Duvelisib attenuates bleomycin‐induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K‐δ and PI3K‐γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin‐induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin‐induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose‐dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.


Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3Kδ and PI3Kγ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL).
In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.

K E Y W O R D S
Duvelisib, myofibroblasts, PI3K/Akt/mTOR signalling pathway, pulmonary fibrosis

| INTRODUC TI ON
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary interstitial disease characterized by diffuse alveolar inflammation and alveolar structural disorder, which eventually leads to pulmonary interstitial fibrosis. 1 As the most common and serious type of idiopathic interstitial pneumonia, it is characterized by the destruction of normal alveolar tissue, excessive deposition of extracellular matrix (ECM) and structural changes, resulting in reduced pulmonary ventilation, blocked gas exchange and irreversible decline of pulmonary function. 2  and Bcl-2 anti-apoptotic protein. 7 PTEN, the main inhibitor of PI3K/ Akt signalling, is down regulated in pulmonary fibrosis in patients with IPF. 8 In vitro model confirmed that PTEN low expression fibroblasts showed higher Akt phosphorylation level, so they were resistant to fibroblast apoptosis caused by collagen matrix contraction.
Low activity autophagy has become an important cause of IPF. 9,10 PI3K/Akt/mTOR signal pathway is the main regulatory pathway in this process. 11 Therefore, PI3K/Akt/mTOR pathway is closely related to autophagy in the process of IPF and plays an important role in IPF.
Duvelisib is an oral dual inhibitor of PI3Kδ and PI3Kγ. PI3K-δ/γ inhibition may directly inhibit malignant T-cell growth, making Duvelisib a promising candidate for patients with peripheral (PTCL) or cutaneous (CTCL) T-cell lymphoma. 12 Because PI3K/Akt/mTOR signalling pathway is closely related to autophagy in the process of IPF, we hypothesized that Duvelisib may have a therapeutic role in pulmonary fibrosis. Therefore, in this study, we verified the effect of Duvelisib in pulmonary fibrosis by in vivo and in vitro experiments.

| BLM administration
A mouse pulmonary fibrosis model was established by intratracheal BLM administration as described previously. 13 Briefly, mice were intratracheally administered BLM (2 U) dissolved in physiological saline (0.9% NaCl). For pulmonary fibrosis models, 48 mice were randomly separated into six groups: (1) NaCl group, (2) BLM group (CMC-Na) is a common solvent for animal administration. We dissolved 2 g of sodium carboxymethyl cellulose in 398 ml of pure water to obtain a CMC-Na solution with a concentration of 0.5%. Then we used 0.5% CMC-Na as the solvent to prepare different concentrations of Duvelisib and Nintedanib. Both drugs can be well dissolved in 0.5% CMC Na. The mice were sacrificed on the 14th day after BLM administration for subsequent analysis.

| Hydroxyproline assay
Collagen content in lung homogenate was determined as previously described. 13 In brief, the right lung of the mouse was isolated and put into 5 ml ampoules and dried. 3 ml of 6 M hydrochloric acid was added into each ampoule. After hydrolyzed, the PH is adjusted to 6.5-8.0. Then the samples were filtered and 1 × PBS volume was fixed to 10 ml. The samples were processed according to the hydroxyproline assay kit. The HYP analysis was carried out basically according to the manufacturer's instructions, each sample 200 μl was transferred into 96-well plate and divided into 3 reproductions.
Absorbance was determined at 577 nm.

| Histological examination
The left lung was fixed in 10% formalin, dehydrated and embedded in paraffin. Tissue sections with a thickness of 4 μM were incubated for 4 h at 60°C and stained with haematoxylin and eosin (H&E) and Masson's trichrome. Images were analysed by Image-Pro Plus version 6.0 to demarcate the entire lung area and automatically calculate the total pixel Pw of the region and then calculate the total pixel Pf of the fibrotic region (fibrosis ratio = fibrotic area total pixel Pf/ total lung total pixel Pw).

| Pulmonary function testing
After the mice were anaesthetised, the trachea was exposed, and a tracheal cannula was placed and fixed. The mice were transferred to a plethysmography chamber for pulmonary function analysis using an Anires2005 system according to the manufacturer's instructions (Beijing Biolab).

