Cyclophosphamide combined with methylprednisolone ameliorates bleomycin-induced pulmonary fibrosis in rats through immunomodulatory mechanisms

doi:10.21203/rs.3.rs-1997090/v1

Download PDF

Research Article

Cyclophosphamide combined with methylprednisolone ameliorates bleomycin-induced pulmonary fibrosis in rats through immunomodulatory mechanisms

https://doi.org/10.21203/rs.3.rs-1997090/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Idiopathic pulmonary fibrosis(IPF) is a distressing lung disorder with poor prognosis and high mortality rates.Immune factors are currently recognized as pathogenic agents throughout the onset of IPF.In clinical studies,the combination of methylprednisolone (MP) and cyclophosphamide (CTX) has great benefits for patients with IPF,but the immune mechanism of improving IPF is not clear.

Results

The lung inflammation and fibrosis model was established by intratracheal instillation of bleomycin (BLM).Pulmonary fibrosis was observed in rats on day 7 after BLM injection. With prolonged fibrosis,we can see that the expression of neutrophils and T lymphocytes was out of control.Our data suggests that immune disorders run through the whole process of pulmonary fibrosis both in the early stage(day 14)and in the advanced stage(day 28).The inflammation and immune disorders were better than before after intervention with medication.

Conclusions

The combination of MP and CTX can alleviate IPF by reducing inflammation,and improving T cell immunity.So CTX combined with MP can modulate immune disorders,which may be an effective anti-fibrosis drug in the treatment of clinical IPF patients.But we should pay attention to dosage and use course of the treatment,in order to prevent the occurrence of toxic side effects.

Idiopathic pulmonary fibrosis

Methylprednisolone and cyclophosphamide combination

T cell immunity

IPF is a distressing lung disorder with poor prognosis and high mortality rates[1].It was characterized pathologically by progressive scarring of lung tissue,increased fibroblast proliferation,excessive accumulation of extracellular matrix(ECM),exacerbation of lung inflammation,diffused destruction of alveolar structure and remodeling of the lung architecture[2].The pathogenesis of IPF is divided into two stages:early inflammatory response and late fibrogenesis. The inflammatory phase is the initial response of the body and is mediated by a variety of inflammatory cells. Multiple pro-inflammatory and fibrogenic cytokines and growth factors were found at 7 and 14 days[3, 4],lung fibroblasts began to form at 14 days[5]and collagen deposition was significantly increased[6].A number of previous studies have shown that the activation and expansion of T cells in IPF patients play an important role in the progression of the disease. The role of the adaptive immune system in this process has been difficult to understand and highly controversial[7].T lymphocytes were represented by CD4,CD8 and regulatory T cells(Tregs),a subpopulation of T cells.The cytokine response of CD4⁺ cells has been examined in a number of studies of IPF patients.CD8⁺ cells were positively correlated with the severity of disease[8]and it also plays an important role in aseptic inflammatory repair/fibrosis effector factors[9].The lower of CD4⁺/CD8⁺and the more CD8⁺ cells,the worse the response to treatment[10].Tregs have immunosuppressive effects that maintain immune tolerance and immune response homeostasis[11].In the BLM-induced pulmonary fibrosis mouse model,Tregs can accumulate in large quantities in lung tissue and worsen the disease.On the contrary,Tregs can also reduce pulmonary fibrosis by inhibiting inflammation and regulating the response of CD4⁺ helper T(Th)cells.Although it has been accepted that disturbed immune homeostasis plays a significant role in the pathogenesis and/or progression of IPF,the underlying mechanisms remains unclear[12].IPF patients have few effective treatment options beyond lung transplantation[13].So far,there are no effective drugs to treat the disease,it is crucial to find possible therapeutic targets to treat IPF.

In autoimmune diseases,CTX is often used to inhibit overactive immune cells[14].The pathogenesis of IPF involves abnormalities in the complex pathways of the immune system,so,CTX may have therapeutic effects on IPF.It was reported that CTX intravenously injection into patients with PF improved pulmonary function[15].While some reports suggested that CTX may cause PF in patients[16, 17]or animals[18].CTX is directly toxic to lung tissue and the strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo[19]. The role of CTX in PF is controversial.

