Nintedanib reduces corticosteroid resistance pulmonary fibrosis induced by bleomycin in mice by increasing the expression of β3 & β6 integrins

Background: Corticosteroid resistance pulmonary fibrosis is a major health problem. This study aimed to determine the effectiveness of nintedanib on corticosteroid resistance pulmonary fibrosis induced by bleomycin in mice. Methods: The mice were divided into five groups 12 mice each. control group, BLM group received single dose of bleomycin (BLM), BLM+MP group received BLM and methylprednisolone (MP), BLM+NIN group received BLM and nintedanib(NIN) and BLM + NIN + MP group. The lung tissues were obtained for biochemical analysis, gene expression and histopathological examination on day 7 and day 28. Results: after 7 days, both NIN groups showed a significant decrease in the levels of interleukin-2, interleukin-4, interferon-gamma, lung tumor necrosis factor-alpha, Malondialdehyde and lung water content with a significant increase in the Glutathione level in lung tissues compared to MP group. After 28 days, both NIN groups showed a significant reduction in hydroxyproline, and Trans-forming Growth Factor beta lung tissues contents compared to MP group, and they showed a positive effect on the expression of β 3 &β6 integrins compared to the negative effect of MP group. Histopathologically, both NIN groups showed significant improvement compared to MP group by H&E and Masson’s trichrome stains. Immunohistochemical staining revealed negative BCL-2 expression in the cytoplasm of bronchiolar epithelium in both NIN groups after 7 and 28 days of treatment. Lung tissue

7 days, both NIN groups showed a significant decrease in the levels of interleukin-2, interleukin-4, interferon-gamma, lung tumor necrosis factor-alpha, Malondialdehyde and lung water content with a significant increase in the Glutathione level in lung tissues compared to MP group. After 28 days, both NIN groups showed a significant reduction in hydroxyproline, and Trans-forming Growth Factor beta lung tissues contents compared to MP group, and they showed a positive effect on the expression of β 3 &β6 integrins compared to the negative effect of MP group. Histopathologically, both NIN groups showed significant improvement compared to MP group by H&E and Masson's trichrome stains. Immunohistochemical staining revealed negative BCL-2 expression in the cytoplasm of bronchiolar epithelium in both NIN groups after 7 and 28 days of treatment. Lung tissue morphometric studies showed significant improvement of pathological changes induced by BLM in both NIN groups. Conclusion: Altogether, our data indicates that nintedanib overcame corticosteroid resistance pulmonary fibrosis induced by bleomycin.

