Copanlisib ameliorates pulmonary fibrosis by modulating cellular autophagy through PI3K/Akt/mTORC1 pathway

Introduction

Idiopathic pulmonary fibrosis (IPF), as a chronic and progressive fibrotic interstitial lung disease (ILD) (1), has a poor prognosis and few effective drugs. The prevalence of IPF is 0.5–4% in Asia, and most patients are over 65 years old (2). The clinical manifestations of IPF include a progressive decline of pulmonary function and deterioration of respiratory symptoms; as a result, the median survival time of patients with IPF has been reported to be 3–5 years. To date, there is still a lack of effective medications for IPF, and the approved treatments that are recommended by international guidelines, pirfenidone and nintedanib, can only slow the progression of the disease rather than stop or even reverse it. Therefore, to improve the prognosis of IPF patients, developing new drugs against pulmonary fibrosis is of the highest priority.

The pathogenesis of IPF is complicated and poorly understood, and it is related to cell viability and recovery, especially in pulmonary fibroblasts and myofibroblasts (3,4). Recurrent subclinical damage to the alveolar epithelium can recruit pulmonary fibroblasts, which proliferate and differentiate into myofibroblasts. Moreover, myofibroblasts are key cells that overproduce collagen, which leads to irreversible harm to the pulmonary parenchyma and ultimately pulmonary function (1). Researchers have shown that these processes involve extracellular matrix (ECM) deposition, epithelial damage, oxidative stress, fibroblast-to-myofibroblast transition (FMT), abnormal immune responses, inflammation and the suppression of cellular autophagy (5). Autophagy is an important degradation process that relies on lysosomes, which can eliminate damaged cells under stress (5). A decreased level of autophagy has been shown to be associated with the pathogenesis of pulmonary fibrosis (6), since it can induce ECM deposition, accelerate the FMT process, and cause dysfunction of the alveolar epithelium and crosstalk with the transforming growth factor-β (TGF-β) pathway. In addition, the PI3K/Akt/mTORC1 pathway is responsible for modulating the level of autophagy (7,8). There are three types of PI3Ks; type I PI3Ks are responsible for cell growth and can be further classified into the α, β, γ and δ subtypes (9). Conte et al. (10) reported that the α and γ subtypes are crucial for the proliferation and differentiation of fibroblasts induced by TGF-β.

Copanlisib, a pan-class I PI3K inhibitor, has been approved for adults experiencing relapsed follicular lymphoma after at least two prior systemic therapies (11,12). Given the important role of the PI3K/Akt/mTORC1 pathway in pulmonary fibrosis, it is possible that copanlisib could reverse pulmonary fibrosis by mitigating the inhibition of autophagy in myofibroblasts.

In this study, we proposed to evaluate copanlisib’s antifibrotic effect and explore the possible underlying mechanism with in vivo and in vitro experiments. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0116/rc).

Methods

Materials

Antibodies targeting fibronectin, alpha smooth muscle actin (α-SMA), collagen I (Col-1), p-PI3K/PI3K, p-Akt/Akt, p-mTORC1/mTORC1, as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were purchased from Affinity Biosciences (Cincinnati, OH, USA). Specifically, the phosphor-epitope used for p-mTORC1 is Ser2448. Antibodies targeting p62 and light chain 3-I/II (LC3-I/II) were obtained from Cell Signaling Technology (Danvers, MA, USA). We also used goat Anti-Mouse IgG (H+L) HRP (ImmunoWay, Plano, TX, USA), DAB Kit (Bioss, Woburn, MA, USA), UltraSensitive^TM SP (Mouse/Rabbit, Fuzhou Maixin Biotech, Fuzhou, China) immunohistochemistry (IHC) Kit (Bioss) and recombinant human TGF-β1 (Peprotech, Cranbury, NJ, USA).

Cell culture

Mouse lung fibroblasts (Mlg, ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; KeyGEN BioTECH, Nanjing, China) supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37 ℃ in an atmosphere containing 5% CO₂.

