Skip to main content
Download PDF

Albendazole ameliorates aerobic glycolysis in myofibroblasts to reverse pulmonary fibrosis

Journal of Translational Medicine volume 22, Article number: 910 (2024) Cite this article

Abstract

Background

Idiopathic pulmonary fibrosis (IPF) is a chronic and lethal lung disorder for which effective treatments remain limited. Recent investigations revealed a potential link between altered glucose metabolism and the activation of fibroblasts, the key cells responsible for generating and depositing extracellular matrix proteins within the lung interstitium during IPF development.

Method

In this study, we aimed to investigate the potential therapeutic impact of albendazole on fibroblast to myofibroblast transition in IPF. We assess albendazole’s effectiveness in attenuating the activation of fibroblasts. We focused on elucidating the mechanism underlying albendazole's impact on TGF-β1-induced aerobic glycolysis in both lung tissues and fibroblasts obtained from patients with IPF and other lung fibrosis types. Furthermore, the antifibrotic effects of oral administration of albendazole were investigated in mouse models of pulmonary fibrosis induced by BLM or SiO2. Human precision-cut lung slices were employed to evaluate the impact of albendazole following TGF-β1 stimulation.

Result

In this work, we demonstrated that albendazole, a first-line broad-spectrum anthelmintic drug, effectively attenuated fibroblast to myofibroblast transition through alleviating TGF-β1-induced aerobic glycolysis dependent on the LRRN3/PFKFB3 signaling pathway. Additionally, LRRN3 expression was downregulated in both lung tissues and fibroblasts from patients with IPF and other types of lung fibrosis. Importantly, the levels of LRRN3 correlated with the progression of the disease. Notably, oral administration of albendazole exerted potent antifibrotic effects in mouse models of pulmonary fibrosis induced by BLM or SiO2, and in human precision-cut lung slices after TGF-β1 stimulation, as evidenced by improvements in lung morphology, reduced myofibroblast formation, and downregulation of α-SMA, collagen type 1 and Fibronectin expression in the lungs.

Conclusion

Our study implies that albendazole can act as a potent agonist of LRRN3 during fibroblast to myofibroblast differentiation and its oral administration shows potential as a viable therapeutic approach for managing IPF.

Introduction

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder characterized by the gradual formation of scars within the lung tissue, leading to impaired gas exchange, respiratory failure, and high mortality rates [1]. Despite the availability of FDA-approved drugs such as pirfenidone and nintedanib, these treatments offer only limited benefits and are unable to halt or reverse disease progression, with a 5-year mortality rate exceeding 70%–80% after diagnosis [2, 3]. This underscores the urgent need for the development of safe and effective therapeutic approaches for IPF.

Despite extensive investigations, the underlying mechanisms of IPF remain elusive. It is generally believed that IPF results from an aberrant wound healing process, leading to the excessive accumulation of extracellular matrix (ECM) in the pulmonary interstitium, ultimately resulting in impaired gas exchange and respiratory failure [4,5,6,7]. Previous research, including our own, has demonstrated that myofibroblasts are identified as the primary cells driving ECM production and deposition in the fibrotic microenvironment [8,9,10]. Notably, the transition of fibroblasts into myofibroblasts is modulated by various factors and recent studies have demonstrated that aerobic glycolysis plays a pivotal role in myofibroblast differentiation, which fuels their functional activities, including proliferation, migration, and collagen synthesis [10, 11]. Inhibiting aerobic glycolysis has been shown to reduce myofibroblast formation and ECM production, while there is still lacking effective and safe drug targeting myofibroblast metabolism during the development of IPF.

Albendazole (ALB), an FDA-approved broad-spectrum anthelmintic drug, has been safely used for decades in both humans and animals [11]. Its pharmacological effects derived from its interaction with the colchicine-sensitive site on β-tubulin in parasites. This interaction disrupts microtubule polymerization, cellular structure, and transport, ultimately leading to paralysis and death of the parasites [12]. Notably, ALB has been found to affect glucose metabolism by inhibiting metabolic enzymes such as malate dehydrogenase and fumarate reductase [13]. Given the potential of repurposing existing drugs for new therapeutic applications, this research seeks to evaluate the therapeutic effects of ALB on myofibroblast metabolism and pulmonary fibrosis.

