Zanubrutinib Attenuated Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF- β 1 Signaling Pathway

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease with high mortality and limited treatment. So far, the only drugs approved for the treatment of IPF are Nintedanib and Pirfenidone. Zanubrutinib, a BTK small molecule inhibitor, is approved for the treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). In this study, we explored the potential effect and mechanisms of zanubrutinib on pulmonary fibrosis in vivo and in vitro. Methods: In the in vivo experiments, different doses of zanubrutinib were administered in a mouse model of bleomycin-induced pulmonary fibrosis, and pathological manifestations and lung function indexes were evaluated. The in vitro experiments were used a TGF- β1 -treated fibroblast model to evaluate the effect of zanubrutinib on the activation and autophagy phenotype of fibroblasts and explored the underlying signaling pathways mechanism. Results: In vivo experiments proved that zanubrutinib effectively attenuated bleomycin (BLM)-induced pulmonary fibrosis in mice. In vitro mechanism study indicated that zanubrutinib could suppress collagen deposition, myofibroblast activation by inhibiting the TGF- β1/Smad pathway and induce autophagy t hrough the TGF- β1/mTOR pathway. Conclusions: Zanubrutinib could alleviate bleomycin- induced lung fibrosis in mice by inhibiting the TGF- β1 signaling pathway.


Background
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and fibrotic interstitial lung disease characterized by fibrosis and inflammation in the lung interstitium and surrounding airspace [1]. Patients with idiopathic pulmonary fibrosis generally use high-resolution chest CT (HRCT) as the diagnostic gold standard and the most obvious pathological manifestations are diffuse or patchy shadows of the lung, as well as bronchial traction and dilation [2]. The median survival time of IPF patients is only 2.41 years and the incidence of IPF is increasing year by year [3]. The specific pathogenesis of IPF has not been clarified so far [4]. It is generally believed that regenerative repair and scar repair are out of balance, and eventually lead to pulmonary fibrosis [5,6]. Currently, there are only two drugs approved for the treatment of IPF, Nintedanib and Pirfenidone, and they cannot significantly delay the survival of patients. There is an urgent need to continue to develop effective therapeutic drugs.
Transforming growth factor-β1 (TGF-β1) was a core regulator in the development of pulmonary fibrosis [7]. Active TGF-β1 may induce myofibroblast activation, migration and proliferation [8]. Phosphorylated smad2 and smad3 as the downstream of TGF-β are motivated by TGF-β1 to regulate fibroblast activation and extracellular matrix (ECM) deposition [9]. Autophagy is an important process involving in cell growth, survival, development and death [10]. mTOR signal is the core regulatory signal pathway of autophagy, inhibiting mTOR signal can induce the formation of autophagy [11]. mTOR signal is mediated by many upstream proteins, TGF-β1 can activate mTOR signal [12]. The overexpression of ECM proteins is a fatal factor in pathogenesis of pulmonary fibrosis, and its levels could be down-regulated by autophagy [13]. Therefore, reducing the activation of fibroblasts and extracellular matrix deposition induced by TGF-β1 and increasing the level of autophagy in fibroblasts are important means to treat fibrosis.
Zanubrutinib (ZB, BGB-3111) is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK). BTK is a key kinase in the B cell receptor (BCR) signal transduction pathway, involved in the proliferation, differentiation and apoptosis of B cells [14]. ZB is a second-generation BTK inhibitor with increased specificity to BTK. The role of this greater selectivity toward BTK aims to increase potency of the drug and reduce off-target effects [15]. ZB is clinically used for the treatment of Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) [16]. It has been reported that Ibrutinib, the first-generation small molecule inhibitor of BTK, can aggravate fibrosis [17].
However, another study reported that the expression of BTK in B cells is increasing in IPF patients [18]. Therefore, the relationship between BTK and pulmonary fibrosis is controversial. Our study evaluated the role of second-generation BTK inhibitor ZB in bleomycin-induced pulmonary fibrosis in mice, and further explored the mechanism of ZB in pulmonary fibrosis in vivo and in vitro.