Cell viability analysis

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) and Cell Counting Kit-8 (CCK-8; Solarbio, Beijing, China) tests were applied to measure cell viability. Mlg cells were seeded in 96-well plates and treated with copanlisib at concentrations ranging from 0 to 2,000 nM for 24 h. For the MTT assay, 20 µL of MTT (5 mg·mL⁻¹) was added to each well for 4 h. Subsequently, 200 µL dimethyl sulfoxide (DMSO) was loaded into each well. The absorbance was then recorded at 570 nm. As regard of CCK-8 assay, 10 µL CCK-8 reagent was added and incubated for 1 h. The absorbance value was tested at 450 nm.

Cell proliferation test

Cell proliferation was also tested via MTT and CCK-8 tests. Mlg cells were co-cultured with copanlisib ranging from 0 to 1,000 nM with or without TGF-β1 (5 ng/mL) for 24 h. The protocol used for the MTT and CCK-8 tests was the same as previously published (13).

Wound closure test

Mlg cells were seeded in a six-well plate and treated with copanlisib, with or without f TGF-β1, for 24 hours. Three parallel lines separated the wound areas in each well. After full cell convergence, a scratch was made in the center of the culture well with a sterile 200 µL pipette tip. The wounds were observed and recorded using an inverted microscope at the intersections of the wound and the marker line at 0, 6, 12, and 24 h post-scratching. The images were analyzed with ImageJ software.

Transwell experiment

A total of 1×10⁵ fibroblasts/mL were suspended in serum-free DMEM; 100 µL of this suspension was placed on the top filter membrane. Varying concentrations of copanlisib and TGF-β1 were then added, incubated for 24 h. Additionally, DMEM with 20% FBS was seeded to the lower chamber of the Transwell plate.

Animals

A protocol was prepared before the study without registration. Male C57BL/6 mice (6–8 weeks of age, body weight 20–25 g) were sourced from Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). All animal experiments were conducted at Peking Union Medical College Hospital. All animal experiments were performed under a project license (No. XHDW-2024-46) granted by the committee for research involving animals at Peking Union Medical College Hospital, in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Mouse pulmonary fibrosis model

The mouse pulmonary fibrosis model was developed by intratracheal BLM administration, as previously described (14). Mice were intratracheally administered 2 U of BLM dissolved in saline (0.9% NaCl). Forty-eight mice were randomly divided randomly into six groups: (I) NaCl group: intratracheal saline administration; (II) BLM group: intratracheal BLM administration; (III) nintedanib group: intratracheal BLM administration with intragastric 100 mg/kg nintedanib; (IV) low-dose copanlisib group: intratracheal BLM administration with intraperitoneal 3 mg/kg copanlisib; (V) medium-dose copanlisib group: intratracheal BLM administration with intraperitoneal 7 mg/kg copanlisib; and (VI) high-dose copanlisib group: intratracheal BLM administration with intraperitoneal 14 mg/kg copanlisib. Mice using copanlisib received intraperitoneally injection of 3, 7, or 14 mg/kg copanlisib suspended in saline every two days. The NaCl and BLM groups were received an equal volume of saline, whereas the positive control group were received 100 mg/kg nintedanib intragastrically daily.

Hydroxyproline (HYP) test

The largest lobe of the right lung from each mouse was isolated and placed in a 5 mL ampoule. After drying, 3 mL of 6 M hydrochloric acid was added and the pH of the mixture was adjusted to 6.5–8.0. Using the hydroxyproline assay kit, we carried out HYP analysis, with 200 µL of each sample transferred to a 96-well plate and divided into 3 replicates. The absorbance was measured at 570 nm.

Pathologic tests

Left lung lobes were harvested and preserved in 10% neutral buffered formalin, followed by a standard dehydration protocol and paraffin infiltration. For histological assessment, sections (5 µm thickness) were heat-fixed at 70 ℃ for 4 hours and subsequently subjected to hematoxylin-eosin (H&E) and Masson’s trichrome staining. Morphometric quantification was performed using ImageJ software to determine the total pixel count of the entire lung area (Pw) and the specifically affected fibrotic regions (Pf). The fibrosis ratio was determined as fibrotic area total pixels (Pf) divided by whole lung total pixels (Pw).

Pulmonary function test (PFT)

The PFT protocol was the same as previously published (13). After anesthesia, the trachea of each mouse was exposed, and intubation with a tracheal cannula was performed. The mice were further moved to a plethysmography chamber to undertake pulmonary function analysis, with an Anires2005 system (Beijing Biolab, Beijing, China).