In this study, we demonstrated that oral administration of ALB effectively ameliorated fibrotic responses in pulmonary fibrosis mouse models induced by bleomycin (BLM) or silicon dioxide (SiO2), as well as in human precision-cut lung slices (hPCLS) stimulated with TGF-β1. These therapeutic effects were evidenced by improvements in lung morphology, reduced myofibroblast formation, and downregulation of α-SMA, Collagen 1, and Fibronectin within the lungs. Mechanistically, ALB remarkably attenuated the aerobic glycolysis of fibroblasts induced by TGF-β1 through the LRRN3/PFKFB3 signaling axis. Moreover, our data mining analysis revealed that LRRN3 was significantly decreased in the lung tissues from patients with IPF and other types of lung fibrosis, and its levels were negatively correlated with disease progression. LRRN3 expression also showed a decreasing trend during fibroblast activation and was negatively correlated with myofibroblast formation. Collectively, our data support that ALB may act as a potent agonist of LRRN3 during fibroblast to myofibroblast differentiation, making oral administration of ALB a promising therapeutic intervention for lung fibrosis in clinical settings.

Materials and methods

Reagents and antibodies

Antibodies against Fibronectin, Collagen 1, α-SMA, PKM2 and PFKFB3 were procured from Proteintech (Wuhan, China). Antibodies specific to HK2 and LDHA were sourced from Abclonal Technology (MA, USA). Antibody specific for LRRN3 was supplied by ThermoFisherScientific (MA, USA). ALB (Supplementary Fig. 1A) was obtained from Yuanye Bio-Technology (Shanghai, China). TGF-β1, BLM, SB431542, SIS3-HCL, 5-Aza and PFK-015 were acquired from MedChemExpress (NJ, China). SiO2 were purchased from Sigma (MO, USA).

BLM or SiO2-induced fibrosis model and treatment

C57BL/6 male mice (8-week-old) were supplied by Beijing Huafukang Bioscience (Beijing, China) and housed under specified pathogen-free (SPF) conditions at the Tongji Hospital with a 12:12-h light/dark cycle. All experimental procedures were executed in compliance with the Animal Care and Use Committee guidelines of Tongji Hospital.

BLM-stimulated fibrosis model: All mice were randomly assigned to six groups: (1) PBS + PBS group, (2) PBS + ALB (200 mg/kg) group, (3) BLM + PBS group, (4) BLM + ALB (100 mg/kg, L-ALB) group, (5) BLM + ALB (200 mg/kg, H-ALB) group, and (6) BLM + Pirfenidone (PFD, 100 mg/kg) group. Anesthetized mice were intratracheally injected with 1.5 mg/kg BLM in 50 μL of PBS using a high-pressure atomizing needle (BJ-PW-M; BioJane Trading Ltd., Shanghai, China) to induce pulmonary fibrosis as previous reported [14]. Mice receiving an equivalent amount of PBS served as control group. The initiation of modeling was designated as day 1. From day 14 onwards, mice in the PBS + ALB group, BLM + ALB group or BLM + PFD group were orally administered ALB (100/200 mg/kg) or PFD (100 mg/kg) once daily for 7 consecutive days. Meanwhile, mice in the BLM + PBS and PBS + PBS groups received the equal amount of PBS. On day 21 following BLM challenge, the mice were euthanized to evaluate pulmonary fibrosis.

