macrophages Increases In Fibrotic ILDs and Is Associated With FVC Decline and Increased Mortality. Front Immunol [Internet]. 2021 Nov 17 [cited 2024 Jul 19];12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuckworth A, Gibbons MA, Allen RJ, Almond H, Beaumont RN, Wood AR, et al. Telomere length and risk of idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease: a mendelian randomisation study. Lancet Respiratory Med. 2021;9(3):285\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGermanguz I, Aranda E, Xiong JC, Kissel N, Nichols A, Gadee E et al. Fibrotic Human Lung Extracellular Matrix as a Disease- Specific Substrate for Models of Pulmonary Fibrosis. Journal of Respiratory Medicine and Lung Disease [Internet]. 2019 Nov 11 [cited 2024 Jul 19];4(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Neill JD, Anfang R, Anandappa A, Costa J, Javidfar JJ, Wobma HM, et al. Decellularization of Human and Porcine Lung Tissues for Pulmonary Tissue Engineering. Ann Thorac Surg. 2013;96(3):1046\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41(4):467\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSgalla G, Iovene B, Calvello M, Ori M, Varone F, Richeldi L. Idiopathic pulmonary fibrosis: pathogenesis and management. Respir Res. 2018;19(1):32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlder JK, Chen JJL, Lancaster L, Danoff S, Su S, chih, Cogan JD et al. Short telomeres are a risk factor for idiopathic pulmonary fibrosis. Proceedings of the National Academy of Sciences. 2008;105(35):13051\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCronkhite JT, Xing C, Raghu G, Chin KM, Torres F, Rosenblatt RL, et al. Telomere Shortening in Familial and Sporadic Pulmonary Fibrosis. Am J Respir Crit Care Med. 2008;178(7):729\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaharaj S, Shimbori C, Kolb M. Fibrocytes in pulmonary fibrosis: a brief synopsis. Eur Respiratory Rev. 2013;22(130):552\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDou F, Liu Q, Lv S, Xu Q, Wang X, Liu S, et al. FN1 and TGFBI are key biomarkers of macrophage immune injury in diabetic kidney disease. Med (Baltim). 2023;102(45):e35794.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature. 1999;402(6761):551\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagpal N, Agarwal S. Telomerase RNA processing: Implications for human health and disease. Stem Cells. 2020;38(12):1532\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X, ming, Nikolic-Paterson DJ, Lan HY. TGF-β: the master regulator of fibrosis. Nat Rev Nephrol. 2016;12(6):325\u0026ndash;38.\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":false,"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, lithium, Nintedanib, Pirfenidone, telomere, TGF-beta1","lastPublishedDoi":"10.21203/rs.3.rs-4777803/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4777803/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn this study, we examined in a proprietary in vitro system the antifibrotic properties of lithium and its modulating effect on telomere maintenance and telomerase activity. Lithium effects were compared with FDA-approved drugs for the treatment of Idiopathic pulmonary fibrosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eLithium in three different concentrations, Nintedanib and Pirfenidone were tested in InMatrico IPF assay (Xylyx Bio) using a decellularized extracellular matrix obtained from a human lung with Idiopathic pulmonary fibrosis added with primary lung fibroblasts. After 72 hours of drug treatment samples were collected for gene expression analysis and protein secretion analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eLithium statistically significantly decreased the expression of two out of three tested pro-fibrotic genes. Nintedanib statistically significantly downregulated the expression of one out of three pro-fibrotic genes. Pirfenidone did not decrease the expression of pro-fibrotic genes tested in this study. Lithium and FDA-approved drugs upregulated the expression of genes related to telomere maintenance. lithium and Nintedanib exhibit a statistically significant upregulating effect on gene related to telomerase activity. Pirfenidone did not show such activity. Lithium in a dose-dependent, statistically significant manner suppresses profibrotic protein secretion. Nintedanib demonstrated a similar activity. Pirfenidone treatment failed to do so.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eLithium in the present study demonstrated the ability to modulate major factors related to lung fibrosis development and progression favorably, better or comparable to the \u0026ldquo;gold standard\u0026rdquo; drugs.\u003c/p\u003e","manuscriptTitle":"Investigation of antifibrotic properties of lithium as a potential treatment agent for idiopathic pulmonary fibrosis and comparing its antifibrotic activity with standard of care drugs for IPF","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-27 08:17:34","doi":"10.21203/rs.3.rs-4777803/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-01T08:41:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-31T11:24:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-31T11:23:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pulmonary Medicine","date":"2024-07-21T17:41:13+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":"6b9069f1-a295-4efd-a679-ce3a86a900d7","owner":[],"postedDate":"August 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-22T12:53:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-27 08:17:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4777803","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4777803","identity":"rs-4777803","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}