Western blot (WB) experiments

Proteins were extracted from lung tissues or cells with radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor cocktail (CT) and sodium fluoride (NaF). After electrophoresis and membrane transfer, primary antibodies against GAPDH, tubulin, α-SMA, fibronectin, col-1 p-PI3K/PI3K, p-Akt/Akt, p-mTORC1/mTORC1 (Ser2448), LC3-I/II, and p62 were used for western blotting. Goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibodies were used. The protein bands were visualized with an enhanced chemiluminescence system (Affinity Biosciences), and GAPDH or tubulin was used as control.

Immunohistochemical staining

The paraffin-embedded lung tissue was dewaxed with xylene and heated in a microwave oven using 0.01 M citrate buffer for 2 minutes. After cooling the sections to room temperature, they were blocked with an IHC kit and incubated with the primary antibody overnight at 4 ℃. The primary antibodies used were rabbit anti-α-SMA, rabbit anti-fibronectin, and rabbit anti-Col-1. Following three Phosphate Buffered Saline with Tween (PBST) washes, the tissue sections were incubated together with the secondary antibody at room temperature for 2 h. Afterward, the tissue sections were stained with a 3,3'-diaminobenzidine (DAB) substrate kit and hematoxylin. We applied neutral balsam to fix the sections and assessed the expression of the proteins with an optical microscope.

Micro-computed tomography imaging and image analysis

To evaluate the extent of pulmonary fibrosis in vivo, we conducted microcomputed tomography (micro-CT). After the mice were anesthetized with inhaled isoflurane, in vivo micro-CT scans were performed in the supine orientation. Total pulmonary volume was stratified by Hounsfield unit (HU) ranges into fully, partially, and non-ventilation regions, which served as quantitative markers for healthy tissue, progressing fibrosis, and consolidated fibrotic areas, respectively.

Statistical analysis

All statistical analysis were conducted in Graphpad Prism 10.0 software (GraphPad Software, Inc., LaJolla, CA, USA) as the means ± standard deviations (SDs). All the data were subjected to one-way statistical analysis. P<0.05 was considered as statistically significant.

Results

Copanlisib ameliorates BLM-induced pulmonary fibrosis in mice

To explore the antifibrotic effect of copanlisib in vivo, we used the BLM-induced pulmonary fibrosis model (Figure 1A). HYP is specific to collagen; as a result, the content of HYP reflects the extent of fibrosis. In our study (Figure 1B), there was a significantly greater level of HYP in BLM-treated mice than in NaCl-treated mice, indicating establishment of the fibrosis model. Nintedanib has been shown to exert significant antifibrotic effects in BLM-induced pulmonary fibrosis, and the effects of copanlisib were also shown to be strongly dose-dependent. In the PFT (Figure 1C,1D), there were significant decreases in forced vital capacity (FVC) and dynamic lung compliance (Cydn) in the BLM group, and the application of nintedanib or copanlisib significantly increased both the FVC and Cydn.

Figure 1 Copanlisib could ameliorate the HYP level and PFT in vivo. (A) The scheme of model administration in this study. (B) Hydroxyproline contents of lung tissues in mice. (C) FVC of mice. (D) Cydn of mice. Results are presented as mean ± SD. ^## P<0.01; ^####, P<0.0001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001 vs. model group. BLM, bleomycin; Cydn, dynamic lung compliance; FVC, forced vital capacity; HYP, hydroxyproline; PFT, pulmonary function test; SD, standard deviation.

To further assess the extent of fibrosis and the location of the fibrotic tissue, we used different pathological stains to evaluate (Figure 2A). H&E staining and Masson’s trichrome staining clearly revealed the fibrotic area in the lung slices, and the collagen were stained blue in Masson’s trichrome staining outcomes. H&E staining revealed a significantly greater fibrotic area after BLM intratracheal administration, and the administration of nintedanib and copanlisib markedly reversed this pathological process, which was also observed by Masson’s trichrome staining. As shown in Figure 2B, the percentage of fibrosis was significantly lower in the nintedanib group and copanlisib group than in the BLM group. Since Col-1, α-SMA and fibronectin are highly expressed in lung fibrosis, we used IHC (Figure 2C,2D) and WB (Figure 2E) to evaluate the expression levels of these proteins in the different groups. The expression rates of α-SMA, Col-1 and fibronectin were much greater in BLM-treated mice than in control mice but were decreased by nintedanib or copanlisib.