SiO2-stimulated fibrosis model: The mice were randomly assigned to six groups: (1) PBS + PBS group, (2) PBS + ALB (200 mg/kg) group, (3) SiO2 + PBS group, (4) SiO2 + ALB (100 mg/kg, L-ALB) group, (5) SiO2 + ALB (200 mg/kg, H-ALB) group, (6) SiO2 + PFD (100 mg/kg) group. Intratracheal instillation of SiO2 (200 mg/kg in 50 mg/mL PBS) or PBS, as previously described [15], was performed. The initiation of modeling was designated as day 1. Starting from day 28, mice in the SiO2 + ALB groups were orally administered with ALB (100/200 mg/kg) or PFD (100 mg/kg) for 14 consecutive days. Meanwhile, mice in the SiO2 + PBS and PBS + PBS groups were exposed to an equal amount of PBS. On day 42, the mice were sacrificed to analyze the phenotypes of IPF.

Histological analysis

The left lungs were dissected and fixed in paraformaldehyde (4%) for 24 h. After fixation, the lungs were subjected to paraffin embedding, followed by sectioning. Sirius red, Masson's trichrome, and Hematoxylin and eosin (H&E) staining were executed on the sections as previous reported [16]. Fibrosis was evaluated using the Ashcroft scoring method on a scale ranging from 0 to 8 [17]. The scores from multiple microscopic fields were averaged to determine the overall fibrotic alterations in each lung section. The results were visualized on a graph at 200-fold magnification. Three independent pathologists carried out the grading in a blinded fashion. The Sirius red staining images were recorded under a polarization microscope at 200-fold magnification.

Human Precision-Cut Lung Slices (hPCLS)

Fresh tissue specimens from non-small-cell lung cancer patients were gently impregnated with 1.5% low-melting agarose at 38 °C through a visible bronchus, Subsequently, the tissues were cooled on ice for a duration of 20 min, as described previously [18]. The obtained tissue samples were then sliced into sections (400 µm thickness) using a Compresstome (VF-300-0Z; Precisionary; oscillation frequency = 5 Hz and cutting speed = 6 μm/s). The hPCLS were cultured in 12-well plates containing 1 mL DMEM-F12 medium without phenol red, supplemented with 0.1% FBS and penicillin/streptomycin, and maintained at 37 °C. The hPCLS were stimulated by TGF-β1 for 24 h and then exposed to ALB (250 nM) for 24 h.

Isolation and culturing of primary human lung fibroblasts

Primary lung fibroblasts were obtained from the lungs of control subjects, following established protocols [19]. Briefly, fresh lung tissues were minced into small fragments, which were then evenly distributed onto 10 cm plates with 2 ml DMEM containing 1% penicillin/streptomycin and 10% FBS. After 24 h, the culture medium was replenished to a total volume of 10 ml DMEM containing 10% FBS and maintained at 37 °C and 5% CO2. Only cells within passages 3 to 5 were employed for subsequent analyses.

To evaluate the effect of ALB on fibroblast differentiation, fibroblasts were exposed to either DMSO, L-ALB (50 nM) or H-ALB (250 nM), followed by stimulation with 10 ng/ml TGF-β1 for 24 h.

To investigate the role of LRRN3 in fibroblast activation, small interfering RNA (siRNA) specific for LRRN3 (5’-GTA CAT CAC TGA AAG TAA A-3’) was obtained from RiboBio (Guangzhou, China). The siRNAs were transfected into primary human lung fibroblasts using Lipofectamine3000 (Invitrogen), following previously described procedures [20]. Subsequently, the transfected cells were exposed to TGF-β1 (10 ng/ml) and a PFKFB3 inhibitor (PFK-015, 100 nM) at specified time points.

Western blot (WB) analysis

RIPA lysis buffer (Beyotime, Shanghai, China) was utilized to homogenize cells and lung tissues. WB was performed according to established protocols [21]. Briefly, proteins were separated through 10% SDS-PAGE and then transferred onto 0.45 μm PVDF membranes. After blocking with 5% milk at room temperature for 60 min, the membranes were incubated overnight at 4 °C with primary antibodies, including anti-PFKFB3 (1:1000), anti-Fibronectin (1:1000), anti-Collagen 1 (1:1,000), anti-α-SMA (1:1000), and anti-β-actin (1:1,000), anti-PKM2 (1:1000), anti-HK2 (1:1000), anti-LDHA (1:1000). After that, HRP-labelled anti-rabbit or anti-mouse IgG secondary antibodies were applied and incubated for 1 h. ImageJ software was used for analysis of gray values.

