Cyclophosphamide combined with methylprednisolone can play a therapeutic role by delaying the aging of pulmonary fibrosis

Cyclophosphamide combined with methylprednisolone can play a therapeutic role by delaying the aging of pulmonary fibrosis | 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 Cyclophosphamide combined with methylprednisolone can play a therapeutic role by delaying the aging of pulmonary fibrosis Qingjie Xu, Manka Zhang, Ming Tang, Zhouping Li, Yin Liu, Tao ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2245802/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Aging is a natural process characterized by a progressive functional impairment and reduced capacity to respond adaptively to environmental stimuli.Idiopathic pulmonary fibrosis(IPF)has been found to increase considerably with age.Immunosenescence,oxidative stress,abnormal shortening of telomeres, apoptosis, and epigenetic changes affecting gene expression have been proposed to contribute to the aging process,and aging-associated diseases. The above indicates that aging can increase the incidence of IPF. So can the occurrence of aging be aggravated after IPF? We examined pathological damage, collagen deposition, oxidative stress and immunosenescence to determine whether bleomycin(BLM)-induced pulmonary fibrosis (PF) accelerates aging in rats. If so, what drugs can inhibit or delay this aging. In clinical studies,the combination of methylprednisolone(MP) and cyclophosphamide(CTX) has shown great benefits in patients with IPF, but its effect on aging resulting from fibrosis is not fully understood. Therefore, we investigated whether MP combined with CTX could delay or inhibit aging in IPF rats. It may provide new targets for the treatment of IPF. Methods: PF rat models were induced by BLM and treated with MP or MP/CTX combination.Transmission electron microscope, hematoxylin and eosin (H&E) and Masson staining were used to measure the morphology of PF. α-SMA and collagen I levels were examined by western blot and immunohistochemistry. Malondialdehyde(MDA),myeloperoxidase(MPO),lutathione peroxidase(GSH-PX) and superoxide dismutase(SOD) levels were determined using commercial kits.T cells were analyzed with flow cytometry. Results: We found that pathological damage, collagen deposition, oxidative stress, and T-cell senescence were increased after BLM-induced PF. The combined use of MP and CTX can alleviate pathological damage, reduce oxidative stress response, such as reducing MDA and MPO levels, and increasing SOD and GSH-PX activities. And inhibition of T cell senescence in lung tissue, such as reduction of CD27 - CD28 - CD4 + T cells in BLM-induced PF. Conclusions: BLM-induced PF aggravated the occurrence of aging in rats. The combination of MP and CTX can inhibit or delay aging, and thus play a therapeutic role in IPF.These findings provide new insights into the mechanism by which MP and CTX act in combination on IPF. Idiopathic pulmonary fibrosis Methylprednisolone and cyclophosphamide combination oxidative stress T cell senescence Figures Figure 1 Figure 2 Figure 3 Highlights Bleomycin-induced pulmonary fibrosis can exacerbate aging. MP combined with CTX can inhibit or delay aging after fibrosis. The evaluation indicators include the following: Pathological damage ,collagen deposition of α-SMA and collagen I levels, the levels of oxidative stress and T cell senescence in pulmonary fibrosis. Background PF is a progressive chronic fibrosing interstitial lung disease[ 1 ],which is characterized by abnormal recruitment of immune cells,epithelial mesenchymal transition(EMT) imbalance, accumulation of extracellular matrix(ECM), and irreversible scar formation in the lung[ 2 ]. PF is a multicellular process,including damaged alveolar epithelium and persistent activation of matrix including fibroblasts and immune cells[ 3 ]. IPF is the most common idiopathic interstitial pneumonia and the one with the worst prognosis,showing a median survival of 2 to 3 years[ 4 ]. Previous studies have shown that the incidence of IPF increases significantly with age. Immunosenescence,oxidative stress, abnormal shortening of telomeres,apoptosis, and epigenetic changes affecting gene expression have been proposed to contribute to the aging process,and aging-associated diseases, including IPF. Modification of both innate and adaptive immune responses, increased oxidative stress with potential consequences in cell viability and predisposition to fibrosis,as well as changes in the balance of specific bone marrow cell populations that are recruited into the lung to promote repair or disrepair[ 5 ]. The number of patients with IPF is large, and aging can lead to an increase in the incidence of IPF. However, there is still a lack of relevant research evidence on whether IPF can aggravate aging.We hypothesized that IPF could also exacerbate aging and that aging could be blocked by medications. So,we seek new therapeutic strategies by studying markers related to aging. Cyclophosphamide(CTX) is a highly potent immunosuppressant that has demonstrated efficacy in inducing and maintaining remission in a range of autoimmune and inflammatory illnesses[ 6 ].In autoimmune diseases, CTX was often used to inhibit overactive immune response[ 7 ] It was reported that CTX intravenously injection into patients with PF improved pulmonary function[ 8 ]. While some reports suggested that CTX may cause PF in patients[ 9 , 10 ] or animals[ 11 ]. And some reports also suggested that CTX did not affect PF progression, even resulting in the increase of 3-month mortality[ 12 , 13 ]. Therefore, the use of CTX in the treatment of IPF is controversial. The double roles of CTX in IPF progression show a question that how to optimize the strategy of CTX to improve the lung injury with fibrosis. Glucocorticoids(GC) are commonly used drugs for IPF because of its anti-inflammatory effect[ 14 ]. Corticosteroid was found to alleviate PF[ 15 ]. MP could significantly attenuated lung injury, and reduced proinflammatory cytokine production and neutrophil infiltration into alveoli[ 16 ]. Prednisolone combined with interferon gamma-1b can increase total lung capacity and arterial oxygen in patients with pulmonary fibrosis[ 17 ].In addition,CTX and MP were combined to treat a case of pulmonary interstitial fibrosis[ 18 ]. High dose prednisolone pulsed combined with CTX could improve or stabilize lung function in patients with severe systemic sclerosing lung disease[ 19 ]. A clinical study indicated that the combination of the CTX and GC were beneficial to PF[ 20 ].So, combination of MP and CTX maybe considered safe in PF[ 21 ]. Taken together, aging increased the incidence of IPF, and we need to clarify the impact of IPF on aging before we can treat IPF.Clinical evidence suggested that the combination of CTX and MP may improve IPF, but due to the toxic side effects of MP and CTX, its efficacy is still controversial, and the therapeutic mechanism needs to be studied. In this study, we determined that IPF exacerbates the onset of aging and that the combination of MP and CTX exerts a therapeutic effect on IPF by blocking aging. Methods Reagents and instruments Reagents: Bleomycin sulfate (MCE, Product number:HY-17565); Cyclophosphamide (Manufacturer: Shanghai Yuanye Biotechnology Co., LTD., Item number:S3056); Methylprednisolone (Pfizer, product number: H20130301); Purified Mouse Anti - Rat Granulocytes monoclonal antibody (manufacturer: BD company, the article number 554905); APC Goat Anti-Mouse Ig polyclonal antibody (BD Company, Product number 550826); FITC anti-Rat CD45, PE anti-Rat CD4, APC anti-Rat CD3, PerCP anti-Rat CD8, RBC Lysis Buffer (10X), Cell Staining Buffer(FBS) Cell washes were purchased from BioLegend. Instruments: micropipettor (Eppendorf, Germany), low-speed horizontal centrifuge (Anhui Zhongke Zhongka, Anhui), flow cytometer Cyto FLEX S (Beckman, USA) Model: BD LSRII, USA. Low speed centrifuge (Model: China Jingli LD5-10B) Animals And Ethics Statements Sprague-Dawley rats(male,6–8 weeks of age,160 ± 10g) were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd. (Beijing,China).Rats were performed a week of adaptation before starting the study.Rats were allowed to eat normal pellet feed and tap water freely throughout the study period, and maintained under standard temperature(22 ± 2°C) and relative humidity(55 ± 5%)with 12h-light/12 h-dark cycles.Rats were divided into four groups(n = 10):(i) blank group, (ii) bleomycin (BLM) group,(iii)BLM-MP group and (iv)BLM-MP + CTX group.All animal experiments were reviewed and approved by the Animal Ethics Committee of the Beijing Medconner Biotechnology Co.,LTD and conform to National Institutes of Health guidelines for the use of rodents. This study was reported in accordance with ARRIVE guidelines. Animal Modeling And Treatment Rats were anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital (45 mg/kg). They were then fixed on rat plates and sterilized by conventional methods. Bleomycin saline (5 mg/kg, 0.05 mL) was injected into the bifurcation of the trachea. The equal volume of normal saline 0.05 mL was injected into the trachea in the sham operation group. The needle was left in the lung for 6 s and the rats were massaged to distribute the drug evenly in the lung. The model was successfully established on the second day. Drug intervention began 7 days after model establishment. The BLM-MP group was intraperitoneally injected with MP (3 mg/kg) every day, the BLM-MP + CTX group was intraperitoneally injected with MP (3 mg/kg) and CTX (8 mg/kg) every day, and the blank and BLM groups were injected with the same volume of normal saline every day. The animals were sacrificed after 3 weeks of drug administration. Pathological Staining The pulmonary tissues of rats were fixed in 4% paraformaldehyde for 24h. After dehydrated, samples were embedded in paraffin, and sectioned to obtain 5µm thickness sections. Then, hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed according to the manufacturer’s instructions (Nanjing Jiancheng, China). For immunohistochemistry, sections were incubated with primary antibody against α-SMA (1:3000, 14395-1-AP, Proteintech, China) and collagen I (1:1000, 14695-1-AP, Proteintech) at 4°C overnight. Negative control was obtained by ignoring the incubation with primary antibody. After that, Goat anti-rabbit HRP labeled secondary antibody (1:1000, ab6721, Abcam, USA) was used for 30min incubation with sections at room temperature. Lastly, sections were developed with 3’3’-diaminobenzidine (DAB, Beyotime, China) for 10min and images were acquired under a light microscope (Leica, Germany). Transmission Electron Microscope The lung tissues were fixed with the mixture of 2.5% glutaraldehyde and 2% polyformaldehyde at 4°C for 1h, then, with 1% osmic acid for 1.5h. Then, tissues were dehydrated with gradient alcohol, embedded and sliced. Subsequently, sections were stained and observed by a H-7500 transmission electron microscope (Hitachi, Japan). Ashcroft Scale According to the scale defined by Ashcroft et al. If there is any difficulty between two odd grades, the field will be given an even score in the middle. In each region, the major degree of fibrosis was recorded as accounting for more than half of the area of the region. The modified scale is defined according to the reference. [ 22 ]. Western blot Total proteins were extracted from lung tissues using radio immunoprecipitation assay (RIPA) buffer with proteinase inhibitor (Solarbio). 40 µg proteins of each sample were separated by 10% SDS-PAGE and proteins were transferred to PVDF membranes (Millipore). The membranes were sealed in 5% skim milk for 1 h at room temperature and incubated with primary antibodies, GAPDH (1:50000, 60004-1-Ig, Proteintech), α-SMA (1:4000, 14395-1-AP, Proteintech) and collagen I (1:2000, 14695-1-AP, Proteintech) at 4 ℃ overnight. Next, membranes were washed with tris buffer containing 0.05% twin-20 (TBST) and incubated with secondary antibody (1:10000, ab6721, Abcam) for 1 h at room temperature. Immunoblots were visualized by an ECL kit (Abcam) followed by quantitative analysis via ImageJ software. Measurement of oxidative stress relating indicators The homogenate supernatant of the lung tissues was used to detect the MDA content, MPO, GSH-PX and SOD activities by commercial kits (MDA:E-BC-K025-M;MPO:E-BC-K074-M;SOD:E-BC-K020-M;GSH-PX:E-BC-K096-M, Elabscience Biotechnology Co., Ltd, China) according to the instructions. Flow cytometry Isolated single cells from rat BALF were stained with CD3 (#201412, BioLegend, San Diego, CA, USA), CD4 (#201505, BioLegend, San Diego, CA, USA), CD8 (#201712, BioLegend), CD27 (#124207, BioLegend, San Diego, CA, USA), CD28 (#200908, BioLegend, San Diego, CA, USA) antibodies for 30 min at 4°C. Then, Flow cytometry was performed by using a Beckman CytoFLEX device and FlowJo software was used for analysis. CD3 gating was performed according to the side light scattering area (SSC-A) and forward light scattering area (FSC-A). CD27, CD28, and CD3 were detected as the next gating cell population of CD4 and CD8. On the basic of CD3 + CD4 + , The proportion of CD27 and CD28 cells was determined. Statistical analysis The measurement data were expressed as mean ± standard deviation (SD), one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used for multiple group comparison by using Graphpad Prism. Differences at p < 0.05 were considered statistically significant. Results PF induced by BLM exacerbated the development of aging in rats We found that pathological damage, collagen deposition, oxidative stress, and T-cell senescence were increased after BLM-induced PF.In BLM treated rats, the alveolar structure was seriously damaged, and partial of alveoli were broken and fused, forming pulmonary bullae, alveolar septum was significantly thickened, and interstitial inflammatory cells infiltrated obviously (Fig. 1 A). In BLM treated rats, as evaluated by Achcroft score(Fig. 1 B). Then, Masson staining was performed to observe collagen fibers. It showed that there were obvious pathological changes in lung tissue, the normal structure of alveoli disappeared, lung tissue was reconstructed, a large number of blue collagen fibers were deposited, and the degree of fibrosis was obvious in PF rats(Fig. 1 C). Then, morphology of lung tissue was observed by transmission electron microscope. In the blank group, the nuclear membrane in lung tissue was clear, the structure of alveolar type II epithelial cells was clear and complete, and there were more lamellar bodies (Fig. 1 D). While, in BLM group,mitochondria of alveolar type II epithelial cells swelled, vacuoles formed, microvilli on the surface disappeared, lamellar bodies were small, intercellular connections were unclear, and collagen and fiber bundles in the stroma increased (Fig. 1 D). Immunohistochemistry and western blot showed BLM significantly up-regulated the expression of α-SMA and collagen I(Fig. 1 E-H). The content of MDA and the activity of MPO in lung tissue of BLM group was significantly higher than that of blank group, SOD and GSH-PX had opposite trends(Fig. 2 ).Compared with blank group, the proportion of CD27 − CD28 − CD3 + T cells and CD27 − CD28 − CD4 + T cells in BLM group were increased, The proportion of CD27 − CD28 − CD8 + T cells had opposite trends(Fig. 3 ). Mp Combined With Ctx Reduced Blm-induced Pf In Rats In BLM-MP group, alveolar structure was improved, alveolar septal thickening and inflammatory cell infiltration were reduced (Fig. 1 A). The combined treatment with MP and CTX further improved lung injury (Fig. 1 A). MP attenuated the development of PF,the combined treatment with MP and CTX had a more fsignificant effect on reducing pulmonary fibrosis as evaluated by Achcroft score. (Fig. 1 B) Then, Masson staining was performed to observe collagen fibers.Compared with BLM group, the destruction of alveolar structure and the proliferation of fibrous tissue in BLM-MP group were significantly reduced, and the blue collagen fibers were also remarkably reduced(Fig. 1 C). These improvements were further enhanced by MP and CTX combination treatment (Fig. 1 C). Morphology of lung tissue was observed by transmission electron microscope.In the group of BLM-MP and BLM-MP + CTX, the pathological manifestations were relieved, microvilli of alveolar type II epithelial cells were decreased, lamellar corpuscles were larger, and inflammatory cell infiltration decreased (Fig. 1 D).. And compared with BLM-MP group, the BLM-MP + CTX group revealed better improvement. MP treatment resulted in the decreases of α-SMA and collagen I expressed in rats with PF (Fig. 1 E- 1 H). CTX further enhanced the inhibitor effect of MP in α-SMA and collagen I (Fig. 1 E- 1 H). Mp Combined With Ctx Improved The Oxidative Stress Response In Blm-induced Pf Rats MDA, MPO, SOD and GSH-PX levels are common indicator of oxidation stress. MP could inhibit the increasing of MDA and MPO, the combination of MP and CTX further enhanced the effect of MP (Fig. 2 A, 2 B). But