Figure 2 Copanlisib could decrease the production of protein related to pulmonary fibrosis in vivo. (A) Lung tissue sections were stained with H&E, Masson trichrome staining and Immunohistochemical staining analysis of α-SMA and Col-1. (B) Statistics of lung fibrosis area among groups. (C) Protein level of α-SMA was verified by IHC in lung tissue sections. (D) Protein level of Col-1 was verified by IHC in lung tissue sections. (E) Protein level of Col-1 was verified by western blot. ^#, P<0.05; ^## P<0.01; ^####, P<0.0001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. model group. BLM, bleomycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin-eosin; IHC, immunohistochemistry; MOD, mean optical density; SMA, smooth muscle actin.

Micro-CT can clearly reveal intrapulmonary changes with noninvasive methods. As shown in Figure 3A, bleomycin (BLM) decreased the normal volume of the lung, and this effect could be reversed by nintedanib in a dose-dependent manner. Furthermore, according to different CT HU values, the lung volume could be further categorized as full ventilation, partial ventilation or no ventilation, which are in accordance with healthy lungs, lungs in the process of fibrosis, and lungs with clear fibrosis, respectively. The results shown in Figure 3B,3C confirmed that the application of nintedanib or copanlisib could significantly reduce the volume of the lung during the process of fibrosis, and a high dose of copanlisib could even considerably restore the full ventilation volume.

Figure 3 Copanlisib could improve the volume of fully ventilation. (A) Representative micro-CT images of mice in different groups. (B) Statistics of volume of partial ventilation among groups. (C) Statistics of volume of fully ventilation among groups. ^#, P<0.05; ^###, P<0.001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001 vs. model group. BLM, bleomycin; CT, computed tomography.

Copanlisib ameliorates the TGF-β1-induced proliferation, migration and activation of pulmonary fibroblasts in vitro

To evaluate the influence of copanlisib on the proliferation and survival of Mlg cells, MTT assays were first performed (Figure 4A,4B). The results showed that copanlisib, even at a concentration of 1,000 nM, did not impair cell survival in the absence (Figure 4A) or presence (Figure 4B) of TGF-β1 stimulation. These findings were further validated by CCK-8 assays (Figure 4C,4D), which demonstrated consistent survival and proliferation patterns under both basal (Figure 4C) and TGF-β1-treated (Figure 4D) conditions. The results showed that copanlisib inhibited the proliferation of TGF-β1-activated Mlg cells at concentrations greater than 62.5 nM. On the basis of the results above, we chose three varying levels (62.5, 125, and 250 nM) at which the proliferation of TGF-β1-activated Mlg cells was suppressed while the survival of the cells was not be affected.

Figure 4 Copanlisib attenuates TGF-β1-induced proliferative activity in Mlg lung fibroblasts. The impact of Copanlisib on Mlg cell viability and proliferation was evaluated using MTT (A,B) and CCK-8 (C,D) assays. (A,C) Basal cytotoxicity of Mlg cells after 24 h of exposure to indicated copanlisib concentrations. (B,D) Mlg cells were treated with 5 ng/mL TGF-β1 and varied copanlisib doses for 24 h to assess its anti-proliferative effects. Values represent mean ± SD (n=3). ^#, P<0.05; ^##, P<0.01; ^###, P<0.001; ^####, P<0.0001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. model group. CCK-8, Cell Counting Kit-8; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide; SD, standard deviation; TGF-β1, transforming growth factor beta 1.

To determine the effect of copanlisib on cell migration, wound closure and Transwell experiments were conducted. In the wound closure assay (Figure 5A), copanlisib dose-dependently inhibited the migration of Mlg cells. After 24 h, there was an approximately 58.4% reduction in migration ability in the high-dose copanlisib group compared with the TGF-β group. Similar results were obtained in Transwell experiments (Figure 5B): copanlisib significantly suppressed the migration of fibroblasts, while the proportion of migrated cells on the bottom membrane with medium and high doses of copanlisib was reduced by 62.7% and 83.8%, respectively. In addition, the shape of the cells also changed to an irregular polygonal shape that was distinct from the typical spindle shape under a high dose of copanlisib.