| EdU assay
Briefly, 1 × 10 5 cells/well were plated in 24-well plates with diameter a 14-mm diameter coverslip. Duvelisib at various concentration was added to the wells for 24 h. Then, 40 μM 5-ethynyl-2′-deoxyuridine (Invitrogen) was added to the wells for an additional 2 h. To fix and improve cell membrane permeability, 4% paraformaldehyde and 0.5% Triton X-100 in PBS were applied for 15 min. Hoechst 33342 was used to label cell nuclei. A fluorescence microscope was used to view the signal.

| Wound healing assay
Wound healing assays were performed as previously described. 13

| Transwell cell migration assay
Transwell assays can not only reveal cell migratory behaviours but also are used to observe variations in cellular morphology. Briefly, 1 × 10 5 fibroblasts/ml were suspended in serum-free DMEM medium, and 100 μl of the cell suspension was seeded on the top filter membrane and added different concentrations of Duvelisib and TGF-β1, followed by incubation for 24 h.
DMEM medium containing 20% FBS is added into the lower chamber of 24-well plate. he cells were fixed with 70% paraformaldehyde for 10 min. Then, a cotton-tipped applicator was used to remove the remaining cells and paraformaldehyde from the top filter membrane. Next, 0.2% crystal violet was added to the low chamber and incubated for 15 min at 25°C. After staining, the remaining crystal violet was carefully removed from the top membrane using a cotton-tipped swab, and the cells were gently washed with water. An inverted microscope was used to view the signal.

| Quantitative real-time PCR
The mRNA expression level of genes was determined by quantitative real-time PCR (qRT-PCR) using primers according to a previously described protocol. 15 Primer pairs of target genes used were as follows:

| Western blot analysis
The proteins were extracted from lung tissues or cells following standard protocols, as described previously. 16 Protein was extracted from lung tissue homogenates or cells using Radio-Immunoprecipitation Assay (RIPA) lysis buffer containing Protease inhibitor cocktail (CT) and sodium fluoride (NaF). After electrophoresis and membrane transfer, the following primary antibodies were used to explore the western blot: GAPDH, α-SMA, Fibronectin, P62, LC3I/II, P-PI3K/PI3K, P-Akt/Akt, P-mTOR/mTOR. The secondary antibodies were goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated antibodies. The protein bands were visualized using an enhanced chemiluminescence system (Affinity Biosciences), GAPDH was used as loading control.

| Immunofluorescence analysis
Mlg cells were seeded in a 24-well chamber. At the end of the treatment, the cells were fixed with a 4% fixative solution for 15 min (Solarbio), permeabilized with 0.2% Triton X-100 for 20 min, and incubated in 5% BSA for 30 min. Mlg cells were incubated overnight with α-SMA primary antibodies. Then, the cells were incubated with FITC-conjugated secondary antibodies. The cells were washed with PBST, and DAPI (Beyotime Biotechnology) was used to stain the nuclei. The fluorescence was examined with a confocal microscope (Nikon).

| Immunohistochemical staining
Paraffin-embedded lung tissue was dewaxed with xylene, and the sections were heated in a microwave oven with antigen-fixing solution (0.01 M citrate buffer) for 20 min. After the sections were cooled to room temperature and blocked with an immunohistochemistry kit, the primary antibody was added and incubated at 4°C overnight. Data are shown as mean ± SD (n = 5). ##p < 0.01, ###p < 0.001, ####p < 0.0001 as compared with control group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with model group.

| Data and statistical analysis
All statistical were performed using Graphpad prism 7.0 software as the means ± SD (GraphPad Software, Inc). All statistical comparisons were analysed by one-way analysis of variance followed by the Tukey-Kramer test to identify significant differences between groups. p < 0.05 was considered to be statistically significant.

| Duvelisib attenuates BLM-induced pulmonary fibrosis in mice
To determine the role of Duvelisib in pulmonary fibrosis in vivo,
Duvelisib, and the experimental results showed that EdU-positive fibroblasts were decreased as the Duvelisib concentration increased ( Figure 2D). In summary, these results indicated that Duvelisib could suppresses TGF-β1-induced proliferation of lung fibroblasts.

| Duvelisib suppresses TGF-β1-induced migration of lung fibroblasts
Wound closure and transwell experiments were used to detect the inhibitory effect of Duvelisib on fibroblast migration. Wound closure assay results showed that Duvelisib inhibited fibroblast migration in a dose-dependent manner ( Figure 3A). Compared with that in the TGF-β1 group, wound closure ability was reduced approximately 62% after treatment with 5 μM Duvelisib for 24 h ( Figure 3A). Figure 3B demonstrates that fibroblast migration was suppressed, and cellular morphology was also altered with Duvelisib treatment. Most cells exhibited an irregular polygonal shape rather than a typical spindle shape after treatment with 5 μM Duvelisib for 24 h ( Figure 3C). The number of migrated cells attached to the bottom membrane after treatment with 2.5 and 5 μM Duvelisib were reduced 47% and 73%, respectively, compared with the TGF-β1 group. These data indicated that Duvelisib could suppresses TGF-β1-induced migration of lung fibroblasts.