SOD and GSH-PX had opposite trends (Fig. 2 C, 2 D). This indicated that the combined application of MP and CTX alleviated the oxidative stress response during PF. Mp Combined With Ctx Treatment Regulates The Immune Senescence Process In Fibrotic Rats Our study shows that the proportion of CD27 − CD28 − CD3 + T cells and CD27 − CD28 − CD4 + T cells in BLM group were increased,which was reduced with MP alone, and was further reduced with the combination of MP and CTX; The proportion of CD27 − CD28 − CD8 + T cells had opposite trends(Fig. 3 ). Because CD27 − CD28 − was associated with immune senescence, this suggests that the combination of MP and CTX inhibited the activation and senescence of immune cells in the process of PF. In conclusion, the combination of MP and CTX can improve the immune function of T cells and reduce immune senescence. Discussion IPF is a progressive, terminal lung disease associated with aging. Fibroblast activation and increased ECM deposition are the key events in IPF pathogenesis[ 23 ]. Within the lung, alterations in the form, concentration and organization of ECM protein during the aging process lead to a reduction in tissue elasticity and physiological declines in lung function[ 24 , 25 ].Molecular, cellular and immunological changes occur concomitantly with aging deleteriously affect the capacity of lung tissue to respond to injury and repair damage[ 26 ].All these indicate that aging may increased susceptibility to PF. In the present study, we first establish a BLM-induced model of pulmonary fibrosis,and assuming that aging is exacerbated after the onset of PF. By Studies of pathology, electron microscopy, oxidative stress and T cell immunity, we confirmed that BLM-induced PF exacerbated the aging process. The pathological and electron microscopic observations showed that the deposition of collagen and ECM was increased, the expression of α-SMA and type collagen I. was up-regulated, the content of MDA and the activity of MPO were increased, the activities of SOD and GSH-Px were decreased, and the senescence of immune cells was aggravated. Without therapeutic intervention, persistent connective tissue deposition leads to scar formation, eventually leading to pulmonary fibrosis and death. The significance of oxidative stress in promoting BLM-induced PF has been reported and plays an important role in the development of PF[ 27 ]. Aging is associated with depletion of antioxidant defense systems[ 28 ], and it is well documented that antioxidant availability is reduced in IPF. Antioxidants may be useful in IPF treatment [ 29 ]. Treatment of mice with antiaging drugs that selectively eliminate senescent cells has been shown to attenuate BLM-induced fibrosis [ 30 ]. Immune mechanisms are critical to tissue injury and repair processes. The immune system undergoes age-related changes, and there is evidence of low-grade inflammation, decreased immune response, and increased autoimmunity with aging. Aging is associated with diminished B and T lymphocyte function, decline in antigen presenting cells, and increases in circulating and local levels of cytokines[ 31 – 33 ]. Yong Chong et al[ 34 ] demonstrated that aging resulted in a marked decrease in the number of CD27 + memory B cells and a marked increase in the number of CD27 − nonsensitized B cells. The accumulation of T cells that do not express CD28, a co-stimulatory molecule for T cells is a hallmark of aging and associated with a diminished immune response [ 35 ]. Changes in T-helper cell populations including the down-regulation of CD28 on T cells occur in IPF[ 36 ]. CD28null T cells have shortened telomeres and represent late-differentiated T cells. Thus, The gradual loss of surface costimulatory molecules CD27 and CD28 is considered to be a phenotypic feature of classical T cell senescence[ 37 – 39 ]. Our experiments further confirmed that the oxidative stress response occurs in the progression of IPF. Our results also showed that the levels of CD27 − CD28 − CD3 + T cells and CD27 − CD28 − CD4 + T cells were increased in IPF rats, while the opposite trend was observed for CD27 − CD28 − CD8 + T cells. Therefore, IPF can accelerate aging. Based on the results, we boldly hypothesize that immunosenescence of PF occurs mainly in CD4 + cells and not in CD8 + cells. However, it may be related to the limitations of this experiment, so a large number of experimental datas are still needed to further confirm. We propose that CD4 + T cells are mainly involved in assisting antigen presentation, promoting antibody secretion, and neutralizing antigens. CD8 + T cells mainly exert cytotoxic effects and damage antigen-carrying cells. During PF, autoproteins are incorrectly recognized as foreign antigens, and CD8 + T cells attack alveolar type II epithelial cells carrying antigens. We try to find a new direction for the treatment of IPF from the perspective of aging represented by oxidative stress and immune senescence. Currently, there is no cure for IPF.MP combined with CTX was effective in the treatment of systemic lupus nephritis. In addition, this combination can be used to treat gastric antral vasodilatation in systemic sclerosis and sjogren's associated chronic interstitial nephritis [ 40 – 44 ]. Previous clinical studies have confirmed the safety and efficacy of MP combined with CTX in the treatment of PF [ 18 – 21 ]. However, the treatment of MP and CTX for IPF has always been controversial, and the focus of the controversy is mostly on drug toxicity. Moreover, the side effects of the two drugs were mainly related to the doses. Therefore, According to the literature,we chose a low-dose combination of the two drugs. We used animal experiments to establish the model of IPF. After the occurrence of IPF, MP combined with CTX was used for intervention, the end point was 21 days after treatment and the effect was achieved. The results showed that MP combined with CTX improved the pathological damage of lung tissue, and played a therapeutic role by reducing the oxidative stress response and adjusting cellular immunity. Therefore, we propose a new perspective on the treatment of IPF from the perspective of aging. In clinical practice, we have also achieved good improvement in the treatment of IPF with MP combined with CTX, but large-scale clinical datas have not yet been formed. Therefore, this experiment provides ideas and directions for further clinical research. Aging is associated with immune dysregulation and extracellular matrix changes. The combination of MP and CTX can regulate lung immunity and extracellular matrix, which may be the mechanism of alleviating cell structural damage. However, the specific mechanism needs to be further studied, such as whether the molecular regulation mechanism of MP combined with CTX is of great significance in the treatment of IPF. Our next experiments will be involved. Limitations: Due to the limitation of experimental conditions, we did not evaluate other senescence markers such as CD57, KLRG1, γH2AX and p38, as well as telomere shortening, etc., and it would have been more interesting to add these markers to make our study more fruitful. We cannot further investigate the more detailed mechanisms of IPF in exacerbating aging, such as molecular regulatory mechanisms. In addition, as immune factors are involved in the development and progression of IPF, further studies are needed to determine the deeper relationship between IPF and immune senescence. These are interesting things that we look forward to the next experiments. Conclusions IPF is an age-related disease, and aging can promote the occurrence of IPF. Our experiment confirmed that IPF also aggravates the occurrence of aging, proving the interaction between aging and IPF. This study treated IPF from the perspective of aging and showed that the combination of MP and CTX reduced collagen deposition, oxidative stress, and altered the number of fibrotic immunosenescence cells in PF, suggesting that MP combined with CTX may improve PF by inhibiting aging. Targeting aging-related pathways may be a viable therapeutic strategy. This study provides theoretical support for its application in PF and the subsequent development of PF therapeutic drugs. List Of Abbreviations PF, pulmonary fibrosis IPF, Idiopathic pulmonary fibrosis BALF, bronchoalveolar lavage fluid BLM, Bleomycin MP, methylprednisolone CTX, cyclophosphamide H&E, hematoxylin and eosin EMT,epithelial mesenchymal transition ECM, extracellular matrix GC, Glucocorticoids GSH-Px, Glutathione peroxidase MDA, malondialdehyde MPO, myeloperoxidase SOD, superoxide dismutase Declarations Ethics approval and consent to participate All animal experiments were reviewed and approved by the Animal Ethics Committee of Beijing Medconner Biotechnology Co., LTD and conform to National Institutes of Health guidelines for the use of rodents. Consent for publication Not Applicable. This article does not contain any studies with human participants performed by any of the authors. Availability of data and materials The datasets used or analyzed 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 an open project of the State Key Laboratory of Molecular Developmental Biology (No.2021-MDB-KF-13). And supported by the State Key Laboratory of Molecular Developmental Biology. Authors' contributions XX guaranteed the integrity of the entire study; QX and MZ designed the study and literature research; MT and ZR defined the intellectual content; TM and YL performed experiment; ZL and KY collected the data; XH and RM analyzed the data; QX wrote the main manuscript and prepared figures. All authors reviewed the manuscript. Acknowledgements We would like to thank the anonymous reviewers who have helped to improve the pape. 