Figure 5 Copanlisib inhibits the TGF-β1-stimulated migration of Mlg lung fibroblasts. (A) Wound healing assays of Mlg cells following treatment with TGF-β1 (5 ng/mL) and graded doses of copanlisib (62.5–250 μM); wound closure was monitored and photographed at 0, 6, 12, and 24 h. (B) Transwell migration capacity of Mlg cells (crystal violet staining) under identical stimulation and treatment conditions for 24 h. Migrated cells were quantified at the study endpoint. Values are expressed as mean ± SD (n=3). ^#, P<0.05 vs. control group. **, P<0.01; ***, P<0.001 vs. model group. SD, standard deviation; TGF-β1, transforming growth factor beta 1.

To evaluate the in vitro antifibrotic effect of copanlisib, we treated Mlg cells with different doses of copanlisib and compared the protein expression levels of Col-1, fibronectin and α-SMA, which are the typical fibroblast activation markers. As shown in Figure 6, copanlisib dramatically decreased the production of fibrosis-related proteins, such as Col-1, fibronectin and α-SMA. Therefore, copanlisib could suppress the proliferation and migration of fibroblasts, differentiation induced by TGF-β1, as well as ECM production.

Figure 6 Copanlisib decreases activation of lung fibroblasts in vitro. Mlg cells were treated with TGF-β1 (5 ng/mL) and copanlisib (62.5, 125, 250 μM) for 24 h. The expression of Col-1 (B), fibronectin (C), and α-SMA (D) was analysed by Western blot analysis (A). Data are shown as mean ± SD (n=3). ^###, P<0.001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. model group. SD, standard deviation; SMA, smooth muscle actin; TGF-β, transforming growth factor beta.

Exploration of the antifibrotic mechanism of copanlisib in vitro

Owing to the importance of the PI3K/Akt/mTORC1 pathway for fibroblast activation and autophagy in our experimental models, we hypothesized that the pan-class I PI3K inhibitor copanlisib can suppress pulmonary fibrosis, by interfering with the PI3K/Akt/mTORC1 pathway and modulating autophagy-related markers. Therefore, we tested the expression of three main proteins and their activated forms in this signaling pathway. As shown in Figure 7A-7D, the levels of phosphorylated PI3K, Akt and mTORC1 (Ser2448), the activated forms, were significantly decreased by copanlisib treatment in a dose-dependent manner, which suggested that copanlisib is associated with the inhibition of the PI3K/Akt/mTORC1 pathway in TGF-β1-activated fibroblasts.

Figure 7 Copanlisib suppresses PI3K/Akt/mTOR signaling and restores autophagy in TGF-β1-stimulated Mlg cells. (A-D) Western blot analysis of the PI3K/Akt/mTOR pathway in Mlg cells treated with 5 ng/mL TGF-β1 and copanlisib (62.5–250 μM) for 3 h. Representative blots (A) and densitometric quantification of phosphorylated PI3K (B), Akt (C), and mTOR (D) are shown. (E-G) Protein levels of autophagic markers LC3-I, LC3-II (F), and p62 (G) were assessed via immunoblotting. GAPDH served as an internal loading control. Data represent mean ± SD (n=3). ^##, P<0.01; ^###, P<0.001; ^####, P<0.0001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. model group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation; SMA, smooth muscle actin; TGF-β, transforming growth factor beta.

Besides, LC3 and p62 are two important markers of autophagy. The ratio of LC3 II/I and the expression level of p62 reflect the status of these markers. As shown in Figure 7E-7G, the addition of TGF-β1 to fibroblast is consistent with the alterations in these proteins, and this trend was dose-dependently modulated by the addition of copanlisib.

Exploration of the antifibrotic mechanism of copanlisib in vivo

To further confirm that copanlisib inhibited the phosphorylation of the PI3K/Akt/mTORC1 signaling pathway in vitro, we performed western blotting on the lung tissues of BLM-treated mice. In BLM-induced mice lung tissues, the phosphorylation of the PI3K/Akt/mTORC1 pathway was increased, and this change was reduced by the addition of copanlisib (Figure 8A-8D). However, nintedanib inhibited the phosphorylation of only mTORC1 and had little effect on p-PI3K or p-Akt.

