Abstract
Background: Pulmonary fibrosis is a chronic progressive disease with limited therapeutic options and inflammatory cytokines play important roles in the pathogenesis of pulmonary fibrosis.
Material and method: Here, we investigated the changes of TGF-β1, IL-8, and IL-17 in the serum of bleomycin-A5-induced rats model of pulmonary fibrosis. 120 healthy male Wistar rats were randomly divided into three groups, the control group (n = 30), the model group (n = 45) and the dexamethasone (DEX) group (n = 45). The rats of both model group and DEX group were injected with Bleomycin-A5 (5 mg/kg) through tracheofistulization to induce pulmonary fibrosis, while the rats of the control group were injected with equivalent physiological saline. After operation, DEX (4 mg/kg) was given to the DEX group rats intraperitoneally once a day. Equivalent saline was administered to rats of both the control group and the model group.
Results: On the 1st, 14th, and 28th day after operation, pathological changes of the lung tissues, and the levels of serum IL-8, TGF-β1, and IL-17 were measured. The concentrations of serum TGF-β1, IL-8, and IL-17 were significantly increased after bleomycin-A5 treatment, especially on the 14th day (p < .01). There was no significant difference between model group and DEX group in the serum level of TGF-β1 and IL-8, but DEX treatment significantly reduce serum IL-17 level (p < .01).
Conclusions: DEX protect bleomycin-A5-induced pulmonary fibrosis in rats through reduced the level of IL-17 in serum.
Introduction
It is believed that pulmonary fibrosis is an epithelial-fibroblastic disease, which is a chronic, progressive, and irreversible condition that occurs in many clinical situations [Citation1,Citation2]. Pulmonary fibrosis is of the induced by a strong inflammatory response to lung injury [Citation3], which is initiated by various immune cells—including neutrophils and eosinophils—that infiltrate lung tissue. Epithelial-mesenchymal transition proteins (collagen and fibronectin) and fibrotic cytokines (transforming growth factor-β and insulin-like growth factors) are critical for the pathogenesis of pulmonary fibrosis [Citation4,Citation5]. The fibroblast and myofibroblast foci secrete excessive amounts of extracellular matrix, mainly collagens, resulting in scarring and destruction of the lung architecture [Citation6]. In the early injury or inflammation, TGF-β promotes the development of inflammation, and increases activity at sites of inflammation, and induces the proliferation of the fibroblasts, leading to severe pulmonary fibrosis [Citation7].
Several reports have showed inflammatory cytokines were associated with the pathologic process of pulmonary fibrosis [Citation8,Citation9], such as tumor necrosis factor-α and IL-1β [Citation10,Citation11]. In recent years, more and more researches have suggested that cytokines, especially interleukins played an important role in the occurrence and development of pulmonary fibrosis [Citation12,Citation13].
Current therapeutic strategies are primarily aimed at controlling the inflammatory processes, often through the oral administration of glucocorticosteroids, which have been the first line of therapy for pulmonary fibrosis [Citation14]. Bleomycin-A5 is widely used to induce pulmonary fibrosis in animal models [Citation15,Citation16]. The intratracheal administration of bleomycin-A5 induces significant inflammatory and fibrosis in murine lungs. In addition, induction of pulmonary fibrosis by bleomycin-A5 is easy to perform and highly reproducible [Citation17].
In the current study, we established the bleomycin-A5-induced pulmonary fibrosis model in rats and analyzed the serum level of inflammatory cytokines, TGF-β1, IL-8 and IL-17 in response to DEX treatment.
Materials and methods
Rats
120 healthy male Wistar rats, weighing from 180–200 g, were purchased from the Lab Animal Center of Shandong University and maintained under specific pathogen-free conditions. They were housed in an air-conditioned animal room (23 °C ± 1 °C; relative humidity 50% ± 10%), fed according to a laboratory diet, and given distilled water. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Main reagents
The bleomycin-A5 hydrochloride, 8 mg per each were purchased from Tianjin Taihe Pharmacy co ltd (cat No.: 070502, Tianjin, China). The Dexamethasone Sodium Phosphate Injection (5 mg per each) wad purchased from Shanghai Tongyong Pharmacy co ltd (Shanghai, China). The ELISA reagent kit was provided by USCN life corporation (Model No. E0063r, Shanghai, China).