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Kalantar H, Sadeghi E, Abolnezhadian F, Goudarzi M, Hemmati AA, Basir Z, Kalantar M. Carnosol attenuates bleomycin-induced lung damage via suppressing fibrosis, oxidative stress and inflammation in rats . Life sciences 2021, 287 :120059. Schafer MJ, White TA, Iijima K,et al. Cellular senescence mediates fibrotic pulmonary disease . Nat Commun 2017,8: 14532. Franceschi C, Campisi J. Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases . J Gerontol A Biol Sci Med Sci. 2014, 69 : S4-S9. Frasca D, Blomberg BB. Inflammaging decreases adaptive and innate immune responses in mice and humans . Biogerontology. 2016, 17 : 7-19 . Weyand CM, Goronzy JJ. Aging of the immune system. Mechanisms and therapeutic targets . Ann Am Thorac Soc .2016, 13 : S422-S428. Chong Y,Ikematsu H,Yamaji K,et al. CD27(+)(memory)B cell decrease and apoptosis-resistantCD27-(naive)B cell increase in aged humans:implications for age-related peripheral B cell developmental disturbances.Int Immunol,2005, 17 (4):383-90 . Moro-Garcia, M.A., Alonso-Arias, R., Lopez-Larrea, C., When aging reaches CD4+ T-cells: phenotypic and functional changes . Front. Immunol. 2013, 4 :107. Gilani, S.R., et al.CD28 down-regulation on circulating CD4 T-cells is associated with poor prognoses of patients with idiopathic pulmonary fibrosis. PLoS One .2010,5(1):e8959. Akbar A N, Henson S M, Lanna A. Senescence of T Lymphocytes: Implications 509 for Enhancing Human Immunity. Trends Immunol, 2016, 37 (12): 866-76. Lanna A, Henson S M, Escors D, Akbar A N. The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells. Nat Immunol, 2014, 15 (10): 965-72. Plunkett F J, Franzese O, Finney H M, Fletcher J M, Belaramani L L, Salmon M, Dokal I, Webster D, Lawson A D, Akbar A N. T he loss of telomerase activity in highly differentiated CD8+CD28-CD27- T cells is associated with decreased Akt (Ser473) phosphorylation . J Immunol, 2007, 178 (12): 7710-19. Lorenzi AR, Johnson AH, Davies G, Gough A. Gastric antral vascular ectasia in systemic sclerosis:complete resolution with methylprednisolone and cyclophosphamide . Annals of the rheumatic diseases. 2001, 60 (8):796-8. Illei GG, Austin HA, Crane M, Collins L, Gourley MF, Yarboro CH, Vaughan EM, Kuroiwa T, Danning CL, Steinberg ADet al. Combination therapy with pulse cyclophosphamide plus pulse methylprednisolone improves long-term renal outcome without adding toxicity in patients with lupus nephritis . Annals of internal medicine 2001, 135 (4):248-257. Barile-Fabris L, Ariza-Andraca R, Olguín-Ortega L, Jara LJ, Fraga-Mouret A, Miranda-Limón JM, Fuentes de la Mata J, Clark P, Vargas F, Alocer-Varela J: Controlled clinical trial of IV cyclophosphamide versus IV methylprednisolone in severe neurological manifestations in systemic lupus erythematosus . Annals of the rheumatic diseases 2005, 64 (4):620-625. Boumpas DT, Austin HA, 3rd, Vaughn EM, Klippel JH, Steinberg AD, Yarboro CH, Balow JE: Controlled trial of pulse methylprednisolone versus two regimens of pulse cyclophosphamide in severe lupus nephritis . Lancet (London, England) 1992, 340 (8822):741-745. Ruiz-Irastorza G, Dueña-Bartolome L, Dunder S, Varona J, Gomez-Carballo C, Dominguez-Cainzos J, Rodrigo-Manjon A, Bueno L, Richez C, Duffau P et al : Eurolupus cyclophosphamide plus repeated pulses of methyl-prednisolone for the induction therapy of class III, IV and V lupus nephritis . Autoimmunity reviews 2021, 20 (10):102898. 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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-2245802","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":154147778,"identity":"aab49ae0-3ab5-444b-a9a0-854b61122ba5","order_by":0,"name":"Qingjie Xu","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qingjie","middleName":"","lastName":"Xu","suffix":""},{"id":154147780,"identity":"9e3854c2-c680-4030-a223-281f0aeacb98","order_by":1,"name":"Manka Zhang","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Manka","middleName":"","lastName":"Zhang","suffix":""},{"id":154147781,"identity":"c719ebd2-1343-4a10-a4d9-e961e4f8cd61","order_by":2,"name":"Ming Tang","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Tang","suffix":""},{"id":154147784,"identity":"80c6b85a-7877-4bb1-af40-585a0c9ceab6","order_by":3,"name":"Zhouping Li","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhouping","middleName":"","lastName":"Li","suffix":""},{"id":154147786,"identity":"9e051747-8cfc-49f3-8838-f6d9be7c56bc","order_by":4,"name":"Yin Liu","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yin","middleName":"","lastName":"Liu","suffix":""},{"id":154147790,"identity":"ba4eac30-a660-4821-b923-ea04ac8e508a","order_by":5,"name":"Tao ma","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"ma","suffix":""},{"id":154147792,"identity":"c87d5890-1a18-45b9-b054-83bfc4811b77","order_by":6,"name":"Zhiguo Rao","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Rao","suffix":""},{"id":154147793,"identity":"f48358f8-1954-490a-9b78-8a9bda32e905","order_by":7,"name":"Keyu Yang","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Keyu","middleName":"","lastName":"Yang","suffix":""},{"id":154147795,"identity":"de2fe3ec-4645-40fa-a4a7-640b068f5e17","order_by":8,"name":"Xiaoxu He","email":"","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxu","middleName":"","lastName":"He","suffix":""},{"id":154147798,"identity":"c926b8ae-6699-4bda-9a2c-87b06267d4d4","order_by":9,"name":"Runlin Ma","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Runlin","middleName":"","lastName":"Ma","suffix":""},{"id":154147800,"identity":"8053d657-97db-4c91-a2b4-78d386e69884","order_by":10,"name":"Xiaoyan Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYLCCBDDJfABIHCBJC1sCVAsz0XbxGBCnxeBG8jGJBzU2if2zez5+/FFzh8Gcvf8Aw88duLVIzkhLNkg4lpY4487ZzdI8x54xWPYcZmDsPYNbC79EjuGDBLbDiQ03cjdIMzYcBtnLwMzYhlsLm0T+hwMJ/w4nzr+R8/jnT5CW+4/xawHawvggse1w4oYbOWwSvGBbmPFrkex5ZmyQ2JdmvPFGmpk1z7HDPAZnkg0O9uLRYnA8+Znkj282svNuJD+++aPmsJzB8YMPH/zEowUGHBugDB4QcYCwBgYGe2IUjYJRMApGwQgFABfBV9/AmT7JAAAAAElFTkSuQmCC","orcid":"","institution":"Aerospace Center Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2022-11-07 07:59:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2245802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2245802/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":29609911,"identity":"28f30173-a5e3-437a-b0a2-ffcf2ca0380b","added_by":"auto","created_at":"2022-11-28 19:41:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":239525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMP and CTX combination treatment improved PF in rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Pathological changes of lung tissue were assessed via H\u0026amp;E staining. (B) Quantification of fibrosis by Ashcroft score. (C)\u003cstrong\u003e \u003c/strong\u003eCollagen deposition in lung tissue was determined by Masson staining. (D)\u003cstrong\u003e \u003c/strong\u003eElectron microscopic morphology of lung tissues. M: mitochondrion, N: nucleus, LC: lamellar corpuscle, TJ: tight junction.\u003cstrong\u003e \u003c/strong\u003e(E, F)\u003cstrong\u003e \u003c/strong\u003eLevels of\u003cstrong\u003e \u003c/strong\u003eα-SMA and collagen I in lung tissue were measured by immunohistochemistry(E) and positive ratio were assessed(F). (G, H) Western blot was performed to determine α-SMA and collagen I (G)and protein grayscale analysis (H)was placed below.