Quantitative RT-PCR assays

Total RNA was extracted from cells and lung tissues using TRIzol reagent (Takara, Dalian, China), following established protocols [21]. The yield and quality of RNA samples were examined using a NanoDrop2000 spectrophotometer (ThermoScientific). Subsequently, cDNA synthesis was executed using HiScript-III-RT-SuperMix (Vazyme). Quantitative RT-PCR assays were conducted on a CFX96 RT-PCR detection system (Bio-Rad) with SYBR qPCR Master Mix (Vazyme). The following cycling conditions were applied: initial denaturation for 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C for and 30 s at 60 °C. The Ct method was employed for data normalization, presenting the mean of the normalized transcript levels. Actb was utilized as the reference for data normalization. The primers used are provided in Supplementary Table 1.

RNA sequencing (RNA-seq) analysis

RNAs were extracted from fibroblasts and lung tissues using the RNA-isolation-kit (QIAGEN). The integrity and quality of the extracted RNA were evaluated using the Nanodrop1000 Spectrophotometer (ThermoFisherScientific) and the Agilent Bioanalyser (DNA-TECH), respectively. Subsequently, the samples were subjected to sequencing on an Illumina HiSeq2000 V3 instrument. The sequencing protocols were performed according to the manufacturer's protocols, utilizing the Illumina-HiSeq-1000-system and HiSeq-control-software. To enable comparison with previously documented RNA-seq data from human IPF tissues (GSE47460), only homologous genes expressed in both human and mouse were selected.

Receiver-operator characteristics (ROC) curve analysis and the area under the ROC curve were used to evaluate the diagnostic effect of LRRN3 for IPF patients and healthy subjects using dataset (GSE47460) in SPSS v23.

We obtained the methylation profiling data GSE135097 from the GEO database, which comprised Illumina 450 k methylation microarray data obtained from primary human lung fibroblasts treated with or without TGF-β1 (10 ng/ml) for either 24 h or 5 days [22]. Subsequent analysis was performed using the ChAMP methylation analysis package in R (v4.2.2) [23]. In brief, raw IDAT files were imported into R (v4.2.2), followed by intensity data quality control and normalization using the BMIQ method. To identify differentially methylated positions (DMPs), we utilized the "champ.DMP" function, focusing on DMPs within the LRRN3 gene. Annotation of these DMPs was done using the "HumanMethylation450 v1.2 Manifest File" released by Illumina, and the "DMP.GUI" function was employed for visualization purposes.

Transwell assay

Fibroblasts treated with DMSO, L-ALB (50 nM) or H-ALB (250 nM) were harvested and suspended in serum-free DMEM. The cells (2 × 104 cells/well) were then reseeded into the upper chamber of transwell, whereas the lower chamber was filled with 10% FBS-containing DMEM. Following reseeding, the cells were left to migrate towards the lower chamber for 24 h. After washing with PBS, the cells were fixed with paraformaldehyde (4%) for 20 min, and subsequently stained with crystal violet for 20 min. The migrated cells were observed under a light microscope (Leica, Wetzlar, Germany) at × 200 magnification. To quantify the migration, the numbers of cells in three randomly selected fields were counted, and the experiments were repeated three times to ensure reliability.

EdU assay

The impact of ALB on the proliferation of fibroblast was evaluated using the Cell-Light-EdU-Apollo488-In-Vitro-Kit (RiboBio) as previous reported [24]. In brief, fibroblasts were treated with DMSO, L-ALB (50 nM) or H-ALB (250 nM) for 24 h. Subsequently, the cells were exposed to 100 μL EdU solution (50 μM) for 2 h and fixed with paraformaldehyde (4%) for 30 min. Permeabilization buffer was then applied, and the cells were incubated for an additional 15 min. A fluorescently labeled Apollo488 solution was subsequently added to the reaction mixture and incubated for another 30 min. Lastly, the cell nuclei were stained with DAPI and examined under a microscope (Nikon Ti, Japan).