Establishment of rats model
The pulmonary interstitial fibrosis rat models were established with the classic method. 120 healthy male Wistar rats were randomly divided into three groups, the control group (n = 30), the model group (n = 45) and the DEX group (n = 45). Intraperitoneally inject the Wistar rats with 10% chloral hydrate (4.5 ml/kg), and after anaethesia fix the rats supine on 15° tilting rat boards. After general skin preserving and antisepsis, cut the medial cervical incision and separate every layer of tissues to expose the trachea. In the model group and DEX group, puncture and inject into the trachea of the rats with about 0.18–0.20 ml bleomycin-A5 Hydrochloride saline dissolution (saline dissolved) with 5 mg/kg, while inject into the trachea of rats in the control with equivalent physiological saline. After the injection quickly intravenously push 0.3–0.5 ml air into trachea and immediately stand the boards, making the rats upright. Circumgyrate and mildly vibrate the boards to make the medicines spreading well in the rats lungs. Suture the skin incision. All the above procedures are all under strict asepsis. After the operation, give the DEX group rats dexamethasone sodium phosphate injection (4 mg/kg) intraperitoneally once a day and equivalent saline to rats of both the control group and the model group once a day.
Animal models treatment
After models preparation, samples were collected on the 1st, 14th 28th day. 10, 15, and 15 rats in control group, the model group and the DEX group were sacrificed every time, respectively. Leave the rats fasting 24 h before collection of serum samples while water is unforbidden. One hour after the last medication, intraperitoneally inject the rats with 10% chloral hydrate (4.5 ml/kg). Fix them supine onto the boards after anesthesia, expose the inferior vena cava to get the blood samples with injectors. Use the centrifuge to separate serum from the samples and store in the fridge at −80 °C. Get the right lungs, fix them with 10% formalin dissolution, routine paraffin embedding, make sections and make HE and Masson staining.
Alveolar catarrh and pulmonary fibrosis score
Alveolar catarrh and pulmonary fibrosis were classified according to the methods reported by Szapiel SV et al [Citation18]. Alveolar catarrh is classified with 4 grades. Grade zero: no alveolar catarrh; GradeI: mild alveolar catarrh: monocytes infiltration, making the alveolar septum broader, limited to local part and parapleural area,; under 20% of the whole lungs involved; alveolar structure still intact; Grade II: moderate alveolar catarrh, 20–50% of the whole lungs are involved; more serious in the parapleural area; Grade III: severe alveolar catarrh; over 50% involved; monocytes were seen in alveolar space occasionally and bleeding caused consolidation. Pulmonary fibrosis is classified with four grades as well. Grade zero: no fibrosis; GradeI:mild fibrosis; under 20% involved; pleural and subpleural pulmonary parenchyma; abnormal alveolar structure; Grade II: 20–50% involved; extending from pleural but still local; Grade III: diffuse pulmonary fibrosis; over 50% involved; fused pulmonary damage together with general abnormal pulmonary parenchyma structure. Transform ranked data to measured data, that is to say grade zero is equal to zero points, gradeIequal to 1 point, grade II equal to 2 points and grade III equal to 3 points.
Measurement of serum TGF-β1, IL-8, and IL-17
The serum levels of TGF-β1, IL-8, and IL-17 were measured by ELISA kit according to manufacturer’s protocol.
Statistical treatment
The difference of normally distributed variables between two groups was evaluated by t-test. Analysis of variance was used to compare normally distributed variables between three or more groups, with SNK-q test for multiple comparisons. The relationship between TGF-β1, IL-8 and IL-17 were determined using Pearson correlation coefficient analysis. Statistical measurements were made using the statistical software SPSS (Version 19.0). p < .05 was considered statistically significant.
Results
Observation of lung samples
The pulmonary fibrosis was successfully established in the model group and the DEX group. On the 14th day after operation, patent hyperemia dots, focal hyperemia or bleeding were seen in the Lungs of model group; on the 28th day lungs atrophy and deformation were observed in most of rats. Similar changes were observed among treatment group. While, lungs of rats the control group were rose pink and smooth on surface during the experiment period.
Lung samples were harvested and HE stained. The alveolar catarrh and pulmonary fibrosis were evaluated under light scope and graded. Compared with model group, the scores of alveolar catarrh and pulmonary fibrosis were not significantly different between model group and DEX group (). There was no sign of alveolar catarrh and pulmonary fibrosis in the control group.