\u003cstrong\u003e \u003c/strong\u003en = 10. Data were represented as mean ± SD. * \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2245802/v1/7860bbbbc2ae0c95493ce327.jpg"},{"id":29609913,"identity":"1b467986-2e09-44e4-a8ff-8b82cec454a6","added_by":"auto","created_at":"2022-11-28 19:41:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMP and CTX inhibited oxidative stress in PF rats. \u003c/strong\u003e(A-D)MDA and MPO content, SOD and GSH-PX activities in lung tissues were assessed via commercial kits. n=10. Data were represented as mean ± SD.\u003csup\u003ens \u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026gt;0.05,* \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2245802/v1/c5114ac04d4bb787185e6f8d.jpg"},{"id":29609912,"identity":"3779715c-59d4-4399-a698-74529d3706ba","added_by":"auto","created_at":"2022-11-28 19:41:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":428938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined treatment of MP and CTX inhibited the immunosenescence of PF in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT cells \u003c/strong\u003ein BALF were determined by flow cytometric analysis.\u003cstrong\u003e \u003c/strong\u003e(Figure 3-1) CD27-CD28-CD3+T cells (A) blank group, (B)BLM group,(C)BIM-MP group,(D)BLM-MP+CTX group. (Figure 3-2) CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells and CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003eT cells. (A1) blank group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells , (B1)BLM group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells,(C1)BIM-MP group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells,(D1)BLM-MP+CTX group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells. (A2) blank group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells , (B2)BLM group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells,(C2)BIM-MP group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells,(D2)BLM-MP+CTX group of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells. (Figure 3-3) Statistical analysis was performed between groups. (A) blank group, (B)BLM group,(C)BIM-MP group,(D)BLM-MP+CTX group.n= 10. Data were represented as mean ± SD.* \u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2245802/v1/1f03eebd2a6c36a4707f78f0.jpg"},{"id":40668770,"identity":"d7d721d5-c639-4a42-afb4-58f5f4063745","added_by":"auto","created_at":"2023-07-27 15:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1693166,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2245802/v1/ae1cab5e-c34b-4057-b284-4fe75992ae14.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cyclophosphamide combined with methylprednisolone can play a therapeutic role by delaying the aging of pulmonary fibrosis","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eBleomycin-induced pulmonary fibrosis can exacerbate aging.\u003c/li\u003e\n \u003cli\u003eMP combined with CTX can inhibit or delay aging after fibrosis.\u003c/li\u003e\n \u003cli\u003eThe evaluation indicators include the following: Pathological damage ,collagen deposition of \u0026alpha;-SMA and collagen I levels, the levels of oxidative stress and T cell senescence in pulmonary fibrosis.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Background","content":"\u003cp\u003ePF is a progressive chronic fibrosing interstitial lung disease[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e],which is characterized by abnormal recruitment of immune cells,epithelial mesenchymal transition(EMT) imbalance, accumulation of extracellular matrix(ECM), and irreversible scar formation in the lung[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PF is a multicellular process,including damaged alveolar epithelium and persistent activation of matrix including fibroblasts and immune cells[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. IPF is the most common idiopathic interstitial pneumonia and the one with the worst prognosis,showing a median survival of 2 to 3 years[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies have shown that the incidence of IPF increases significantly with age. Immunosenescence,oxidative stress, abnormal shortening of telomeres,apoptosis, and epigenetic changes affecting gene expression have been proposed to contribute to the aging process,and aging-associated diseases, including IPF. Modification of both innate and adaptive immune responses, increased oxidative stress with potential consequences in cell viability and predisposition to fibrosis,as well as changes in the balance of specific bone marrow cell populations that are recruited into the lung to promote repair or disrepair[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The number of patients with IPF is large, and aging can lead to an increase in the incidence of IPF. However, there is still a lack of relevant research evidence on whether IPF can aggravate aging.We hypothesized that IPF could also exacerbate aging and that aging could be blocked by medications. So,we seek new therapeutic strategies by studying markers related to aging.\u003c/p\u003e \u003cp\u003eCyclophosphamide(CTX) is a highly potent immunosuppressant that has demonstrated efficacy in inducing and maintaining remission in a range of autoimmune and inflammatory illnesses[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].In autoimmune diseases, CTX was often used to inhibit overactive immune response[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] It was reported that CTX intravenously injection into patients with PF improved pulmonary function[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While some reports suggested that CTX may cause PF in patients[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] or animals[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. And some reports also suggested that CTX did not affect PF progression, even resulting in the increase of 3-month mortality[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, the use of CTX in the treatment of IPF is controversial. The double roles of CTX in IPF progression show a question that how to optimize the strategy of CTX to improve the lung injury with fibrosis.\u003c/p\u003e \u003cp\u003eGlucocorticoids(GC) are commonly used drugs for IPF because of its anti-inflammatory effect[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Corticosteroid was found to alleviate PF[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. MP could significantly attenuated lung injury, and reduced proinflammatory cytokine production and neutrophil infiltration into alveoli[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Prednisolone combined with interferon gamma-1b can increase total lung capacity and arterial oxygen in patients with pulmonary fibrosis[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].In addition,CTX and MP were combined to treat a case of pulmonary interstitial fibrosis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. High dose prednisolone pulsed combined with CTX could improve or stabilize lung function in patients with severe systemic sclerosing lung disease[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A clinical study indicated that the combination of the CTX and GC were beneficial to PF[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].So, combination of MP and CTX maybe considered safe in PF[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, aging increased the incidence of IPF, and we need to clarify the impact of IPF on aging before we can treat IPF.Clinical evidence suggested that the combination of CTX and MP may improve IPF, but due to the toxic side effects of MP and CTX, its efficacy is still controversial, and the therapeutic mechanism needs to be studied. In this study, we determined that IPF exacerbates the onset of aging and that the combination of MP and CTX exerts a therapeutic effect on IPF by blocking aging.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eReagents and instruments\u003c/p\u003e \u003cp\u003eReagents: Bleomycin sulfate (MCE, Product number:HY-17565); Cyclophosphamide (Manufacturer: Shanghai Yuanye Biotechnology Co., LTD., Item number:S3056); Methylprednisolone (Pfizer, product number: H20130301); Purified Mouse Anti - Rat Granulocytes monoclonal antibody (manufacturer: BD company, the article number 554905); APC Goat Anti-Mouse Ig polyclonal antibody (BD Company, Product number 550826); FITC anti-Rat CD45, PE anti-Rat CD4, APC anti-Rat CD3, PerCP anti-Rat CD8, RBC Lysis Buffer (10X), Cell Staining Buffer(FBS) Cell washes were purchased from BioLegend.