Figure 8 Copanlisib blunts BLM-induced PI3K/Akt/mTOR signaling activation and rescues autophagic flux in vivo. (A) Western blot analysis of lung tissue lysates was performed to assess the phosphorylation status of the PI3K/Akt/mTOR axis. Corresponding densitometric ratios of p-PI3K/PI3K (B), p-AKT/AKT (C), and p-mTOR/mTOR (D) are presented. GAPDH was employed as a loading control for grayscale normalization. (E) Protein levels of LC3 (F) and p62 (G) were verified by Western blot in lung tissues. GAPDH was used as an internal reference in densitometric analysis. Data are shown as mean ± SD (n=3). ^##, P<0.01; ^###, P<0.001; ^####, P<0.0001 vs. control group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 vs. model group. BLM, bleomycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SD, standard deviation.

To assess the level of autophagy-related markers in vivo, we used western blotting to evaluate the expression levels of LC3 and p62 in the lung tissues of BLM-treated mice. The results (Figure 8E-8G) showed that BLM was associated with a reduction in these autophagy-related markers in the lung tissue, and nintedanib had a slight effect on their expression. However, copanlisib at medium and high doses led to changes consistent with increased autophagic activity, which was associated with the attenuation of fibrosis.

Discussion

Currently, only a few treatment options are available for IPF. Nintedanib, a multitarget tyrosine kinase inhibitor, has been approved for the treatment of IPF (15), systematic sclerosis-related ILD (SSc-ILD) (16), and progressive fibrosing ILD (PF-ILD) (17). Moreover, pifernidone has been approved for IPF (18), and its inhaled form is in a phase II clinical trial (19). However, as this is a challenging and difficult to manage disease, more potent treatment options are needed. Approximately 61 drugs are currently in clinical trials, most of which target fibroblasts as effector cells in the fibrosis process. Similarly, we aimed to target fibroblasts with a pan-class I PI3K inhibitor for antifibrosis treatment. In this study, we applied the pan-class I PI3K inhibitor copanlisib in a BLM-treated mouse model to identify an effective drug for pulmonary fibrosis through western blotting, IHC, PFT and micro-CT. Copanlisib exerted similar or superior antifibrotic effects on BLM-induced pulmonary fibrosis compared to nintedanib: it inhibited the expression of fibrosis-related proteins, increased the ventilation volume, and consequently ameliorated pulmonary fibrosis. Furthermore, we hypothesized that copanlisib fights against pulmonary fibrosis by ameliorating the inhibition of autophagy in fibroblasts by profibrotic factors. Given that copanlisib is on the market, further validation of the efficacy of copanlisib in IPF in a clinical trial is possible and could provide novel options for IPF treatment in the future.

In research on pulmonary fibrosis, studies have focused on fibroblasts as the primary effector cells, epithelial cells as the main initiator cells, and macrophages as the key participating cells. Therefore, multiple drugs targeting fibroblasts are currently under development (20,21). In other words, the ability to inhibit the proliferation and differentiation of fibroblasts could reflect the effects of drugs on pulmonary fibrosis. As a result, our study revealed that copanlisib inhibited fibroblast proliferation and differentiation, as shown in Figures 4,5. Furthermore, the WB results also verified that the administration of copanlisib could reduce the degree of fibrosis. A similar conclusion was reached by in vivo pathological sectioning and western blotting (Figure 2), which revealed that the administration of copanlisib markedly reversed pulmonary fibrosis under BLM stimulation. Moreover, in the clinical course of IPF, PFT and thoracic high-resolution CT are among the most important examinations used to evaluate the extent and prognosis of IPF. FVC and diffusing capacity of the lungs for carbon monoxide (D_LCO) are key measurements in PFT, since patients with IPF often suffer from restrictive ventilation disorders (2). In addition, Cydn can also reflect lung compliance, which can be impaired by fibrosis. Consequently, our study used PFT and micro-CT to evaluate the efficacy of copanlisib in BLM-induced mice, and the results showed that the administration of copanlisib markedly preserved pulmonary function (Figure 1) and lung volume (Figure 3), even better than nintedanib, a drug marketed for the treatment of IPF.