Glucocorticoids(GC) are commonly used drugs for PF[20]. GC was found to alleviate PF[21]. MP could significantly attenuated lung injury,and reduced proinflammatory cytokine production and neutrophil infiltration into alveoli[22].In addition,CTX and MP were combined to treat a case of pulmonary interstitial fibrosis[23].High dose prednisolone pulsed CTX could improve or stabilize lung function in patients with severe systemic sclerosing lung disease[24].A clinical study indicated that the combination of the CTX and glucocorticoids were beneficial to PF[25].Moreover, combination of MP and CTX was considered safe in PF[26].

In this study,we divided it into two treatment nodes:the early stage(14 days)and the late stage(28 days)after the establishment of pulmonary fibrosis. We used neutrophils, CD3⁺, CD4⁺, CD8⁺ and CD4⁺CD25⁺Foxp3⁺ cells,which on behalf of Tregs as representatives for immune cell experiments. The aim was to investigate the immunomodulatory effects of CTX combined with MP on different stages of BLM-induced PF in rats,and to explore its possible therapeutic effects.

The PF model on rats was successfully established

7 days after intratracheal injection of BLM,the PF model was successfully established through gross observation and pathological staining of lung specimens(Fig. 1A-F).

CTX combined with MP can reduce damage and collagen deposition in BLM-induced PF

In BLM treated rats of the early stage,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.In BLM + MP group,alveolar structure was improved,alveolar septal thickening and inflammatory cell infiltration were reduced.The combined treatment with MP and CTX further improved lung injury.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.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.These improvements were further enhanced by MP and CTX combination treatment(Fig. 2A-K).HE pathological staining showed that the degree of advanced fibrosis was more obviously than that of early stage.CTX combined MP reduced the lung inflammatory damage,the inflammatory score can demonstrates this.Masson staining showed that collagen deposition increased significantly after BLM induction.Compared with the early stage,the degree of fibrosis was also significantly aggravated.The lung tissue structure was clear,collagen deposition was reduced after the treatment with CTX combined MP,semi-quantitative analysis through Image J can be used for evaluation(Fig. 3A-H).

CTX combined with MP improved immune response in rats PF

CTX combined with MP reduced the total number of granulocytes and neutrophils in BALF,and alleviated the inflammatory response(Fig. 4A-F).

In the early stage, BLM inhibited the immune response of T cells, and the expression of CD3⁺, CD4⁺ cells and CD4/CD8 decreased significantly, while the expression of CD8⁺ cells increased. After MP intervention, CD3⁺, CD4⁺ cells and CD4/CD8 ratio increased, while CD8⁺ cells decreased. CTX combined with MP intervention further strengthened the effect of MP. Thus, CTX combined with MP enhanced the immunomodulatory effects of T cells (Fig. 5A-P).

In the advanced stage,the expression of CD3⁺,CD4⁺ cells and CD4/CD8 showed the same trend as in the early stage.The number of Tregs decreased after BLM induction,MP can up-regulate the number of Tregs,inhibit inflammation and regulate the response of Th cells.The treatment of CTX combined MP had a more obvious response to immune regulation and indirectly alleviated pulmonary fibrosis.These results suggest that MP can inhibit immune overactivation,but the effect was limited.CTX combined with MP can further regulate the immune state (Fig. 6A-U).

In view of the above treatment of MP and CTX combined with MP on BLM-induced fibrosis rats,the immunoregulatory effect on T cells was summarized as shown in Table 1.

Table 1

+ represents significant comparison with the BLM group,- represents meaningless compared with the BLM group,* represents statistical difference between the two drug therapy groups,ns means there is no statistical difference between the two drug therapy groups
Drug therapy			CD3⁺T		CD4⁺T		CD8⁺T		CD4/CD8⁺
BLM-MP	+		-		-		+		-
BLM-MP + CTX	++		+		+		++		+
Comparison in two groups	*		ns		*		*		*
A: early stages (14 day) Drug therapy	Treg	CD3⁺T		CD4⁺T		CD8⁺T		CD4/CD8⁺
BLM-MP	-	+		-		+		-
BLM-MP + CTX	+	++		+		++		+
Comparison in two groups	ns	*		ns		*		*
B: late stage(28 day)