Immunofluorescence

Fibroblasts were fixed with paraformaldehyde (4%) for 5 min, followed by 3 washes with PBS. Following this, the cells were exposed to a solution of 10% normal donkey serum in PBS for a duration of 1 h. Following this step, the cells were incubated overnight at 4 °C with primary antibodies targeting Collagen 1 (1:100), α-SMA (1:100), LRRN3 (1:100), or PFKFB3 (1:100), as previously described [25]. After the primary antibody incubation, the cells were further exposed to Alexa 488- or 594-conjugated antibodies (Invitrogen). Lastly, the cell nuclei were stained with DAPI and examined under a microscope (Nikon Ti).

Real-time cell metabolism assay

The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF24 analyzer. Briefly, fibroblasts (5 × 104/well) were resuspended in sterile XF base media containing 10 mM d-glucose (pH 7.4), and then seeded on 0.001% poly-l-lysine-pre-coated XFp cell culture plates. The cells were left to settle at 37 °C for 30 min. Real-time assessments of ECAR and OCR were obtained following the manufacturer's instructions. OCR was determined under the conditions as follows: (i) baseline; (ii) 1 μM oligomycin; (iii) 0.3 μM FCCP; and (iv) 0.5 μM antimycin + 0.5 μM rotenone. For the quantification of ECAR, 2-DG, a glycolysis inhibitor, was introduced to halt glycolytic acidification. The values of ECAR and OCR were adjusted relative to the overall protein content.

Statistical analysis

Statistical differences between groups were compared using Prism software (Prism 8.lnk; GraphPad Software, CA, USA). For comparisons between two groups, the two-tailed t-test was employed for normally distributed data and homogeneity of variance, while the two-tailed Mann–Whitney test was used for non-normally distributed data. When comparing multiple groups, either one-way or two-way analysis of variance (ANOVA) with Tukey's multiple comparison test was applied for normally distributed data, while the Kruskal–Wallis test with Dunn's post hoc tests was utilized for non-normally distributed data. All experiments were conducted with a minimum of three independent replications. The data are presented as mean ± SD/SEM. p < 0.05 was deemed statistically significant.

Result

Albendazole attenuates the profibrotic phenotype of primary human pulmonary fibroblasts

To experimentally assess the antifibrotic potential of ALB in the lungs, we initially treated primary human pulmonary fibroblasts with varying doses of ALB for 24 h, and its toxicity was then evaluated. Our results demonstrated a low toxicity of ALB towards fibroblasts, with the viability of fibroblasts remained above 80%, When the concentration of albendazole reached 250 nM (Supplementary Fig. 1B). Subsequently, fibroblast-to-myofibroblast transition was stimulated by TGF-β1 following ALB treatment. As expected, TGF-β1-treated fibroblasts exhibited characteristics of myofibroblasts, as revealed by high expression of myofibroblast biomarkers (Fibronectin, Collagen 1, and α-SMA) at protein (Fig. 1A) and RNA levels (Fig. 1B–D). However, ALB treatment significantly abrogated the expression of Fibronectin, Collagen 1, and α-SMA (Fig. 1A–D). Furthermore, fibroblasts treated with ALB at a concentration of 250 nM exhibited lower expression of myofibroblast markers compared to those treated with 50 nM of ALB after TGF-β1 administration, suggesting the transition from fibroblast to myofibroblast is dose-dependently inhibited by ALB. In line with these data, the immunostaining of Collagen 1 yielded similar results (Fig. 1E).