Introduction:
Pulmonary fibrosis is one of disease of the lower respiratory tract (Green, 2002). It may be as Idiopathic fibrosis which is unknown cause of disease or may be as a secondary effect from other causes, such as; the environmental inhalation such as; pollutants and smoking, Some typical connective tissue diseases. Infections, such as TB, SARS-CoV-2 and COVID-19, some drugs such as bleomycin, methotrexate, and Radiation therapy (Meyer, 2017).
In year of 2020, Millions of people affected by COVID-19. It is an outbreak virus affected the respiratory tract. COVID-19 induced pulmonary fibrosis resistance to corticosteroid. The risk factors of post COVID-19 pulmonary fibrosis are elderly patient, illness severity, prolong stay in ICU and patient on mechanical ventilation, smoking and chronic alcoholism (Ojo et al., 2020).
Glucocorticoid is an effective anti-inflammatory therapy for several chronic inflammatory and immune diseases, but there are several disease resistances to corticosteroid such as interstitial pulmonary fibrosis. Furthermore, the long term of corticosteroids use has been shown to be linked with a major number of comorbidities (Gross and Hunninghake, 2001). Molecular mechanisms of glucocorticoid resistance have currently been identified. The oxidative stress leads to significantly decrease in activity and expression of HDAC2 which causes resistant to the action of glucocorticoid. Patients with glucocorticoid resistance should use alternative antiinflammatory treatments as well as drugs that may reverse the molecular mechanism of glucocorticoid resistance (Barnes, 2010).
Bleomycin is an anti-tumour agent that produces toxicity of pulmonary tissue leading to sever progressive pulmonary fibrosis. The pathogenesis of acute pulmonary toxicity induced by bleomycin is associated with production of reactive oxygen species. The oxidants can lead inflammatory reactions in the lung. For example, the oxidation of arachidonic acid is the early step in the metabolic cascade that produces active mediators like, prostaglandins and leukotrienes. Cytokines such as IL-1, macrophage inflammatory protein-1, PDGF, and TGF-are released from alveolar macrophages of bleomycin toxicity, resulting in fibrosis. Damage and stimulation of alveolar epithelial cells may result in the release of cytokines and growth factors that stimulate proliferation of myofibroblasts and secretion of a pathologic extracellular matrix, leading to fibrosis (Hay et al., 1991). Nettelbladt and co-workers (1990) found that there is no effect of methylprednisolone treatment on bleomycin induced lung fibrosis in rat model.
The current work employed a bleomycin-induced mouse model, which is the most widely used and internationally known animal model for studying pulmonary fibrosis mechanisms (Jenkins et al., 2017). The intra-tracheal instillation of BLM in mice induces pulmonary injury characterized by an inflammatory response followed by fibrosis resembling what is happening in humans (Moeller et al., 2008, Zhao et al., 2019. Nintedanib is an intracellular inhibitor of tyrosine kinases that targets PDGF receptors a/b, FGF receptors 1-3, VEGF receptors 1-3, TGF-b, c-Abelson and Src family kinases. Nintedanib exhibits significant therapeutic effects on modulating myofibroblast differentiation and extracellular matrix (ECM) secretion in vitro (Richeldi et al., 2014). In this study assessment of potential therapy of nintedanib either alone or combined with methylprednisolone on bleomycin induced corticosteroid resistance pulmonary fibrosis in mice was done.

Materials and methods:
Animals: Sixty of albino mice male adult with age of 8 to 10 weeks (20-30gram) (Kilkenny, Parsons et al. 2009). Were obtained the mice from the Animal house from pharmacy faculty, king Abdul-Aziz University, Jeddah, K.S.A. Animals were kept under a 12-hours light/dark cycle at room temperature (22±2°C) and 55±5% humidity, with provided food and water free. Under followed animal care Committee Regulations. The animals were approved by Institutional Animal Ethical Committee for King Abdul-Aziz University, faculty of pharmacy.

Drugs and chemicals:
Bleomycin sulfate powder was purchased from Shanghai Huirui Chemical Technology Co., Ltd, nintedanib powder with purity of 99% was purchased from Shanghai Huirui Chemical Technology Co., Ltd and methylprednisolone powder was purchased from Pfizer, New York were suspended in a 0.5% solution of carboxymethylcellulose sodium as a vehicle.
Formaldehyde 100% was purchased from Zoad international Co for the medical supplier, KSA forceps to one side, toward the mandible to visualize the vocal cords; then, lower the pipet tip loaded with bleomycin into the back of the oral cavity to deliver the liquid through the vocal cords during inspiration. Wait to hear a gasp, which confirms endotracheal delivery of the liquid.
Control animals were received an equal volume of sterile PBS instead of the bleomycin solution.
The tongue was released and carefully dislodge the upper incisors from the suspension thread.
The mice were placed under a heating lamp or pad until it recovers from the anesthesia (Liu et al., 2017a).

Outcome measure
After scarification the mice, the lung rapidly removed then, left lung for histological examination and right lung for analysis by ELIZA, PCR, wet/dry ratio.