| Duvelisib attenuates TGF-β1-induced activation of lung fibroblasts
To further examine whether Duvelisib could inhibit the activation of TGF-β1-induced fibroblasts, we treated Mlg cells with TGF-β1 (5 ng/ ml) and Duvelisib (1.25, 2.5, and 5 μM) to assess the protein expression levels of the typical fibroblast activation marker α-SMA and the extracellular matrix (ECM) proteins fibronectin. As shown in Figure 4A Accordingly, these results indicated that Duvelisib suppressed TGF-β1-induced myofibroblast differentiation and reduced ECM production in myofibroblasts.

| Duvelisib enhances TGF-β1-inhibited autophagy in lung fibroblasts
In the process of autophagosome formation, a large number of proteins are required to participate in the formation of autophagosome membrane, such as LC3 and p62. The ratio of LC3II/I can estimate the level of autophagy. 17 P62 is an autophagy substrate in cells and the higher the expression of P62, the more serious the autophagy damage. 18

| Duvelisib inhibits TGF-β1-induced activation of PI3K/Akt/mTOR signal pathway in lung fibroblasts
Because Duvelisib is a PI3K inhibitor and PI3K/Akt/mTOR pathway is closely related to fibroblasts activation and autophagy in the process of IPF, we detected the expression of three main related proteins of this signal pathway: PI3K, Akt, mTOR in Mlg and PPF cells by Western blotting. As shown in Figure 6A ##p < 0.01, ###p < 0.001, ####p < 0.0001 as compared with control group. *p < 0.05, **p < 0.01, ****p < 0.0001 as compared with model group.
Akt and mTOR in BLM-PPF ( Figure 6B). These results revealed that Duvelisib restrained the activation of PI3K/Akt/mTOR signalling in active fibroblasts.

| Duvelisib attenuates BLM-induced fibroblast activation and enhances autophagy in vivo
In order to further explore the effect of Duvelisib on BLM induced pulmonary fibrosis, we extracted RNA and protein from mouse lung tissues, carried out immunohistochemistry experiment on lung tissue sections, and determined expression of α-SMA and fibronectin.
The results showed that compared with NaCl group, the expression of α-SMA and fibronectin of BLM group was significantly increased.
After Duvelisib treatment, the expression of α-SMA and fibronectin decreased significantly, and the inhibitory effect of Duvelisib in high-dose group was equivalent to Nintedanib ( Figure 7A,C). The immunohistochemistry experiment of α-SMA and fibronectin showed the same results ( Figure 7D). These results indicated that Duvelisib can inhibit the activation of fibroblasts in vivo. Additionally, the autophagy-related proteins LC3 and p62 were detected by western blot, and their expression levels were improved by Duvelisib treatment in a dose-dependent manner ( Figure 7B). The immunohistochemistry experiment of p62 showed the same results ( Figure 7E).
These data indicated that Duvelisib could promote autophagy in vivo.

| Duvelisib inhibits activation of PI3K/ Akt/mTOR signal pathway in vivo
We have demonstrated that Duvelisib attenuated pulmonary fi-