Animal Model of Pulmonary Fibrosis
Male C57BL/6 mice (6-to 8-week old; 20-25 g body weight) were bought from Charles River Laboratories (Beijing, China). All animal care and experimental procedures complied with guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Nankai University (Permit No. SYXK 2014-0003). Mice were housed under controlled temperature (22-26°C), humidity (60 ± 2%) and a 12 h light-dark cycle. During the whole experiment, the mice were free access to food and water.
The modeling method of the animal model of pulmonary fibrosis is according to the procedures previously described [19]. In briefly, 42 mice were randomly divided into 6 groups: control group, BLM group, BLM + Nintedanib group (100 mg/kg), BLM + Zanubrutinib group (5 mg/kg), BLM + Zanubrutinib group (10 mg/kg), BLM + Zanubrutinib group (20 mg/kg). Nintedanib was used as the positive control. For control group saline was injected intratracheally. Intratracheal injection of 2.5 U/kg BLM (Hanhui Pharmaceuticals CO., LTD, China) in other groups. Zanubrutinib and nintedanib were given by gavage once a day from the seventh day of BLM injury. The mice were sacrificed under anesthesia 14 days after the BLM injection to evaluate the degree of pulmonary fibrosis.

Western Blotting
All proteins were extracted from cells and lung tissues as reported previously [20]. After incubating with 5% skimmed milk at room temperature for 30 minutes, incubate the primary antibody overnight at 4 degrees. Then, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam, UK) for 2 h at room temperature and tested with chemiluminescence reagent (Affinity, USA). The following primary antibodies (Table 1) were used to detect the protein expression levels.

MTT Assay
The MTT assay can be used to detect the toxicity of Zanubrutinib to cells. NIH3T3 cells were seeded in 96-well plates. After incubation of TGF-β1 (5 ng/mL) with/without Zanubrutinib (12.5-1600nM) for 24 h, 20 μL MTT (0.5 mg/mL) was added. After four hours, use a pipette to remove the supernatant, add 120ul of DMSO to dissolve the precipitated crystals. Then, the OD540 nm was measured to reflect the relative number of cell.

Wound-Healing Assays
Spread NIH3T3 and Mlg cells in a six-well plate for Wound-Healing Assays. Use a 200ul pipette tip to draw a line in the middle of each hole of the six-well plate. After washing with phosphate buffer, add TGF-β 1 (5 ng/mL) or Zanubrutinib (100 nM, 200 nM, 400 nM) and take images at 0h, 6h, 12h, 24h to observe cell migration by using a light microscope (Nikon, Tokyo, Japan).

Immunofluorescence
This experiment used the NIH3T3 cell line. All the steps are the same as previously reported [21]. For microtubule-associated protein 1 light chain 3 (LC3)-B plasmid transfection, according to the supplier's instructions (SinoBiological, Beijing, China), 1.5 mg GFP-LC3 and 2 mg Cherry-GFP-LC3 plasmids incubate with PEI in serum-free Opti for 20 minutes at room temperature respectively. Then they were transfected into NIH3T3 cells. After adding TGF-β1 (5 ng/mL) or Zanubrutinib (100 nM, 200 nM, 400 nM) for 24 hours, fix the cells and stain the nucleus with DAPI. The fluorescent was examined with the confocal microscope (Nikon, Japan).

Immunohistochemistry
Use the UltraSensitiveTM SP (Mouse/Rabbit) IHC Kit and DAB Kit (Maxim, Fuzhou, China) according to the instructions to detect the expression of the measured makers .
Randomly select three fields of view for each slice for positive area statistics.

Hydroxyproline content determination
Hydroxyproline was tested according to the methods as described previously [22].
Adjust the PH to 7 after dissolving the three lung lobes on the right of the mice with hydrochloric acid. The results were calculated as µg hydroxyproline per right weight using hydroxyproline standards (Sigma, USA).

Histological examination
The left lung of the mice is filled with 10% formaldehyde and embedded in paraffin to prepare tissue sections with a thickness of 5μM. Tissue sections for H&E and Masson staining (Solarbio, Shanghai, China). Images were collected using a fluorescence microscope (Nikon, Japan). Pulmonary fibrosis area was evaluated as described previously [23].