Figure 1. Comparison of scores of alveolar catarrh (A) pulmonary fibrosis (B) in the 3 groups. The score of alveolar catarrh and pulmonary fibrosis was zero in the control group, and there was no significant difference between model group and DEX group.
TGF-β1, IL-8 and IL-17 levels in serum
The concentrations of TGF-β1, IL-8 and IL-17 were analyzed in the 3 groups at different time and the data were shown in . As was shown, the concentrations of TGF-β1, IL-8 and IL-17 in serum all increased at 14th day and then slightly decreased in model group and DEX group, and were significantly higher than those of the control group, especially on the 14th day.
Table 1. Serum levels of TGF-β1, IL-8, and IL-17 in the 3 groups.
As was shown in , there was no significant difference between model group and DEX group in the serum concentration of TGF-β1, IL-8 and IL-17 at any times points, while the serum level of cytokines in model group and DEX group were higher than those of control group, and the difference was of statistical significance (p < .05). Compared with control group, the serum level of IL-17 was lower in DEX group at 14th and 28th days, but there was no significant difference in the serum levels of TGF-β1 and IL-8. Intriguingly, we found IL-17 was positively correlated with TGF-β1 (r = 0.849, p < .001, ) and IL-8 (r = 0.834, p < .001).
Figure 2. The serum levels of TGF-β1 (A), IL-8 (B), and IL-17 (C) at different time points. *p < .05.
Figure 3. The serum level of IL-17 was significantly correlated with TGF-β1 (A) and IL-8 (B).
Discussion
Pulmonary fibrosis is the common pathological change, which is benign but has relatively bad prognosis. It will end up with respiratory failure to death because of alveolar-capillary functional unit loss little by little. Various factors are associated with pulmonary fibrosis, including genetic factors, environmental exposure, smoking, gastroesophageal reflux disease, diabetes mellitus, infectious agents, and commonly prescribed drugs, such as bleomycin-A5 [Citation3]. Although pulmonary fibrosis has been investigated for decades, the exact molecular mechanism is still unclear to us. In recent years, more and more researches have suggested that the cytokines played an important role in pulmonary fibrosis [Citation13,Citation19].
In the current study, we analyzed serum level of 3 important inflammatory cytokines in bleomycin-A5-induced rats pulmonary fibrosis model. Compared with control group, the serum level of TGF-β1, IL-8 and IL-17 significantly increased in rats with pulmonary fibrosis. DEX has been used to treat pulmonary fibrosis for decades. In the current study, we found there was no significant difference between the cathrra and pulmonary fibrosis scores in the model group and the DEX group. While DEX (4 mg/kg) decreased the serum IL-17 of pulmonary fibrosis rats compared with model group, suggesting DEX inhibited immune response of rats in the DEX group.
IL-17 has been discovered as a pre-inflammation cytokine, a homodimer protein, produced by activated peripheral T cells. Researchers suggest that IL-17 is mainly derived from activated human CD4 + T memory cells, and then is also found that IL-17 can also be produced by TCR + CD4-CD8- thymus cells, epithelial cells and endothelial cells [Citation14,Citation20].
Studies show that IL-17 is the most important effector produced by Th17 cells. IL-17 can induce the expressions of other inflammatory cytokines (e.g., TNF, IL-6 and chemokines) to mediate the local infiltration of inflammatory cells and induce tissue damage [Citation21]. As an important agent between T lymphocytes and neutrophils, IL-17 takes part in the recruitment of neutrophils in the airway under inflammation condition, by stimulating the bronchial epithelial, capillary endothelial cells and fibroblasts synthesizing and releasing adherence factors (IL-8, GCP-2, MIP-2 and biological factors, etc). It has been proved in vitro that IL-17 could promote human respiratory epithelial cells and venous endothelial cells secreting NCF-A and follows the time-concentration dependence [Citation22]. IL-17 plays an important role in systemic anti-infection immunity, tumor immunology, transplantation immunity and autoimmune disorders. In recent years researches about IL-17 have increased in respiratory inflammations, chronic obstructive pulmonary disease, bronchial asthma and all respiratory disorders, but relatively less in the pathogenesis of pulmonary fibrosis home and abroad. Bleomycin-A5-induced pulmonary fibrosis can be divided into two stages: early stage with inflammation and later stage with fibrosis. According to the above research, we can suppose that IL-17 plays a role in the early stage of pulmonary fibrosis pathogenesis [Citation23].