| DISCUSS ION
Idiopathic pulmonary fibrosis is a fatal chronic interstitial lung disease that is usually triggered by recurrent, multifocal lung parenchymal injury. 19 Patients with IPF only have a median survival F I G U R E 5 Duvelisib relieved blocked autophagy in lung fibroblasts in vitro. (A) The total protein of Mlg cells was extracted for Western blot to detect the protein expression level of autophagy markers LC3I, LC3II and p62. (B) The total protein of NaCl/BLM-PPF cells was extracted for Western blot to detect the protein expression level of autophagy markers LC3I, LC3II and p62. GAPDH was used as a loading control in grayscale analysis. Data are shown as mean ± SD (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 as compared with control group. **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with model group. Duvelisib, a PI3K-targeted inhibitor that inhibits p110δ and p110γ activity, has been approved for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma (SLL). 21 In this study, we used in vivo and in vitro disease models to explore the efficacy and mechanism of Duvelisib on pulmonary fibrosis. The results found that Duvelisib could regulate lung fibroblasts proliferation, migration, activation and autophagy by inhibiting the PI3K/Akt/mTOR signalling pathway, thereby attenuating pulmonary fibrosis.
Bleomycin (BLM) is a clinical chemotherapeutic drug used to treat malignant tumours, and high-dose administration can cause lung injury and fibrosis. 22 Therefore, BLM is widely used to induce lung fibrosis in animals. 23,24 Intratracheal administration of BLM caused lung inflammation in the first week and pulmonary fibrosis in the second and third weeks. 25  Myofibroblasts are currently recognized as the main effector cells of IPF, with significantly enhanced proliferation and ECM production ability, forming a large number of lesions in the lung interstitium. 28 In addition to hyperactivation, myofibroblasts also exhibit a phenotype of insufficient autophagy, which is also involved in regulating ECM formation. 9,29 Studies have shown that increasing the autophagic clearance of type 1 collagen by lung fibroblasts can reduce the invasiveness of IPF fibroblasts. 30 Different studies have also confirmed that autophagy activity is reduced in the lung tissues of IPF patients. 31,32 PI3K/Akt/mTOR is a classic autophagy signalling pathway, and studies have shown that the PI3K/Akt/mTOR signalling pathway is closely related to pulmonary fibrosis. 33 Changes in the PI3K/Akt/mTOR signalling axis enable IPF fibroblasts to maintain a pathological phenotype of collagen overproduction by inhibiting autophagy. 34 Decreased expression of FoxO3a, a direct target of Akt, inhibits the production of the autophagy marker LC3B on the F I G U R E 6 Duvelisib inhibited activation of PI3K/Akt/mTOR signalling pathway in lung fibroblasts in vitro. (A) Mlg cells were treated with TGF-β1 (5 ng/ml) and Duvelisib (1.25, 2.5, 5 μM) for 3 h and the phosphorylation levels of PI3K, Akt and mTOR were analysed by Western blot. (B) NaCl/BLM-PPF cells were treated with Duvelisib (1.25, 2.5, 5 μM) for 3 h and the phosphorylation levels of PI3K, Akt and mTOR were analysed by Western blot. GAPDH was used as a loading control in grayscale analysis. Data are shown as mean ± SD (n = 3). ##p < 0.01, ###p < 0.001, ####p < 0.0001 as compared with control group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with model group.
collagen matrix, thereby inhibiting the autophagic response to collagen in IPF fibroblasts. 35 Inhibition of mTOR activation stimulates autophagy, which is characterized by increased beclin1 and LC3 levels and autophagosome formation. 36 Novel small-molecule inhibitors targeting the PI3K/mTOR pathway have recently entered clinical trials for the treatment of IPF. 37 Duvelisib is a PI3K-targeted inhibitor that simultaneously inhibits the activities of PI3Kδ and PI3Kγ. 37 In previous studies, it was generally believed that p110α and p110β of PI3K were widely distributed, while p110δ and p110γ were mainly expressed in lymphocytes. However, studies by Enrico Conte et al. . ##p < 0.01, ###p < 0.001, ####p < 0.0001 as compared with control group. **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared with model group.
showed that both p110δ and p110γ were expressed in human lung fibroblasts, and blocking the activities of p110δ and p110γ could also inhibit Akt phosphorylation, α-SMA expression and collagen deposition. 6 In this study, we proved that Duvelisib could inhibit the activation of PI3K/Akt/mTOR signalling pathway in vitro and in vivo, then inhibits the proliferation, migration and activation of lung fibroblasts, alleviates the blockage of autophagy in fibroblasts, and thus attenuates the progression of pulmonary fibrosis.
In conclusion, our study showed that Duvelisib has a therapeutic effect in an animal model of bleomycin-induced pulmonary fibrosis, indicating that Duvelisib is promising as a potential drug for the treatment of IPF. F I G U R E 8 Duvelisib inhibited activation of PI3K/Akt/mTOR signalling pathway in lung fibroblasts in vivo. The phosphorylation levels of PI3K, Akt and mTOR in lung tissues were detected by Western blot. Data are shown as mean ± SD (n = 3). ###p < 0.001, ####p < 0.0001 as compared with control group. *p < 0.05, ***p < 0.001, ****p < 0.0001 as compared with model group

ACK N OWLED G EM ENT
Thanks to Wen Ning (Nankai University) for her generous offer of Mlg.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
No data are available in this study.