Biochemical parameters measurement, after 28 days post treatments
Measurement of transforming growth factor beta (TGF-β) and hydroxyproline contents were assessed by using the Mouse ELISA (Enzyme-Linked Immunosorbent Assay) kits is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Mouse in Tissue Homogenates was performed as previously described (Sisson et al., 2010)& (Saraiva et al., 2018). The Enzyme Linked Immunosorbent (ELIZ) kits: Lung Transforming growth factor-β1 (TGF-β1) in mice purchased from Picokine Co. Bio-vision's hydroxyproline ELISA Kit in mice purchased from bio-vision's Inc.

Tissue homogenate
The lung tissue rinsed in ice-cold PBS (0.01M, pH=7.4) to eliminate excess blood thoroughly.
The 100mg tissue was rinsed with 1x PBS, homogenized in 1 ml of 1x PBS and stored immediate at -20°C. Next of two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 5000×g at 2 -8°C to get the supernatant for immediately assayed or stored at -20°C or -80°C to avoid loss of bioactivity and contamination. Centrifuge the samples again before the assayed. Avoid multiple freeze-thaw cycles.
Wet-to-dry lung weight ratio at day 7 & at day 28 post-treatments: The lung wet-to-dry (W/D) weight ratio was used as an index of lung water accumulation after endotracheal instillation of bleomycin. To measure the lung water content and edema, the animals were killed under deep ether anesthesia, and the lung weight was measured directly after its removal (wet weight). The lung tissue was then dried in an oven at 60°C for 5 days and reweighed as dry weight. The W/D weight ratio was calculated by dividing the wet by the dry weight (Matsuyama et al., 2008).
Gene expression of β3and β6 integrins profiling by real-time quantitative polymerase chain reaction (qPCR) at day 28 post treatment: Lung tissue was removed from each mouse using a dissecting scissor and forceps. The dissecting was performed by the same investigator for all animals to limit discrepancy in the sample collection. Tissue from three mice was pooled for each biological replicate (12 animals per group were used to obtain 3 biologicals replicates). The lung tissues were transferred to liquid nitrogen directly. Lung tissue was homogenized in a homogenizer on ice and total RNA was isolated using Thermo Scientific GeneJET RNA Purification Kit #K0731 (USA). RNA samples with 1.8 to 2.0 of OD260/280 ratio were accepted for analysis. 1 µg of total RNA was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA). RT-qPCR was performed as previously described (Bi et al., 2016, Bi et al., 2019.

Showed the sequences of gene of Integrins b3 &b6
Mouse Gene Orientation Sequence

Lung histopathology:
The left lung specimens from each animal were fixed in 10 % formalin solution. Fixation was followed by dehydration, clearing, and embedding in paraffin. Serial sections of 5 µm thickness were cut then stained using the following stains and measurements were performed by a pathologist blinded to the study groups. H&E and trichrome blue slides were reviewed and subjectively analyzed for general signs of inflammation and fibrosis. As the same mentioned in histological analysis and lung damage severity scores acute after one week and chronic after 4 weeks (Aubin Vega et al., 2019)&(Polosukhin et al., 2012. Visualization and photographing of slides were done using an Olympus light BX61microscope.
Immuno-histochemical analysis for BCL-2 at day 7 & at day 28 post-treatment: The left lung specimens from each animal after one week and after 4 weeks of bleomycin and treatment administration were fixed in 10 % formalin solution. Fixation was followed by dehydration, clearing, and embedding in preparation of 5um paraffin section slides. Immunohistochemical technique for detection of Bcl-2 expression was performed using labeled streptavidin biotin technique (Zymed) Cat No. 18-0193 with the monoclonal antibody (Bcl-2).
The localization of Bcl-2 protein was demonstrated as yellowish brown color area (Ahmed and Anwar, 2004).
Morphometric measurement after 7 days, the mean thickness of the interalveolar septa in (µm) and the mean alveolar space surface area (µm 2) after 7 days As the same mentioned in morphometric analysis (Zakaria et al., 2021). Blind histological analysis was performed on the lung paraffin sections from mice after 7 days for estimation of parenchymal distortion and airway (peribronchial) inflammation using specific semi-quantitative scores: Analysis of Lung parenchymal distortion: It was assessed by analysis of ten sequential non-overlapping tissue fields using x200 magnification. Each tissue field was scored using a 0-to-4-point system. Mean scores for all fields were calculated for each mouse (Polosukhin et al., 2012). Analysis of airway inflammation: It was estimated by individual assessment of each airway in the tissue section using a 0-to-3-point system. Mean scores for all analyzed airways were calculated for each animal (Polosukhin et al., 2012).