Fig. 1
figure 1

The effects of albendazole on the differentiation, proliferation, and migration of fibroblasts. A WB analysis of Fibronectin, Collagen 1, and α-SMA expression in fibroblasts. Left side: Representative results of WB. Right side: Bar chart depicting WB data. RT-PCR assessment of FN1 (B), COL1A1 (C), and ACTA2 (D) expression in fibroblasts. E Immunofluorescence of Collagen 1 in fibroblasts. F Representative data for EdU staining in fibroblasts. G Representative data for Transwell assays in fibroblasts. The data are represented as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

In addition, we investigated the influence of ALB on fibroblast proliferation using EdU staining and observed a decline in the number of EdU-positive cells upon ALB treatment (Fig. 1F). We further examined the effect of ALB on fibroblast migration using Transwell assay, which demonstrated a significant reduction in fibroblast migration across the Transwell membrane (Fig. 1G). Taken together, these data demonstrate that ALB could inhibit the differentiation, proliferation, and migration of fibroblasts in a concentration-dependent manner.

Albendazole reprograms the aerobic glycolysis in fibroblasts induced by TGF-β1

ALB has been shown to regulate glucose metabolism, and previous reports have indicated the role of aerobic glycolysis in fibroblast activation [26, 27]. In light of this, we attempted to evaluate the effect of ALB on the glycolytic program in lung fibroblasts induced by TGF-β1. To this end, we employed the Seahorse X24 Extracellular Flux Analyzer to assess the ECAR and OCR of TGF-β1-treated fibroblasts. Our results revealed that TGF-β1 treatment could promote glycolysis in fibroblasts, as evidenced by a sustained increase in ECAR (Fig. 2A, B), whereas OCR, an indicator of mitochondrial oxidative phosphorylation, was decreased following ALB treatment (Fig. 2C, D). Consequently, the ratios of ECAR/OCR, which serve as a measure of aerobic glycolysis, were higher in TGF-β1-treated fibroblasts compared to those treated with DMSO (Fig. 2E). Importantly, ALB treatment significantly reduced ECAR values and reversed the TGF-β1-induced decrease in OCR, indicating that ALB could mitigate TGF-β1-induced aerobic glycolysis in fibroblasts (Fig. 2A–E). To further validate these findings, we directly measured lactate levels, the primary metabolite of glycolysis, which were significantly reduced upon ALB treatment (Fig. 2F). Additionally, we investigated the levels of glycolytic enzymes in the aforementioned fibroblasts, and found that the expression of phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) was decreased after ALB treatment in a dose-dependent manner, whereas pyruvate kinase M2 (PKM2), Lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2) were not affected (Fig. 2G). Consistently, the immunostaining of PFKFB3 showed similar results (Fig. 2H). Above all, these results suggest that ALB may attenuate TGF-β1-induced aerobic glycolysis in fibroblasts by downregulating PFKFB3 expression.

Fig. 2
figure 2

Albendazole attenuates the aerobic glycolysis in TGF-β1 treated fibroblast. A ECAR was recorded in fibroblasts after the addition of glucose (10 mM) oligomycin (1 µM) and 2-DG (50 mM). B Bar graph of the basal levels of ECAR. C OCR was determined in fibroblasts after the addition of oligomycin (1 µM) FCCP (0.5 µM) and rotenone/antimycin A (0.5 µM). D Bar graph of the basal levels of OCR. E Mean ratios of ECAR/OCR. F Relative levels of lactate in the supernatant of fibroblasts. G WB analysis of PFKFB3, HK2, LDHA, and PKM2 expression in fibroblasts. Left side: Representative image of WB data. Right side: Bar chart depicting WB data. H Immunofluorescence of PFKFB3 in fibroblasts. The data are represented as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Albendazole orchestrates aerobic glycolysis via enhancing LRRN3/PFKFB3 signaling

To elucidate the underlying mechanism by which ALB attenuates TGF-β1-induced aerobic glycolysis and fibroblast differentiation, RNA-seq analysis was executed to compare mRNA expression patterns between fibroblasts exposed to TGF-β1 and those exposed to TGF-β1 and ALB. In total, 153 genes with significant differential expression were identified (Fig. 3A). To further contextualize our findings, we compared these genes with those that had previously been found to be altered in IPF lung tissues (GSE47460) and BLM-induced pulmonary fibrosis model (Fig. 3A). Interestingly, we identified top five genes (LRRN3, VCAN, SERPINB2, MCTP1, and RELN) that were commonly dysregulated across these datasets (Fig. 3B). We then performed RT-PCR to confirm the results, and found that only LRRN3 exhibited downregulation upon TGF-β1 stimulation and was restored to normal levels following treatment with ALB (Fig. 3C and Supplementary Fig. 2A–D). Consistently, the immunostaining of LRRN3 yielded similar results (Fig. 3D), indicating that LRRN3 may be a target of ALB.