Autophagy involves initiation, nucleation, elongation, maturation, fusion and degradation (5). Markers reflecting alteration in autophagic level and function include microtubule-associated protein 1A/1B-LC3, a ubiquitous protein distributed ubiquitously in mammalian tissues and cultured cells. The cytoplasmic form of which (LC3-I) is converted to LC3-phosphatidylethanolamine conjugate (LC3-II) during autophagy, and the LC3-II/LC3-I ratio can consequently reflect autophagic function (22,23); in addition, p62 interacts with autophagic substrates, delivering them to autophagosomes for degradation (24), with p62 itself degraded when autophagy is induced. As a consequence, a decrease in p62 levels should be observed when autophagy is elevated (25). Recently, autophagy in fibroblasts has been shown to be related to IPF. Abdel Fattah et al. (26) reported that mice with autophagy deficiency exhibit exacerbated inflammation and fibrosis in the context of BLM-induced fibrosis. Accordingly, the inability to fuse autophagosomes with lysosomes, elevated intracellular p62 expression, and accumulation of ubiquitinated proteins were also found in IPF lung tissues (6). Considering the discoveries above, Klionsky et al. (5) proposed that the antifibrotic effects attributed to autophagy are as follows: (I) increased resilience of alveolar epithelial cells to apoptosis; (II) attenuation of TGF-β-mediated fibroblast differentiation; and (III) inhibition of the inflammatory cascade. As a result, several studies (27,28) have suggested that the activation of autophagy can counter pulmonary fibrosis. In our study, we measured these markers and observed a significant decrease in the LC3-II/I ratio alongside an elevation of p62 levels in the lung tissues of BLM-treated mice and TGF-β1-stimulated lung fibroblasts (Figures 7,8), which is suggestive of a modulation toward increased autophagic activity in these models.

In mammalian cells, autophagy initiation is regulated by the Unc-51-like kinase 1 (ULK1) or ULK2 complex (13), modulated by mammalian target of rapamycin complex 1 (mTORC1) through the phosphorylation of ULK1 (29,30). In addition, the PI3K/Akt pathway can activate mTORC1 by suppressing tuberous sclerosis complex 2 (TSC2), which inhibits the activity of mTORC1 (31). Our study measured the phosphorylation levels of key nodes in this axis, and found that the phosphorylation of the PI3K/Akt/mTORC1 pathway was increased in BLM-induced mice lung tissues and TGF-β-stimulated fibroblasts, and this effect was suppressed by copanlisib (Figures 7,8). Together with the modulation of autophagy-related markers observed after treatment with copanlisib, these findings suggested that the pan-class I PI3K inhibitor copanlisib is associated with the suppression of the PI3K/Akt/mTORC1 pathway and the modulation of the autophagy pathway, which may contribute to amelioration of pulmonary fibrosis under specific experimental conditions (BLM or TGF-β stimulating in vivo and in vitro models). When viewed alongside the observed changes in LC3 and p62, these findings suggest a potential mechanistic link where the suppression of PI3K

Our study still has several limitations: first, as copanlisib is a pan-class I PI3K inhibitor, its anti-fibrotic effects likely stem from a comprehensive suppression of the PI3K/Akt/mTOR signaling cascade. While the reduction in p-mTOR (Ser2448) is highly suggestive of mTORC1 modulation, further studies using complex-specific inhibitors or downstream readouts (such as p-p70S6K for mTORC1 or p-Akt Ser473 for mTORC2) would be required to fully dissect the relative contributions of mTORC1 and mTORC2 to the observed phenotype. Second, the current study lacks direct measurements of autophagic flux, such as tandem mRFP-GFP-LC3 reporter. We intend to further investigate the spatiotemporal dynamics of autophagy in our future research. Third, although our current study is highly suggestive of a mechanistic link between modulation of the PI3K/mTORC1 pathway and autophagy flux, further research involving autophagy dependency is needed to establish a direct dependency. Finally, we did not isolate which specific isoform of PI3K dominate the phenotype found in our study, and further study will be working on discrimination of the phenotype of PI3K.

Conclusions

In conclusion, copanlisib exerts antifibrotic effects on fibroblasts by alleviating the inhibition of autophagy through the PI3K/Akt/mTORC1 pathway and is a promising candidate for treating pulmonary fibrosis.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0116/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0116/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0116/prf

Funding: This study was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (No. 2023ZD0509500) and Beijing Natural Science Foundation (No. 7242102).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1-0116/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. XHDW-2024-46) granted by the committee for research involving animals at Peking Union Medical College Hospital, in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

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