Although inhibition of inflammatory response is considered an important clinical strategy for the treatment of IPF[27], the role of inflammatory response in pulmonary fibrosis has been controversial. Recent studies have proved that anti-inflammatory treatment is not effective for all patients with IPF. Patients with IPF have abundant lymphocytic structures, mainly composed of CD3 and DCS, which promote fibroblast differentiation [28]. The role of immune factors in fibrosis has attracted increasing attention. In the present study, we detected a significant increase in inflammatory cells in the early BALF of BLM-treated rats, especially neutrophils, which are known to be the major inflammatory cells. This is consistent with the conclusion in previous studies that early fibrosis is dominated by inflammation. By analyzing T lymphocytes, we confirmed that BLM inhibited the expression of CD3⁺, CD4⁺ cells and significantly increased the expression of CD8⁺ cells. This suggests that immune factors are also present in the early stages of fibrosis. And there is a link between inflammation and immunity. The immune function is further suppressed in the late stage of fibrosis, in addition to the decrease of CD3⁺ and CD4⁺ cells, the number of Tregs also decreased significantly, while CD8⁺ cells had the opposite effect.So,immune disorder runs through the whole process of fibrosis.

At present, there is no unified and effective treatment for IPF. New drugs and technologies are still lacking in a large number of prospective studies. New use of old drugs as a drug development strategy has become a new direction of drug development. This can shorten the development time and reduce the cost and risk of research and development. In addition, pharmacokinetic and safety data are more detailed and can be quickly entered into clinical evaluation.

GC is the most important regulating hormone in stress response.In the primary lung fibroblasts and endothelial cells,GC affected fibroblasts through pulmonary TGF-β signaling[29],and was the main and basic drug in the treatment of IPF[12]. GC may inhibit inflammatory and fibrotic processes by anti-inflammatory and inhibiting the synthesis of lymphokines[30].Early application of GC can reduce alveolar inflammation,delay the development of pulmonary fibrosis,improve symptoms or stop the progression of symptoms,then improve the lung function.However,the clinical application of GC was limited.Long-term application of GC was easy to cause pulmonary bacterial and fungal infection,and can cause hyperglycemia,peptic ulcer,induce or aggravate infection,severe osteoporosis,even adrenal cortical dysfunction.As a result,there was considerable debate in the medical community about whether patients with IPF should use hormone therapy for a long time.MP is the most commonly used representative drug in GC and is often used in clinical practice.Therefore,MP was selected for intervention in our experiment.

CTX is a highly potent immunosuppressive agent that has been shown to be effective in inducing and maintaining remission in a range of autoimmune and inflammatory diseases. Its anti-inflammatory pathway is different from GC and can be used in patients who cannot tolerate GC or do not respond to GC. During the process of GC withdrawal, the condition sometimes went back and forth, and the DC could be smoothly decreased after the addition of CTX. Complete nonspecific interstitial pneumonia/fibrosis recovered completely with the addition of CTX to corticosteroids, which are commonly used in patients with collagen vasculopathy-related NSIP[31]. We observed a trend toward improvement: after 1 year of treatment, 57% of patients in the combination group were improved or stable compared with 32% in the prednisolone group. CTX + prednisolone group had obvious advantages. These suggest that adding CTX to corticosteroid therapy may be a useful treatment. However, CTX has a range of serious toxicities that make its use problematic and should only be prescribed by specialists. Most patients experience nausea and thinning hair. In addition, CTX can lead to hemorrhagic cystitis, bone marrow suppression, an associated increased risk of opportunistic infections, and reactivation of latent infections such as tuberculosis. Studies have shown that among IPF patients with rapid exacerbation of respiratory failure, patients treated with intravenous high-dose CTX have a trend of lower survival [32]. However, there is a lack of strong clinical data for any of the above conclusions or ideas, and most studies are retrospective studies with small patient populations. Reports on CTX use are limited to a few anecdotal reports and two controlled studies[33, 34]. Therefore, the use of CTX for IPF requires further exploration of appropriate dosing and usage, alone or in combination with other therapies, which makes it therapeutic without side effects. We further defined the therapeutic effect of CTX on IPF by its effect on BLM-induced fibrotic rat T lymphocytes. A review of the extensive literature found that the long-term toxicity of CTX was related to the cumulative dose, mode of administration, and route of administration. Studies have shown that low-dose CTX can improve survival in patients with[35] metastatic colorectal cancer. It has also been shown that low-dose CTX can promote immunity and enhance immune cell function. On the contrary, high doses of CTX can inhibit DNA replication, promote apoptosis, reduce immune cell function, and make the body in an immunosuppressive state. Combined with the literature, we used a small dose (8mg/kg) of intravenous injection for intervention, and divided the treatment into early and late stages. The modulatory effect of CTX combined with MP on immune function in rats with pulmonary fibrosis was further confirmed by its effects on neutrophils and T lymphocytes.