Evaluation of Pulmonary Function
Pulmonary function analysis of mice using Anires2005 system (Biolab, Beijing, China). The system will automatically measure lung function parameters including forced vital capacity (FVC), dynamic compliance (Cydn), expiratory resistance (Re) and inspiratory resistance (Ri).

Statistical Analysis
GraphPad Prism 6.0 was used for Statistical analysis. Differences between experimental and control group were assessed by Student's t test. Significant differences among multiple groups were detected by one-way ANOVA. Significance was described as follows: # represent the difference between control/NaCl and TGF-β 1/BLM-treated group, and * represents the difference between the TGF-β 1/BLM-treated and the ZB -treated group: * p <0.05, ** p < 0.01, *** p < 0.001; # p <0.05, ## p < 0.01, ### p < 0.001

ZB alleviates BLM-induced pulmonary fibrosis in mice.
In order to evaluate the anti-fibrotic effect of ZB and explore the mechanism, we verified the anti-fibrotic effect of ZB in BLM-induced animal model of pulmonary fibrosis. From day 7 to day 14 after BLM injury, ZB was administered per day and Nintedanib acted as a positive control ( Figure 1A). Compared with the model group, the hydroxyproline content and fibrosis area in the administration group were significantly reduced, and the highest dose was almost equal to the positive drug ( Figure 1B-C). H&E and Masson staining of lung tissue pathological sections also showed that ZB can improve the deposition of collagen in lung tissues and restored lung structure damage caused by BLM ( Figure 1D). The change of lung function is also an important indicator for evaluating pulmonary fibrosis. Our experimental results showed that ZB can alleviate the decline of forced vital capacity (FVC) and dynamic compliance (Cdyn), and decreased inspiratory resistance (Ri) and expiratory resistance (Re) (Figure 1E).

ZB down-regulates TGF-β1/Smad signal pathway in fibroblasts.
TGF-β1 is the most important driving factor of pulmonary fibrosis, and the activation of TGF-β1/Smad signaling pathway will promote the processing of fibrosis [25]. ZB can reduce the expression level of p-smad2 and p-Smad3 induced by TGF-β1 in NIH3T3 and Mlg cells (Figure 4A-B). Immunofluorescence results also showed that

ZB promotes autophagy by inhibiting the TGF-β1/mTOR signaling pathway in fibroblasts.
Autophagy is a physiological process that maintains cell homeostasis and is also closely related to pulmonary fibrosis. In pulmonary fibrosis, autophagy triggered by TGF-β1 can enhance the apoptosis resistance of fibroblasts, induces epithelial-mesenchymal transition to promote the processing of fibrosis [26]. ZB can induce autophagy and reduce the expression of P62 in NIH3T3 and Mlg cells ( Figure   5A-B). Autophagy inhibitor chloroquine (CQ) can inhibit autophagy and increase the expression level of P62, but ZB can alleviate the inhibitory effect of chloroquine in NIH3T3 and Mlg cells (Figure 5C-D). The LC3B-GFP and LC3B-mCherry-GFP plasmids were transferred into the NIH3T3 cells to evaluate the effect of ZB on autophagic flux. Experimental results show that ZB can significantly increase the number of autophagosomes (GFP+) and autophagolysosomes (Cherry+GFP-) induced by TGF-β1 (Figure 5E-F). The formation of autophagy is regulated by many protein signals, among which mTOR protein is the core regulator [27]. Experimental results show that in NIH3T3 and Mlg cells, TGF-β1 can activate mTOR and downstream signaling proteins, and ZB can significantly inhibit the expression of p-mTOR, p-ULK1, p-p70 S6K and pS6RP induced by TGF-β1 (Figure 6A-B). All in all, ZB can induce autophagy and increase the number of autophagolysosomes by inhibiting the TGF-β1/mTOR signaling pathway. Cherry-GFP-LC3 plasmids (F) were transfected into NIH3T3 cells.