Measurement of area percentage of PCL-2 immunostaining after 7 days.
The intensity of immunohistochemical staining was graded semiquantitative as follows: grade 0 = no staining present or less than 10% of the cells are positive; grade 1 = 10% of the cells are positive; grade 2 = more than 10% and less than 50% of the cells are positive and grade 3 = more than 50% of the cells are positive (Safaeian et al., 2008).

Morphometric analysis after 28 days: measurement of the area percent of collagen fibers in
Masson trichrome stain as same mentioned (Zakaria et al., 2021).They were measured by using the NIH Image J (v1.50) program and measurement of subepithelial connective tissue volume density (VVsub) in Masson trichrome stain after 28 days. Airway wall remodeling was evaluated by measurement of VV sub as the difference in the area, delimited by the basement membrane and the outer edge of the airway adventitia, divided by the length of subepithelial basement membrane (Polosukhin et al., 2012). They were measured from x 40 photomicrographs using Digimizer 4.3.2. image analysis software (MedCalc Software bvba, Belgium).

Statistical analysis
Statistical comparisons among experimental groups were performed by one way of variance

The effect of nintedanib (NIN) and methylprednisolone (MP) either alone or in combination on transforming growth factor beta and hydroxyproline contents in lung tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after 28-days post treatments:
Figure 3 showed significant reduction in the contents of TFG-β and hydroxyproline in bleomycin group treated with NIN alone or in combined with MP compared to bleomycin group treated with MP alone (P < 0.05). No significant differences were detected between the bleomycin treated group with NIN either alone or combined with MP.

The effect of nintedanib and methylprednisolone either alone or in combination on the mean alveolar space surface area (µm 2 ) in lug tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after day 7 post-treatments:
The bleomycin group and Bleomycin group treated with methylprednisolone showed significant (P<0.0001) decrease in the mean alveolar space surface area as compared to the treated group with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone.
Interestingly, there was significant increase (P<0.01) in the mean alveolar space surface area in the group of mice treated with nintedanib and methylprednisolone compared to the group of bleomycin treated with nintedanib alone as showed in table 1.

The effect of nintedanib and methylprednisolone either alone or in combination on the scoring of lung parenchymal degeneration and the scoring of air way inflammation in lug tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after day 7 post-treatments:
Table1 showed non-significant (P=0.17) difference between bleomycin group and bleomycin treated mice with methylprednisolone. Moreover, the treated group with nintedanib alone and the group of mice treated with nintedanib methylpredinsolone showed significant (P=0.0001, P<0.0001; respectively) decrease in the scoring of lung parenchymal degeneration as compared to the group of bleomycin treated with methylprednisolone. No significant difference between treated mice with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone.

The effect of nintedanib and methylprednisolone either alone or in combination on the scoring of air way inflammation in lug tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after day 7 post-treatments:
In table 1, the bleomycin treated mice with methylprednisolone showed highly significant (P<0.0001, P<0.0001; respectively) increase in the airway inflammation scoring as compared to the treated group with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone. No significant (P=0.17) difference between treated mice with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone.  The expression of BCL-2 was significantly upregulated in bronchiolar cells, alveolar epithelial