Our study showed that MP alone reduced neutrophil expression in the early stages, which was further reduced when combined with CTX. T cell function was also significantly higher in BLM group. Due to the interaction between neutrophils and B cells, CTX combined with MP not only inhibits inflammatory response, but also regulates the immune response of T cells and B cells, suggesting that CTX combined with MP has better therapeutic effect than MP alone. The overall changes of T cells in the late stage were the same as those in the early stage. However, they have different effects on CD3, CD4 and CD8, and also improve their immune function by increasing the number of Tregs. This further confirmed that the immunomodulatory effect of CTX combined with MP was superior to MP alone. CTX combined with MP can alleviate pulmonary fibrosis. This confirms that the pathogenesis and immune mechanisms of the different stages of fibrosis are different. CTX combined with MP therapy has different immunomodulatory mechanisms in different stages of fibrosis.

Limitations of this study:1.The detection of neutrophils in alveolar lavage fluid was performed only in the early stage of pulmonary fibrosis,but not in the late stage,which could not further clarify the role of inflammation in late fibrosis.2.Due to the limitations of reagent purchase and experimental conditions,Tregs were detected only in the late stage,not in the early stage,and only the number of cells was detected,so phenotype and microenvironment could not be detected.The regulatory effect of CTX on immune function needs to be further studied.

CTX combined with MP promotes immune function in BLM-induced pulmonary fibrosis rats. Therefore, combined use of CTX and MP can improve the therapeutic effect of pulmonary fibrosis by improving immune function.

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 12 h-light/12 h-dark cycles. Rats were divided into four groups (n = 10): (i) blank group, (ii) 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% Pentobarbital Sodium (45 mg/kg).Then,they were fixed on the rat plate and disinfected with conventional methods.Rats were injected with a special needle for tracheal spray from the left corner of the mouth of the rats and placed near the trachea,BLM in normal saline solution (5mg/kg,0.05mL)was injected into the bifurcation,and equal volume normal saline(0.05mL)was injected into the trachea of the sham group.The needle stayed in the lung for 6s and massaged rats to make the drug evenly distributed in the lungs.MP (3mg/kg) was injected intraperitoneally every day in BLM + MP group,and MP(3mg/kg)and CTX(8mg/kg) were injected intraperitoneally daily in BLM + MP + CTX group for 21 days.The BLM group and blank group were injected with the same amount of normal saline every day.

Pathological staining

The pulmonary tissues of rats in each group 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)staining and Masson’s trichrome staining were performed in according with the manufacturer’s instructions(Nanjing Jiancheng Bioengineering Institute,China).

Flow cytometry

Isolated single cells from rat BALF were stained with CD3(#201414,BioLegend, San Diego,CA,USA),CD4(#201509,BioLegend),CD8(#200610,BioLegend),CD25 (#202114, BioLegend),FOXP3(ab215206,Abcam)antibodies for 30 min at 4°C.Flow cytometry was performed with a Beckman CytoFLEX device and FlowJo software was used for analysis.

Statistical analysis

The measurement data were expressed as mean ± standard deviation (SD).For multiple group comparison,one-way analysis of variance(ANOVA)followed by Tukey’s post hoc test was performed using Graphpad Prism.Differences at p < 0.05 were considered statistically significant.

BALF, bronchoalveolar lavage fluid

BLM, Bleomycin

CTX, cyclophosphamide

MP, methylprednisolone

IPF, Idiopathic pulmonary fibrosis

PF, pulmonary fibrosis

Tregs,Regulatory T cells

Th,CD4⁺helper T cells

DCS,Dendritic cells

CTL,CD8⁺cytotoxic T cells

ANOVA,One-way analysis of variance

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.