\u003c/p\u003e \u003cp\u003eInstruments: micropipettor (Eppendorf, Germany), low-speed horizontal centrifuge (Anhui Zhongke Zhongka, Anhui), flow cytometer Cyto FLEX S (Beckman, USA) Model: BD LSRII, USA. Low speed centrifuge (Model: China Jingli LD5-10B)\u003c/p\u003e\n\u003ch3\u003eAnimals And Ethics Statements\u003c/h3\u003e\n\u003cp\u003eSprague-Dawley rats(male,6\u0026ndash;8 weeks of age,160\u0026thinsp;\u0026plusmn;\u0026thinsp;10g) were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd. (Beijing,China).Rats were performed a week of adaptation before starting the study.Rats were allowed to eat normal pellet feed and tap water freely throughout the study period, and maintained under standard temperature(22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and relative humidity(55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%)with 12h-light/12 h-dark cycles.Rats were divided into four groups(n\u0026thinsp;=\u0026thinsp;10):(i) blank group, (ii) bleomycin (BLM) group,(iii)BLM-MP group and (iv)BLM-MP\u0026thinsp;+\u0026thinsp;CTX group.All animal experiments were reviewed and approved by the Animal Ethics Committee of the Beijing Medconner Biotechnology Co.,LTD and conform to National Institutes of Health guidelines for the use of rodents. This study was reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003ch3\u003eAnimal Modeling And Treatment\u003c/h3\u003e\n\u003cp\u003eRats were anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital (45 mg/kg). They were then fixed on rat plates and sterilized by conventional methods. Bleomycin saline (5 mg/kg, 0.05 mL) was injected into the bifurcation of the trachea. The equal volume of normal saline 0.05 mL was injected into the trachea in the sham operation group. The needle was left in the lung for 6 s and the rats were massaged to distribute the drug evenly in the lung. The model was successfully established on the second day. Drug intervention began 7 days after model establishment. The BLM-MP group was intraperitoneally injected with MP (3 mg/kg) every day, the BLM-MP\u0026thinsp;+\u0026thinsp;CTX group was intraperitoneally injected with MP (3 mg/kg) and CTX (8 mg/kg) every day, and the blank and BLM groups were injected with the same volume of normal saline every day. The animals were sacrificed after 3 weeks of drug administration.\u003c/p\u003e\n\u003ch3\u003ePathological Staining\u003c/h3\u003e\n\u003cp\u003eThe pulmonary tissues of rats were fixed in 4% paraformaldehyde for 24h. After dehydrated, samples were embedded in paraffin, and sectioned to obtain 5\u0026micro;m thickness sections. Then, hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining were performed according to the manufacturer\u0026rsquo;s instructions (Nanjing Jiancheng, China). For immunohistochemistry, sections were incubated with primary antibody against α-SMA (1:3000, 14395-1-AP, Proteintech, China) and collagen I (1:1000, 14695-1-AP, Proteintech) at 4\u0026deg;C overnight. Negative control was obtained by ignoring the incubation with primary antibody. After that, Goat anti-rabbit HRP labeled secondary antibody (1:1000, ab6721, Abcam, USA) was used for 30min incubation with sections at room temperature. Lastly, sections were developed with 3\u0026rsquo;3\u0026rsquo;-diaminobenzidine (DAB, Beyotime, China) for 10min and images were acquired under a light microscope (Leica, Germany).\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscope\u003c/h3\u003e\n\u003cp\u003eThe lung tissues were fixed with the mixture of 2.5% glutaraldehyde and 2% polyformaldehyde at 4\u0026deg;C for 1h, then, with 1% osmic acid for 1.5h. Then, tissues were dehydrated with gradient alcohol, embedded and sliced. Subsequently, sections were stained and observed by a H-7500 transmission electron microscope (Hitachi, Japan).\u003c/p\u003e \u003cp\u003eAshcroft Scale\u003c/p\u003e \u003cp\u003eAccording to the scale defined by Ashcroft et al. If there is any difficulty between two odd grades, the field will be given an even score in the middle. In each region, the major degree of fibrosis was recorded as accounting for more than half of the area of the region. The modified scale is defined according to the reference. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWestern blot\u003c/p\u003e \u003cp\u003eTotal proteins were extracted from lung tissues using radio immunoprecipitation assay (RIPA) buffer with proteinase inhibitor (Solarbio). 40 \u0026micro;g proteins of each sample were separated by 10% SDS-PAGE and proteins were transferred to PVDF membranes (Millipore). The membranes were sealed in 5% skim milk for 1 h at room temperature and incubated with primary antibodies, GAPDH (1:50000, 60004-1-Ig, Proteintech), α-SMA (1:4000, 14395-1-AP, Proteintech) and collagen I (1:2000, 14695-1-AP, Proteintech) at 4 ℃ overnight. Next, membranes were washed with tris buffer containing 0.05% twin-20 (TBST) and incubated with secondary antibody (1:10000, ab6721, Abcam) for 1 h at room temperature. Immunoblots were visualized by an ECL kit (Abcam) followed by quantitative analysis via ImageJ software.\u003c/p\u003e \u003cp\u003eMeasurement of oxidative stress relating indicators\u003c/p\u003e \u003cp\u003eThe homogenate supernatant of the lung tissues was used to detect the MDA content, MPO, GSH-PX and SOD activities by commercial kits (MDA:E-BC-K025-M;MPO:E-BC-K074-M;SOD:E-BC-K020-M;GSH-PX:E-BC-K096-M, Elabscience Biotechnology Co., Ltd, China) according to the instructions.\u003c/p\u003e \u003cp\u003eFlow cytometry\u003c/p\u003e \u003cp\u003eIsolated single cells from rat BALF were stained with CD3 (#201412, BioLegend, San Diego, CA, USA), CD4 (#201505, BioLegend, San Diego, CA, USA), CD8 (#201712, BioLegend), CD27 (#124207, BioLegend, San Diego, CA, USA), CD28 (#200908, BioLegend, San Diego, CA, USA) antibodies for 30 min at 4\u0026deg;C. Then, Flow cytometry was performed by using a Beckman CytoFLEX device and FlowJo software was used for analysis. CD3 gating was performed according to the side light scattering area (SSC-A) and forward light scattering area (FSC-A). CD27, CD28, and CD3 were detected as the next gating cell population of CD4 and CD8. On the basic of CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e, The proportion of CD27 and CD28 cells was determined.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe measurement data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test was used for multiple group comparison by using Graphpad Prism. Differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePF induced by BLM exacerbated the development of aging in rats\u003c/h2\u003e \u003cp\u003eWe found that pathological damage, collagen deposition, oxidative stress, and T-cell senescence were increased after BLM-induced PF.In BLM treated rats, the alveolar structure was seriously damaged, and partial of alveoli were broken and fused, forming pulmonary bullae, alveolar septum was significantly thickened, and interstitial inflammatory cells infiltrated obviously (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In BLM treated rats, as evaluated by Achcroft score(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Then, Masson staining was performed to observe collagen fibers. It showed that there were obvious pathological changes in lung tissue, the normal structure of alveoli disappeared, lung tissue was reconstructed, a large number of blue collagen fibers were deposited, and the degree of fibrosis was obvious in PF rats(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Then, morphology of lung tissue was observed by transmission electron microscope. In the blank group, the nuclear membrane in lung tissue was clear, the structure of alveolar type II epithelial cells was clear and complete, and there were more lamellar bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). While, in BLM group,mitochondria of alveolar type II epithelial cells swelled, vacuoles formed, microvilli on the surface disappeared, lamellar bodies were small, intercellular connections were unclear, and collagen and fiber bundles in the stroma increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Immunohistochemistry and western blot showed BLM significantly up-regulated the expression of α-SMA and collagen I(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H). The content of MDA and the activity of MPO in lung tissue of BLM group was significantly higher than that of blank group, SOD and GSH-PX had opposite trends(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).Compared with blank group, the proportion of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003eT cells and CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003eT cells in BLM group were increased, The proportion of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells had opposite trends(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMp Combined With Ctx Reduced Blm-induced Pf In Rats\u003c/h3\u003e\n\u003cp\u003eIn BLM-MP group, alveolar structure was improved, alveolar septal thickening and inflammatory cell infiltration were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The combined treatment with MP and CTX further improved lung injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). MP attenuated the development of PF,the combined treatment with MP and CTX had a more fsignificant effect on reducing pulmonary fibrosis as evaluated by Achcroft score. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) Then, Masson staining was performed to observe collagen fibers.Compared with BLM group, the destruction of alveolar structure and the proliferation of fibrous tissue in BLM-MP group were significantly reduced, and the blue collagen fibers were also remarkably reduced(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These improvements were further enhanced by MP and CTX combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Morphology of lung tissue was observed by transmission electron microscope.In the group of BLM-MP and BLM-MP\u0026thinsp;+\u0026thinsp;CTX, the pathological manifestations were relieved, microvilli of alveolar type II epithelial cells were decreased, lamellar corpuscles were larger, and inflammatory cell infiltration decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).. And compared with BLM-MP group, the BLM-MP\u0026thinsp;+\u0026thinsp;CTX group revealed better improvement. MP treatment resulted in the decreases of α-SMA and collagen I expressed in rats with PF (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). CTX further enhanced the inhibitor effect of MP in α-SMA and collagen I (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e\n\u003ch3\u003eMp Combined With Ctx Improved The Oxidative Stress Response In Blm-induced Pf Rats\u003c/h3\u003e\n\u003cp\u003eMDA, MPO, SOD and GSH-PX levels are common indicator of oxidation stress. MP could inhibit the increasing of MDA and MPO, the combination of MP and CTX further enhanced the effect of MP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). But SOD and GSH-PX had opposite trends (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This indicated that the combined application of MP and CTX alleviated the oxidative stress response during PF.\u003c/p\u003e\n\u003ch3\u003eMp Combined With Ctx Treatment Regulates The Immune Senescence Process In Fibrotic Rats\u003c/h3\u003e\n\u003cp\u003eOur study shows that the proportion of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells and CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003eT cells in BLM group were increased,which was reduced with MP alone, and was further reduced with the combination of MP and CTX; The proportion of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003eT cells had opposite trends(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Because CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003e was associated with immune senescence, this suggests that the combination of MP and CTX inhibited the activation and senescence of immune cells in the process of PF. In conclusion, the combination of MP and CTX can improve the immune function of T cells and reduce immune senescence.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIPF is a progressive, terminal lung disease associated with aging. Fibroblast activation and increased ECM deposition are the key events in IPF pathogenesis[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Within the lung, alterations in the form, concentration and organization of ECM protein during the aging process lead to a reduction in tissue elasticity and physiological declines in lung function[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].Molecular, cellular and immunological changes occur concomitantly with aging deleteriously affect the capacity of lung tissue to respond to injury and repair damage[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].All these indicate that aging may increased susceptibility to PF. In the present study, we first establish a BLM-induced model of pulmonary fibrosis,and assuming that aging is exacerbated after the onset of PF. By Studies of pathology, electron microscopy, oxidative stress and T cell immunity, we confirmed that BLM-induced PF exacerbated the aging process. The pathological and electron microscopic observations showed that the deposition of collagen and ECM was increased, the expression of α-SMA and type collagen I. was up-regulated, the content of MDA and the activity of MPO were increased, the activities of SOD and GSH-Px were decreased, and the senescence of immune cells was aggravated. Without therapeutic intervention, persistent connective tissue deposition leads to scar formation, eventually leading to pulmonary fibrosis and death.\u003c/p\u003e \u003cp\u003eThe significance of oxidative stress in promoting BLM-induced PF has been reported and plays an important role in the development of PF[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Aging is associated with depletion of antioxidant defense systems[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and it is well documented that antioxidant availability is reduced in IPF. Antioxidants may be useful in IPF treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Treatment of mice with antiaging drugs that selectively eliminate senescent cells has been shown to attenuate BLM-induced fibrosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Immune mechanisms are critical to tissue injury and repair processes. The immune system undergoes age-related changes, and there is evidence of low-grade inflammation, decreased immune response, and increased autoimmunity with aging. Aging is associated with diminished B and T lymphocyte function, decline in antigen presenting cells, and increases in circulating and local levels of cytokines[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Yong Chong et al[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] demonstrated that aging resulted in a marked decrease in the number of CD27\u003csup\u003e+\u003c/sup\u003ememory B cells and a marked increase in the number of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003e nonsensitized B cells. The accumulation of T cells that do not express CD28, a co-stimulatory molecule for T cells is a hallmark of aging and associated with a diminished immune response [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Changes in T-helper cell populations including the down-regulation of CD28 on T cells occur in IPF[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. CD28null T cells have shortened telomeres and represent late-differentiated T cells. Thus, The gradual loss of surface costimulatory molecules CD27 and CD28 is considered to be a phenotypic feature of classical T cell senescence[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur experiments further confirmed that the oxidative stress response occurs in the progression of IPF. Our results also showed that the levels of CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003e T cells and CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells were increased in IPF rats, while the opposite trend was observed for CD27\u003csup\u003e\u0026minus;\u003c/sup\u003eCD28\u003csup\u003e\u0026minus;\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells. Therefore, IPF can accelerate aging. Based on the results, we boldly hypothesize that immunosenescence of PF occurs mainly in CD4\u003csup\u003e+\u003c/sup\u003e cells and not in CD8\u003csup\u003e+\u003c/sup\u003e cells. However, it may be related to the limitations of this experiment, so a large number of experimental datas are still needed to further confirm. We propose that CD4\u003csup\u003e+\u003c/sup\u003e T cells are mainly involved in assisting antigen presentation, promoting antibody secretion, and neutralizing antigens. CD8\u003csup\u003e+\u003c/sup\u003e T cells mainly exert cytotoxic effects and damage antigen-carrying cells. During PF, autoproteins are incorrectly recognized as foreign antigens, and CD8\u003csup\u003e+\u003c/sup\u003e T cells attack alveolar type II epithelial cells carrying antigens. We try to find a new direction for the treatment of IPF from the perspective of aging represented by oxidative stress and immune senescence.