cells, interstial myofibroblasts and inflammatory cells after bleomycin instillation as compared to
Data Values were mean ± standard error of mean (SEM); N = number of animals. BLM; bleomycin, MP; methylprednisolone, NIN; nintedanib. (%) = mean percentage change compared to control group. ANOVA oneway, accompanied by a multiple comparison test by Tukey. * Control group compared to other groups. # BLM group compared to treatments group. $ BLM+MP group compared to BLM+NIN and BLM+(MP+NINI). @ BLM+NIN compared to BLM+ (MP + NIN) group.
the bleomycin-treated mice with nintedanib alone and bleomycin-treated mice with methylprednisolone and nintedanib. Interestingly, the expression of BCL-2 was significantly (P<0.0001) upregulated in inflammatory cells in bleomycin-treated mice with nintedanib alone as compared to bleomycin-treated mice with methylprednisolone and nintedanib. No significant (P>0.05) difference in the expression of BCL-2 in the bronchiolar epithelium, alveolar epithelial cells, and interstial myofibroblasts in bleomycin-treated mice with nintedanib alone as compared to bleomycin-treated mice with methylprednisolone and nintedanib as showed in table 2. The effect of nintedanib and methylprednisolone either alone or in combination on the area percentage of collagen fibers in lung tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after day 28 post treatments: The bleomycin group and bleomycin group treated with methylprednisolone showed significant increase in the mean area percentage of collagen fibers as compared to the treated group with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone.
Interestingly, there was significant increase between treated mice with nintedanib alone as compared to the group of mice treated with nintedanib and methylprednisolone as showed in table 3.

The effect of nintedanib and methylprednisolone either alone or in combination on the mean of the subepithelial connective tissue volume density (VVsub) (mm) in lung tissue in a mice model of corticosteroid resistance pulmonary fibrosis induced by bleomycin after day 28 post treatments:
The bleomycin group and bleomycin group treated with methylprednisolone showed significant increase in the mean subepithelial connective tissue volume density (VVsub) as compared to the treated group with nintedanib alone and to the group of mice treated with nintedanib and methylprednisolone. Interestingly, there was significant increase between treated mice with nintedanib alone as compared to the group of mice treated with nintedanib and methylprednisolone as showed in table 3.