Author Contributions

XX guaranteed the integrity of the entire study; QX and WZ designed the study and literature research; MT defined the intellectual content; MZ and YL performed experiment; ZLand YC collected the data; 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 paper

Yi cheng He,Fei Li,Chao Zhang,et al.Therapeutic Effects of the Bcl-2 Inhibitor on Bleomycin-induced Pulmonary Fibrosis in Mice.Frontiers in Molecular Biosciences (2021)8:645–846.doi:10.3389/fmolb.2021;645-846.
Todd,N.W,Luzina,I.G,and Atamas,S.P.Molecular and Cellular Mechanisms of Pulmonary Fibrosis.Fibrogenesis Tissue Repair.2012;5(1),11.doi:10.1186/1755-1536-5-11.
Kendall,R.T.,and Feghali-Bostwick,C.A. Fibroblasts in Fibrosis:Novel Roles and Mediators.Front.Pharmacol.2014;5,123.doi:10.3389/fphar.2014.00123.
Li,G.,Jin,F.,Du,J.,He,Q.,Yang,B.,and Luo,P.Macrophage-secreted TSLP and MMP9 Promote Bleomycin-Induced Pulmonary Fibrosis.Toxicol.Appl.Pharmacol.2019;366:10–16. doi:10.1016/j.taap.2019.01.011
Yicheng He,Fei Li 1,Chao Zhang,et al.Therapeutic Effects of the Bcl-2 Inhibitor on Bleomycin-induced Pulmonary Fibrosis in Mice.Frontiers in Molecular Biocsiences. 2021;10(7).doi:10.3389/fmolb.2021.645846
Peng,R.,Sridhar,S.,Tyagi,G.,Phillips,J.E.,Garrido,R.,Harris,P.,et al..Bleomycin Induces Molecular Changes Directly Relevant to Idiopathic Pulmonary Fibrosis:a Model for "active" Disease.2013;PLoS One 8 (4),e59348. doi:10.1371/journal.pone.0059348.
Meagan W.Moore and Erica L.Herzog.Regulatory T Cells in Idiopathic Pulmonary Fibrosis Too Much of a Good Thing?The American Journal of Pathology.2016;186(8):1978–81.
Atamas SP,Yurovsky VV,Wise R,et al.Production of type 2 cytokines by CD8⁺lung cells is associated with greater decline in pulmonary function in patients with systemic sclerosis.Arthritis Rheum.1999;42(6):1168–78.
Brodeur TY,Robidoux TE,Weinstein JS,et al.IL-21 Promotes Pulmonary Fibrosis through the Induction of Profibrotic CD8⁺T Cells.Journal of immunology (Baltimore,Md.: 1950).2015;195(11):5251-60.
Fireman E,Vardinon N, Burke M, et al.Predictive value of response to treatment of T-lymphocyte subpopulations in idiopathic pulmonary fibrosis.Eur Respir J.1998;11(3):706–11.
Sakaguchi S.Naturally arising CD4⁺ regulatory T cells for immunologic self-tolerance and negative control of immune responses.Annu Rev Immunol.2004;22:531–62.
Ziliang Hou,Qiao Ye,Meihua Qiu,et al.Increased activated regulatory T cells proportion correlate with the severity of idiopathic pulmonary fibrosis.Respiratory Research.2017;18:170.DOI 10.1186/s12931-017-0653-3.
Algar FJ,Espinosa D,Moreno P,Illana J,Cerezo F,Alvarez A,et al.Results of lung transplantation in idiopathic pulmonary fibrosis patients.Transplant Proc.2010;42:3211–3.
Beretta L,Caronni M,Raimondi M,et al.Oral cyclophosphamide improves pulmonary function in scleroderma patients with fibrosing alveolitis: experience in one centre.Clin Rheumatol.2007;26(2):168–72.
Baughman,R.P.and E.E.Lower.Use of intermittent, intravenous cyclophosphamide for idiopathic pulmonary fibrosis.Chest.1992;102(4):1090–4.
Segura A,Yuste A,Cercos A,et al.Pulmonary fibrosis induced by cyclophosphamide.Ann Pharmacother.2001;35(7–8):894–7.
Abdel Karim,F.W.,et al.Pulmonary fibrosis after prolonged treatment with low-dose cyclophosphamide.A case report.Oncology.1983;40(3):174–6.
Lee,Y.C. and J.P. Kehrer,Increased pulmonary collagen synthesis in mice treated with cyclophosphamide.Drug Chem Toxicol.1985;8(6):503–12.