Fig. 3
figure 3

Albendazole facilitates LRRN3/PFKFB3 signaling in TGF-β1 induced fibroblasts. A Among 153 differentially expressed genes in ALB-treated fibroblasts, 56 genes are dysregulated in IPF patients and lung fibrosis mice. B Heatmap of the 5 genes exhibiting the most significant disparities among 56 commonly dysregulated genes in ALB-treated fibroblasts. C RT-PCR assessment of LRRN3 expression in fibroblasts. D Immunofluorescence of LRRN3 in fibroblasts. E WB analysis of Fibronectin, Collagen 1, α-SMA, and PFKFB3 expression in fibroblasts after TGF-β1, ALB and LRRN3 siRNA exposure. Left panel: representative WB data. Right panel: bar graph of the WB data. F WB analysis of Fibronectin, Collagen 1, α-SMA, and PFKFB3 expression in fibroblasts after TGF-β1, ALB, LRRN3 siRNA and PFK-015 exposure. Left panel: Representative image of WB data. Right panel: Bar graph of the WB data. The data are represented as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

To further verify this hypothesis, we first designed LRRN3 siRNA and evaluated its knockdown efficiency in fibroblasts through RT-PCR analysis (Supplementary Fig. 3). Subsequently, we transfected LRRN3 siRNA into TGF-β1 + ALB-treated fibroblasts. Consistent with the above data, ALB treatment significantly attenuated TGF-β1-induced fibroblast transition (Fig. 3E). However, this therapeutic effect was significantly diminished after LRRN3 siRNA transfection (Fig. 3E). Furthermore, we observed that the downregulation of PFKFB3 induced by ALB was reversed after LRRN3 siRNA transfection, implying that the impact of ALB on fibroblasts relies on LRRN3 expression (Fig. 3E). In order to strengthen our hypothesis, we treated the TGF-β1 + ALB + LRRN3 siRNA-induced fibroblasts with PFK-015 (a PFKFB3 inhibitor). Intriguingly, we found that the effects of LRRN3 siRNA on fibroblasts were reversed upon treatment with PFK-015 (Fig. 3F). This was evident by a reduced expression of myofibroblast markers. In summary, our findings provide compelling evidence that ALB functions as an agonist of LRRN3 during the transition from fibroblast to myofibroblast, thereby alleviating aerobic glycolysis through modulation of the LRRN3/PFKFB3 signaling pathway.