Discussion:
In this study assessment of potential therapy of nintedanib either alone or combined with methylprednisolone on bleomycin induced corticosteroid resistance pulmonary fibrosis in mice was done. In this model many inflammatory mediators and cytokines are released as IL-2, IL-4, IFN-y and TNF-α. The interleukin-2 (IL-2) is important in the development of fibrosis by supported cell mediated immunity and in general promote tissue restoration (Keane, 2008). In present study, there was a significant reduction in the level of IL-2 by 68.5%in lung tissue in the bleomycin group treated with MP compared to bleomycin group in addition there was more significant reduction in the IL-2 content by 84% in the lung tissue in nintedanib alone and in combination with methylprednisolone compared to bleomycin group on day 7 post treatment.
Moreover, the interleukin -4 (IL-4) is important in inflammation and fibrosis by more involved with antibody-mediated immunity, and tend to promote fibroblast activation, matrix development and, therefore, fibrosis (Keane, 2008 to BLM group. Additionally, activation of alveolar macrophages purified from the lung of mice instilled with bleomycin release pro-inflammatory mediators such as TNF-α as well as MIP-2, responsible for the persistence of inflammation and the development of fibrosis (Barbarin et al., 2005). In current study, the BLM+MP group significantly decreased the level of TNF-α by 67.15% in lung tissue compared to BLM group in addition there was more significant reduction in the level of TNF-α in lung tissue in BLM+NIN & BLM+MP+NIN by 74.7% & 77.1% respectively compared to BLM group. In Japanese article found that the level of cytokines in the lung tissue were studied with the reverse-transcriptase polymerase chain reaction. Levels of promotor cytokines, such as IL-2, IL-4, INF-y and TNFα were significantly higher in lung tissue in the bleomycin group. The expression of these cytokines in the glucocorticoid group was low, especially the peak value, but the expression of IL-4 was high in the bleomycin group and was not reduced in the glucocorticoid group (Hosoya, 1997). But, they found that significantly decreased in inflammatory cytokines after nintedanib treatment because it is act as antiinflammatory effect as in study (Kim et al., 2018). Also, Ubieta, Thomas et al found that nintedanib inhibited the release of cytokines such as IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12p70 and IL-13, which may cause a clinical benefit in pulmonary fibrosis in interstitial lung diseases.
Also, IL-2 has able to bind to lung fibroblasts, and proliferation of fibroblast induced by IL-2.
IL-4 act directly on lung fibroblast to induce a fibro-genic response. However, IFN-γ, which is also reduced by nintedanib, exhibits potent antifibrotic activity by inhibiting synthesis of collagen in fibroblasts (Ubieta et al., 2021). Moreover, the oxidative stress is defined as extra production of reactive oxygen species (ROS) or decreases in antioxidants. The oxidative stress plays a role in lung fibrosis. Malondialdehyde (MDA) is one of the end products of polyunsaturated fatty acids peroxidation in the cells. An increase in free radicals causes overproduction of MDA in lung injury. Glutathione is a low molecular weight antioxidant found in normal lungs, is reduced in the epithelial lining fluid and fibrotic foci in pulmonary fibrosis (Cheresh et al., 2013). In current study, there was a little reduction in the level of MDA by 46.77 in lung tissue in BLM+MP group compared to bleomycin group in addition there was more significant reduction in the level of MDA by 77.4% & 80%in lung tissue in groups of BLM+NIN& BLM+MP+NIN compared to bleomycin group and there was a significant reduction in level of MDA in group of BLM+NIN& BLM+MP+NIN compared to BLM+MP group. Also, there was a significant increase in level of GSH by 540% in lung tissue in BLM+MP group compared to bleomycin group in addition there was a significant increase in level of GSH by 759.75% & 789.64% in lung tissue in BLM+NIN& BLM+MP+NIN groups compared to bleomycin group. And there was a significant increase in level of GSH in BLM+NIN& BLM+MP+NIN groups compared to BLM+MP group. Also, the effect of nintedanib and methylprednisolone in combination in level of GSH was much better than the effect of nintedanib alone. The present study is agree with they found that the production of MDA in lung tissues was increased and GSH production in lung tissues was decreased in after bleomycin treatment (Liu et al., 2017b). Furthermore, TGFβ is play important role in fibrosis by stimulating fibroblast proliferation and the synthesis of extracellular matrix proteins, recruiting inflammatory cells through MCP-1 (CCL2) and inhibiting T-cell responses with contribute to healing of wound and fibrosis and Hydroxyproline also plays important role in the collagen synthesis and stability. (Wilson and Wynn, 2009). In present study, there was a significant decrease in the level of TGF-β in BLM+MP group by58.4 % compared to bleomycin group, in addition there was more significant reduction in content of TGF-β in lung tissue in BLM+NIN& BLM+MP+NIN groups by 66% compared to bleomycin group after 28 days post treatment.