Hoyt,D.G. and Lazo J.S.Acute pneumocyte injury,poly(ADP-ribose) polymerase activity,and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide.Biochem Pharmacol.1994;48(9):1757–65.
Krinsky,G.Low-dose corticosteroid therapy for idiopathic pulmonary fibrosis. Radiology,1994;192(2):582.
Webb,D.R.and Currie G.D.Pulmonary fibrosis masking polymyositis.Remission with corticosteroid therapy.Jama.1972;222(9):1146–9.
Song,L.C.,et al.,Effects of different corticosteroid doses and durations on smoke inhalation-induced acute lung injury and pulmonary fibrosis in the rat.Int Immunopharmacol.2019;71:392–403.
Ziesche R,Hofbauer E,Wittmann K,Petkov V,Block LH.A preliminary study of long-term treatment with interferon gamma-1b and low-dose prednisolone in patients with idiopathic pulmonary fibrosis. New England Journal of Medicine.1999;341(17):1264–9.
Fort,J.G,Scovern H,and Abruzzo JL.Intravenous cyclophosphamide and methylprednisolone for the treatment of bronchiolitis obliterans and interstitial fibrosis associated with crysotherapy.J Rheumatol.1988;5(5):850–4.
Wanchu A,Suryanaryana BS,Sharma S,Sharma A,Bambery P.High-dose prednisolone and bolus cyclophosphamide in interstitial lung disease associated with systemic sclerosis:a prospective open study.Int J Rheum Dis.2009;12(3):239–42.
Naccache JM,Jouneau S,Didier M,et al.Cyclophosphamide added to glucocorticoids in acute exacerbation of idiopathic pulmonary fibrosis (EXAFIP):a randomised,double-blind, placebo-controlled,phase 3 trial.Lancet Respir Med.2022;10(1):26–34.
Hara A,Sakamoto N,Ishimatsu Y,et al.S100A9 in BALF Is a Candidate Biomarker of Idiopathic Pulmonary Fibrosis. Respir. Med.2012;106(4),571–580.
Vuga LJ,Tedrow JR,Pandit KV,Tan J,Kass DJ,Xue J,et al.C-X-C motif chemokine 13(CXCL13)is a prognostic biomarker of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med.2014;189:966–74.
Schwartze JT, Becker S, Sakkas E, et al.Glucocorticoids recruit Tgfbr3 and Smad1 to shift trans forming growth factor-β signaling from the Tgfbr1/Smad2/3 axis to the Aevrl1/Smad1 axis in lung fibroblasts.J Biol Chem.2014;289(6):3262–75.
Wahl,S.M.Altman,L.C,and Rosenstreich,D.L.Inhibition of in vitro lymphokinc synthesis by glucocorticosteroids.J.Immun. 1975;115:476–81.
ki Nanki,Jiro Fujita,Yasufumi Yamaji,et al.Nonspecific Interstitial Pneumonia/Fibrosis Completely Recovered by Adding CTX to Corticosteroids.Internal Medicine.2002;41(10):867–70.
Keishi Oda,Kazuhiro Yatera,Yoshihisa Fujino,et al.Efficacy of concurrent treatments in idiopathic pulmonary fibrosis patients with a rapid progression of respiratory failure: an analysis of a national administrative database in Japan.Oda et al.BMC Pulmonary Medicine.2016;16:91.
Kolb M,Kirschner J,Riedel W,et al.CTX pulse therapy in idiopathic pulmonary fibrosis.Eur Respir J.1998;12:1409–14.
Hubbard R,Johnston I,Britton J.Survival in patients with cryptogenic fibrosing alveolitis:a population-based cohort study.Chest.1998;113:396–400.
Scurr MJ,Pembroke T,Bloom A,et al.Low-dose CTX induces anti-tumor T-cell responses which associate with survival in metastatic colorectal cancer.Clin Cancer Res.2017;23(22):6771–80.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Cyclophosphamide combined with methylprednisolone ameliorates bleomycin-induced pulmonary fibrosis in rats through immunomodulatory mechanisms

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Introduction

Results

Discussion

Conclusion

Methods

Animals and ethics statements

Animal modeling and treatment

Pathological staining

Flow cytometry

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1