\u003c/p\u003e \u003cp\u003eCurrently, there is no cure for IPF.MP combined with CTX was effective in the treatment of systemic lupus nephritis. In addition, this combination can be used to treat gastric antral vasodilatation in systemic sclerosis and sjogren's associated chronic interstitial nephritis [\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Previous clinical studies have confirmed the safety and efficacy of MP combined with CTX in the treatment of PF [\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the treatment of MP and CTX for IPF has always been controversial, and the focus of the controversy is mostly on drug toxicity. Moreover, the side effects of the two drugs were mainly related to the doses. Therefore, According to the literature,we chose a low-dose combination of the two drugs. We used animal experiments to establish the model of IPF. After the occurrence of IPF, MP combined with CTX was used for intervention, the end point was 21 days after treatment and the effect was achieved. The results showed that MP combined with CTX improved the pathological damage of lung tissue, and played a therapeutic role by reducing the oxidative stress response and adjusting cellular immunity. Therefore, we propose a new perspective on the treatment of IPF from the perspective of aging. In clinical practice, we have also achieved good improvement in the treatment of IPF with MP combined with CTX, but large-scale clinical datas have not yet been formed. Therefore, this experiment provides ideas and directions for further clinical research.\u003c/p\u003e \u003cp\u003eAging is associated with immune dysregulation and extracellular matrix changes. The combination of MP and CTX can regulate lung immunity and extracellular matrix, which may be the mechanism of alleviating cell structural damage. However, the specific mechanism needs to be further studied, such as whether the molecular regulation mechanism of MP combined with CTX is of great significance in the treatment of IPF. Our next experiments will be involved.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLimitations:\u003c/h2\u003e \u003cp\u003eDue to the limitation of experimental conditions, we did not evaluate other senescence markers such as CD57, KLRG1, γH2AX and p38, as well as telomere shortening, etc., and it would have been more interesting to add these markers to make our study more fruitful. We cannot further investigate the more detailed mechanisms of IPF in exacerbating aging, such as molecular regulatory mechanisms. In addition, as immune factors are involved in the development and progression of IPF, further studies are needed to determine the deeper relationship between IPF and immune senescence. These are interesting things that we look forward to the next experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIPF is an age-related disease, and aging can promote the occurrence of IPF. Our experiment confirmed that IPF also aggravates the occurrence of aging, proving the interaction between aging and IPF. This study treated IPF from the perspective of aging and showed that the combination of MP and CTX reduced collagen deposition, oxidative stress, and altered the number of fibrotic immunosenescence cells in PF, suggesting that MP combined with CTX may improve PF by inhibiting aging. Targeting aging-related pathways may be a viable therapeutic strategy. This study provides theoretical support for its application in PF and the subsequent development of PF therapeutic drugs.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003ePF, pulmonary fibrosis\u003c/p\u003e\u003cp\u003eIPF, Idiopathic pulmonary fibrosis\u003c/p\u003e\u003cp\u003eBALF, bronchoalveolar lavage fluid\u003c/p\u003e\u003cp\u003eBLM, Bleomycin\u003c/p\u003e\u003cp\u003eMP, methylprednisolone\u003c/p\u003e\u003cp\u003eCTX, cyclophosphamide\u003c/p\u003e\u003cp\u003eH\u0026amp;E, hematoxylin and eosin\u003c/p\u003e\u003cp\u003eEMT,epithelial mesenchymal transition\u003c/p\u003e\u003cp\u003eECM, extracellular matrix\u003c/p\u003e\u003cp\u003eGC, Glucocorticoids\u003c/p\u003e\u003cp\u003eGSH-Px, Glutathione peroxidase\u003c/p\u003e\u003cp\u003eMDA, malondialdehyde\u003c/p\u003e\u003cp\u003eMPO, myeloperoxidase\u003c/p\u003e\u003cp\u003eSOD, superoxide dismutase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were reviewed and approved by the Animal Ethics Committee of Beijing Medconner Biotechnology Co., LTD and conform to National Institutes of Health guidelines for the use of rodents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable. This article does not contain any studies with human participants performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed 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 an open project of the State Key Laboratory of Molecular Developmental Biology (No.2021-MDB-KF-13). And supported by the State Key Laboratory of Molecular Developmental Biology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXX guaranteed the integrity of the entire study; QX and MZ designed the study and literature research; MT and ZR defined the intellectual content; TM and YL performed experiment; ZL and KY collected the data; XH and RM analyzed the data; QX wrote the main manuscript and prepared figures. 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The above indicates that aging can increase the incidence of IPF. So can the occurrence of aging be aggravated after IPF? We examined pathological damage, collagen deposition, oxidative stress and immunosenescence to determine whether bleomycin(BLM)-induced pulmonary fibrosis (PF) accelerates aging in rats. If so, what drugs can inhibit or delay this aging. In clinical studies,the combination of methylprednisolone(MP) and cyclophosphamide(CTX) has shown great benefits in patients with IPF, but its effect on aging resulting from fibrosis is not fully understood. Therefore, we investigated whether MP combined with CTX could delay or inhibit aging in IPF rats. It may provide new targets for the treatment of IPF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003ePF rat models were induced by BLM and treated with MP or MP/CTX combination.Transmission electron microscope, hematoxylin and eosin (H\u0026amp;E) and Masson staining were used to measure the morphology of PF. α-SMA and collagen I levels were examined by western blot and immunohistochemistry. Malondialdehyde(MDA),myeloperoxidase(MPO),lutathione peroxidase(GSH-PX) and superoxide dismutase(SOD) levels were determined using commercial kits.T cells were analyzed with flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We found that pathological damage, collagen deposition, oxidative stress, and T-cell senescence were increased after BLM-induced PF. The combined use of MP and CTX can alleviate pathological damage, reduce oxidative stress response, such as reducing MDA and MPO levels, and increasing SOD and GSH-PX activities. And inhibition of T cell senescence in lung tissue, such as reduction of CD27\u003csup\u003e-\u003c/sup\u003eCD28\u003csup\u003e-\u003c/sup\u003e CD4\u003csup\u003e+ \u003c/sup\u003eT cells in BLM-induced PF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e BLM-induced PF aggravated the occurrence of aging in rats. The combination of MP and CTX can inhibit or delay aging, and thus play a therapeutic role in IPF.These findings provide new insights into the mechanism by which MP and CTX act in combination on IPF.\u003c/p\u003e","manuscriptTitle":"Cyclophosphamide combined with methylprednisolone can play a therapeutic role by delaying the aging of pulmonary fibrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-28 19:41:38","doi":"10.21203/rs.3.rs-2245802/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61f58e23-62ee-4b6b-97db-a10ed22609ca","owner":[],"postedDate":"November 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-27T14:59:31+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-28 19:41:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2245802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2245802","identity":"rs-2245802","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}