Also, there was a significant decrease in the level of hydroxyproline in BLM+MP group by 77.2% compared to bleomycin group, in addition there was more significant reduction in content of hydroxyproline in lung tissue in BLM+NIN& BLM+MP+NIN groups by 88% compared to bleomycin group after 28 days post treatment. Khalil et al found that the corticosteroid therapy administered in the advanced stages of the disease would likely not suppress the TGF-b production by alveolar macrophages. The relative resistance to corticosteroid therapy in pulmonary inflammatory and fibrotic responses seen in many human lung diseases may be caused by the corticosteroid insensitivity of TGF-b production by alveolar macrophages (Khalil et al., 1993). The water content in lung tissue after 28 days post treatment, it was a significant reduce in water content in bleomycin group with nintedanib either alone or in combination with methypredisolone compared to bleomycin group in addition there was no significant difference between bleomycin group and bleomycin group treated with MP. Our finding agree with They found that dexamethasone treatment did not prevent bleomycin-induced edema (Aubin Vega et al., 2019). Similarly, to dexamethasone, another corticosteroid did not elicit any beneficial effect on lung edema in bleomycin mice. Also, our finding agree with pervious study found the effect of Nintedanib in a rat model of lung fibrosis induced by bleomycin and they showed a significant decrease in water content compared to bleomycin group (Pittelli et al., 2017). Our study's histological findings showed that methylprednisolone was unable to enhance the histological outcomes of the bleomycin model of lung fibrosis after 7 and 28 days in the current research.
This was in line with prior research by (Dik et al., 2003, Bahtouee et al., 2018, Aubin Vega et al., 2019. MP therapy increased the expansion of alveolar airspaces and the decrease in lung compliance, according to (Langenbach et al., 2007). Also, Aubin Vega et al. investigated the effect of corticosteroids on the bleomycin outcomes during the acute exudative phase after 3 and 7 days. They found that exposure to bleomycin and dexamethasone exhibited severe injury scores, alveolar damage at day 3 and at day 7. They stated that dexamethasone failed to reduce inflammatory cell infiltration and alveolar epithelial injury induced by bleomycin.
Dexamethasone affected the expression of β3and β6-integrins, key proteins of alveolar repair (Aubin Vega et al., 2019). Previous research has demonstrated that long-term corticosteroid treatment, either before or at the same time as bleomycin treatment, reduced the development of lung fibrosis in rats (Phan et al., 1981, Cross et al., 1985, Shaker and Sourour, 2011. Moreover, in the present study, methylprednisolone does not reverse the bleomycin-repair impairment, but it further worsened the bronchiolar epithelial condition. The epithelial lining of the bronchiolar passages showed marked disorganization and degeneration in which their epithelial lining exhibited vacuolation with eukaryotic nuclei. Their lumen appeared obliterated by detached epithelial cells. Dexamethasone has been found to have a negative influence on repair processes in bronchiolar epithelial cells, which is consistent with our findings , Kadmiel et al., 2016. According to these findings, bleomycin-induced pulmonary fibrosis is resistant to suppression by concurrent MP therapy. In current study, we used the QPCR for expression of integrins. Integrins are membrane-bound proteins that play an important role in maintenance and remodeling of ECM by transmitting signals from the ECM to control cell function. In the case of the fibroblast, integrin expression can influence the expression of intracellular structural or contractile proteins, such as alpha-smooth muscle actin (αSMA), and thus the myofibroblast phenotype, as well as extracellular structural proteins, such as collagen (Merna et al., 2015). In present study, negative effect of methylprednisolone alone compared to control group, but the effect of nintedanib alone and in combination with methylprednisolone was little increased in β3& β6 integrins gene in lug tissue compared to bleomycin group. We discovered that nintedanib reduced BLM-induced alveolar inflammation and pulmonary fibrosis. In comparison to the BLM group, the mice in the nintedanib-treated and nintedanib and MP groups had reduced inflammatory cell infiltration and collagen deposition.