ZB inhibits ECM deposition and induces autophagy via regulating TGF-β1 signaling pathway in vivo.
In order to better prove the anti-fibrosis efficacy of ZB, we performed immunohistochemistry, Western Blot and Quantitative real-time PCR to detect a-SMA and extracellular matrix (ECM) deposition maker Fibronectin, Collagen Ⅰ in lung tissues. The results showed that ZB can reduce the protein and mRNA expression levels of Fibronectin, Collagen Ⅰ and a-SMA in vivo (Figure 7A-C). The results of immunohistochemistry showed that the expression levels of P62 and p-Smad3 in the administration group were significantly reduced compared with the model group ( Figure 7C). The Western Blot results of the lung tissues of each group showed that ZB can reduce the phosphorylation level of the protein on the TGF-β1/Smad and TGF-β1/mTOR pathway including p-Smad3, p-Smad2, p-mTOR and p-S6RP ( Figure   7D). At the same time, Western Blot results showed that ZB reduced the expression of autophagy marker P62 (Figure 7D). In conclusion, ZB could attenuate BLM-induced pulmonary fibrosis via regulating TGF-β1 signaling pathway in vivo. Smad3, p-Smad2, Smad2, P62, p-mTOR, mTOR, p-S6RP and S6RP. GAPDH was used as an internal reference in densitometric analysis. Data was noted as the means ± SD, n=5. # represent the difference between NaCl and BLM-treated group, ##P<0.01, ###P< 0.001, ####P< 0.0001. * represent the difference between BLM-treated and ZB-treated group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Discussion
IPF is a disease of unknown cause, characterized by diffuse alveolitis and alveolar structural disorders, which eventually lead to pulmonary interstitial fibrosis [2]. IPF has an insidious onset, with more than 200 pathogenic factors, and an average survival period of 2.41 years [3]. The mortality rate is higher than that of most cancers [28]. At present, the only drugs approved by the FDA for the treatment of IPF are Nintedanib and Pirfenidone, but they can only delay the decline of lung function and cannot reverse the progression of the disease. Therefore, there is an urgent need to develop better drugs [29]. In this study, the experimental results showed that ZB can alleviate bleomycin-induced lung fibrosis in mice.
TGF-β1 is an important regulatory factor for the occurrence of fibrotic diseases.
It can regulate fibroblasts transform into myofibroblasts, and induce collagen synthesis by interacting with the downstream Smads [32]. The formation process of pulmonary fibrosis involves the classic TGF-β1/Smads signaling pathway and the non-classical TGF-β signaling pathway. The TGF-β1 signaling pathway is involved in the occurrence and development of pulmonary fibrosis [33]. Our experimental results showed that ZB can inhibit the activation of TGF-β1/Smad pathway caused by TGF-β1 to alleviate fibrosis. Recent studies have also shown that mTOR-dependent autophagy plays an important role in the development of pulmonary fibrosis.
It has been reported that Ibrutinib, the first-generation small molecule inhibitor of BTK, can aggravate fibrosis by aggravating inflammation [17]. Compared with our study, Ibrutinib (10 mg/kg) was administered by oral gavage daily since the beginning till sacrifice and ZB was given by gavage once a day from the seventh day after BLM injury. The fibrosis process caused by bleomycin includes the early inflammatory phase (days 2-7) and the late fibrotic phase (days 7-14) [35]. At the same time, ibrutinib could increase the apoptosis of bleomycin-induced epithelial cell, but cannot increase the differentiation of myofibroblasts in vitro and also has off-target effects.
Other studies have shown that ibrutinib can reduce the expression of activated NF-κB and NLRP3 inflammasomes and reduce inflammation by targeting BTK [36].
Therefore, the relationship between BTK and inflammation, fibrosis is controversial and needs further research.

Conclusions
Our study found that ZB attenuated BLM-induced pulmonary fibrosis in mice, inhibited myofibroblast migration and activation by suppressing the TGF-β1/Smad signaling, and induced autophagy via regulating TGF-β1/mTOR pathways. Although the specific target of ZB to treat pulmonary fibrosis has not yet been elucidated, ZB may be used as a potential drug candidate for the treatment of pulmonary fibrosis.

Consent for publication
Not applicable.

Availability of data and materials
Data available on request from the authors.

Funding
This study was supported by The National Natural Science Foundation of China

Competing Interests
The authors declare that they have no conflict of interest.