To summary, in the current study we show that DEX could reduce serum level of IL-17 in rats of bleomycin-A5-induced pulmonary fibrosis.
Acknowledgements
This work has no funding.
Disclosure statement
The authors have declared none conflict of interests.
References
- Kaur G, Narang RK, Rath G, et al. Advances in pulmonary delivery of nanoparticles. Artif Cells Nanomed Biotechnol. 2011;2:75–96.
- Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest. 2007;117:524–529.
- Zisman DA, Keane MP, Belperio JA, et al. Pulmonary fibrosis. Methods Mol Med. 2005;117:3–44.
- Liu RM, Desai LP. Reciprocal regulation of TGF-β and reactive oxygen species: a perverse cycle for fibrosis. Redox Biol. 2015;6:565–577.
- Warburton D, Shi W, Xu B. TGF-β-Smad3 signaling in emphysema and pulmonary fibrosis: an epigenetic aberration of normal development? Am J Physiol Lung Cell Mol Physiol. 2013;304:L83–L85.
- Gross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N Engl J Med. 2001;345:517–525.
- Lee CM, Park JW, Cho WK, et al. Modifiers of TGF-β1 effector function as novel therapeutic targets of pulmonary fibrosis. Korean J Intern Med. 2014;29:281–290.
- Conte E, Fagone E, Gili E, et al. Preventive and therapeutic effects of Thymosin beta4 N-terminal fragment Ac-SDKP in the bleomycin model of pulmonary fibrosis. Oncotarget. 2016;7:33841–33854.
- Oh K, Seo MW, Kim YW, et al. Osteopontin potentiates pulmonary inflammation and fibrosis by modulating IL-17/IFN-gamma-secreting T-cell Ratios in bleomycin-treated mice. Immune Netw. 2015;15:142–149.
- Rice KM, Manne N, Kolli MB, et al. Curcumin nanoparticles attenuate cardiac remodeling due to pulmonary arterial hypertension. Artif Cells Nanomed Biotechnol. 2015;03:1909–1916.
- Kolb M, Margetts PJ, Anthony DC, et al. Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 2001;107:1529–1536.
- Wilson MS, Wynn TA. Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol. 2009;2:103–121.
- Joshi BH, Hogaboam C, Dover P, et al. Role of interleukin-13 in cancer, pulmonary fibrosis, and other T(H)2-type diseases. Vitam Horm. 2006;74:479–504.
- Yao Z, Spriggs MK, Derry JM, et al. Molecular characterization of the human interleukin (IL)-17 receptor. Cytokine. 1997;9:794–800.
- Chen XL, Li WB, Zhou AM, et al. Role of endogenous peroxynitrite in pulmonary injury and fibrosis induced by bleomycin A5 in rats. Acta Pharmacol Sin. 2003;24:697–702.
- Shi K, Jiang J, Ma T, et al. Dexamethasone attenuates bleomycin-induced lung fibrosis in mice through TGF-β, Smad3 and JAK-STAT pathway. Int J Clin Exp Med. 2014;7:2645–2650.
- Adamson IY, Bowden DH. The pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Am J Pathol. 1974;77:185–197.
- Szapiel SV, Elson NA, Fulmer JD, et al. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. Am Rev Respir Dis. 1979;120:893–899.
- Shoki AH, Mayer-Hamblett N, Wilcox PG, et al. Systematic review of blood biomarkers in cystic fibrosis pulmonary exacerbations. Chest. 2013;144:1659–1670.
- Molet S, Hamid Q, Davoine F, et al. IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol. 2001;108:430–438.
- Chen X, Vodanovic-Jankovic S, Johnson B, et al. Absence of regulatory T-cell control of TH1 and TH17 cells is responsible for the autoimmune-mediated pathology in chronic graft-versus-host disease. Blood. 2007;110:3804–3813.
- Rath G, Hussain T, Chauhan G, et al. Fabrication and characterization of cefazolin-loaded nanofibrous mats for the recovery of post-surgical wound. Artif Cells Nanomed Biotechnol. 2015;18:1783–1792.
- Tan HL, Rosenthal M. IL-17 in lung disease: friend or foe? Thorax. 2013;68:788–790.