This came in accordance with previous study (Wollin et al., 2013, Rangarajan et al., 2016, Redente et al., 2018, Chen et al., 2020. Chen et al. looked into the function of nintedanib in bleomycin-induced pulmonary fibrosis, comparing lung sections from pulmonary fibrosis mice who received nintedanib by oral gavage with those from mice with pulmonary fibrosis who did not. They discovered lung inflammation and fibrosis seven days after bleomycin-induced pulmonary fibrosis, which were substantially decreased by nintedanib therapy. They also discovered that animals given nintedanib had considerably lower lung damage scores, as well as reduced pulmonary fibrosis as measured by Masson's trichrome staining and the Ashcroft score seven days following bleomycin administration. Moreover, Wollin et al. investigated nintedanib at 30 mg/kg or 60 mg/kg as a preventive treatment (from day 0 to day 14) and as a therapeutic treatment (from day 7 to 21) in a mouse model of lung inflammation and fibrosis by a single intratracheal administration of bleomycin. They found that therapeutic therapy at 60 mg/kg had similar inhibitory effects as preventative treatment, while the effect magnitude was lower at 30 mg/kg. They found that nintedanib successfully decreased pulmonary inflammation and fibrosis in mice whether given as a preventative or therapeutic therapy. Nintedanib's anti-inflammatory and anti-fibrotic properties may have an influence on the progression of fibrotic lung disorders such IPF (Wollin et al., 2013). Apoptosis is a form of cell death that plays a critical function in the maintenance of cellular homeostasis. The Bcl-2 family of proteins is one of the most essential regulators of the apoptosis process (Dewson and Kluck, 2010). Bcl-2 protein is an intracellular membrane-associated protein that inhibits cell death when overexpressed (Nemec and Khaled, 2008, Youle and Strasser, 2008, Dewson, 2011. Bcl-2 family members appear to play a critical role in the pathogenesis of inflammation, apoptosis, and fibrosis produced by different causes in interstitial lung disorders, according to an increasing body of data (Safaeian et al., 2014). In the current study, in group II (BLM group) and group III (BLM group treated with MP) there was upregulation of positive immune reaction in the inter-alveolar septa and the interstitium of the lung. While few positive immune reactions were seen in the epithelium lining the bronchiole lumen. High expression of the anti-apoptotic gene Bcl-2 and low expression of the pro-apoptotic gene Bax prevented apoptosis in lung fibroblasts following bleomycin instillation, which is a major characteristic of chronic fibrotic illnesses, according to our findings, which are similar with previous research (Safaeian et al., 2009, Aguilar et al., 2009, Zhou et al., 2013, Hu and Zhu, 2020. Furthermore, the current findings were consistent with those of Safaeian et al., who discovered BCL-2 immunoreactivity in a variety of cells, including bronchiolar epithelial cells and lymphocytes, macrophages, neutrophils, alveolar epithelial cells, and myofibroblasts, and found that their interactions are linked to the development of interstitial fibrosis after bleomycin instillation (Safaeian et al., 2008). Predescu et al., on the other hand, detected no alterations in Bcl-2 and Bcl-xL during Fas-mediated apoptosis in primary lung fibroblasts (Predescu et al., 2017). The extracellular matrix-producing myofibroblasts that collect in fibrotic lung lesions gain resistance to apoptosis in pulmonary fibrosis Horowitz, 2006, Potter-Perigo et al., 2010). Nintedanib has been found to inhibit fibroblast proliferation, migration, and transformation, although its effects on apoptosis have yet to be investigated .
In the present study, the treatment with nintedanib and the combined treatment of methylprednisolone and nintedanib were more effective in a model of pulmonary fibrosis. There was an apparent decrease of the positive immune reaction for BCL-2 in inter alveolar septa with negative BCL-2 of bronchiolar epithelium. The present results were similar to the findings of studies by (Milara et al., 2018). Milara et al. discovered that JAK2 and STAT3 are activated in IPF in a rat model of bleomycin-induced lung fibrosis, and that their dual inhibition could be an appealing strategy for inhibiting fibroblast migration, preventing increases in fibroblast senescence and Bcl-2 expression, and improving impaired autophagy. Rangarajan et al. discovered that nintedanib enhances the apoptotic clearance of fibrocytes and lung-resident myofibroblasts, slowing the development of TGF-induced pulmonary fibrosis. In fibroblasts isolated from IPF lungs, nintedanib was reported to activate autophagy (Rangarajan et al., 2016).
Furthermore, nintedanib can prevent fibrocyte migration, lowering the amount of fibrocytes in the lungs during bleomycin-induced pulmonary fibrosis (Sato et al., 2017b).

In conclusion:
Altogether, our data indicates that the nintedanib overcame on the corticosteroid resistance pulmonary fibrosis induced by bleomycin because the bleomycin group treated with nintedanib alone and bleomycin group treated with methylprednisolone and nintedanib were able to improve the alveolar damage and decrease the collagen deposition with decrease edema in the lung and reduce the inflammatory cells.