LRRN3 is associated with disease severity in pulmonary fibrosis

To explore the potential role of LRRN3 in the severity of pulmonary fibrosis, we performed an analysis using publicly accessible datasets of lung samples from pulmonary fibrosis patients. Specifically, we reanalyzed the GSE2052 dataset, comprising of the samples from 13 IPF patients and 11 controls (Fig. 4A). Additionally, we analyzed the GSE47460 dataset, including the samples from 168 IPF patients, 10 uncharacterized pulmonary fibrosis patients, 14 patients with fibrosis associated with cardiovascular disease (CVD), 17 patients with nonspecific interstitial pneumonia, 6 patients with desquamative interstitial pneumonia, 30 patients with hypersensitive pneumonitis, and 108 controls (Fig. 4B). Our analysis revealed an obvious reduction in the mRNA levels of LRRN3 in pulmonary fibrosis lungs compared with control samples (Fig. 4A, B). Notably, a positive correlation between LRRN3 expression and lung function was observed (Fig. 4C–E). We also examined the performance of LRRN3 in diagnostic effect for IPF. ROC analysis revealed that LRRN3 serves as a potential diagnostic biomarker with AUC = 0.832, Best cutoff = 9.52 (Supplementary Fig. 4). The GSE47460 dataset provided further evidence suggesting that LRRN3 might contribute to the differentiation of fibroblast, as we observed a negative correlation between LRRN3 expression and ACTA2 (Fig. 4F) or various collagen genes (COL7A1, COL9A3, COL15A1, COL17A1, COL4A6, COL5A2, COL10A1, COL14A1 and COL27A1) (Fig. 4G–J and Supplementary Fig. 5A–E). In IPF cases, there was a notable increase in the quantity of α-SMA+LRRN3− cells, whereas among Control subjects, there was a distinct elevation in the abundance of α-SMA−LRRN3+ cells (Fig. 4K). To further investigate this association, we performed RNA-Seq analysis on fibroblast samples obtained from donors (n = 4), IPF patients with slow progression (n = 6), and IPF patients with rapid progression (n = 4) using the GSE44723 dataset (Fig. 4L). Consistently, our results demonstrated a marked reduction in LRRN3 expression in slow-progressing IPF patients compared to donors (Fig. 4L). Moreover, rapid-progressing IPF patients exhibited an even greater reduction in LRRN3 expression (Fig. 4L). Interestingly, the expression of LRRN3 was gradually decreasing along with TGF-β1 treatment (Fig. 4M). Collectively, our findings imply that LRRN3 plays a vital role in myofibroblast formation during IPF progression.

Fig. 4
figure 4

LRRN3 is downregulated in pulmonary fibrosis. A Relative LRRN3 expression in lung tissues from the dataset of GSE2052. B Relative LRRN3 expression in lung tissues from the dataset of GSE47460. The correlation between LRRN3 expression and DLCO (% Predicted) (C), FVC (% Predicted) (D) and FEV1 (% Predicted) (E) in the dataset of GSE47460. The correlation between the expression of LRRN3 and ACTA2 (F), COL7A1 (G), COL9A3 (H), COL15A1 (I) and COL17A1 (J) in the dataset of GSE47460. K Immunofluorescence of LRRN3 and ɑ-SMA in the lung tissues of IPF patients (n = 3) and Control subject (n = 3). L: Relative LRRN3 mRNA levels in fibroblasts from the dataset of GSE44723. M RT-PCR assessment of LRRN3 expression in fibroblasts after TGF-β1 stimulation for different time points. Data are presented as Mean ± SD (A, B, L) or Mean ± SEM (M). *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Albendazole treatment prevents from BLM-stimulated pulmonary fibrosis

The above in vitro results suggested that ALB might be a potential effective clinical drug for treatment of pulmonary fibrosis, therefore, to evaluate the therapeutic effects of ALB in vivo, we first adopted the animal model of BLM-induced pulmonary fibrosis, which is the most widely used experimental pulmonary fibrosis model [28]. On day 14 following BLM induction, we treated C57BL/6 J mice with ALB at a dosage of 100 or 200 mg/kg by intragastric gavage for 7 consecutive days and then sacrificed (Fig. 5A). Histological evaluation of the lungs revealed extensive collagen accumulation and pronounced structural disruption in mice exposed to BLM (Fig. 5B), which are characteristic features of pulmonary fibrosis. However, treatment with ALB markedly attenuated these pathological changes, suggesting its potential therapeutic efficacy in reversing the established pulmonary fibrosis (Fig. 5B). Notably, the efficacy of ALB antifibrotic effects was found to be dose-dependent, with the 200 mg/kg dose leading to a lower Ashcroft score compared with the 100 mg/kg dose (Fig. 5C). Consistent with the histological findings, mice treated with ALB showed dramatical reduction in the levels of fibrotic biomarkers at both protein (Fig. 5D) and RNA levels (Fig. 5E–G). Importantly, we observed that the levels of Lrrn3 were diminished following BLM exposure. However, treatment with ALB led to a reversal of this effect, indicating its potential role in modulating Lrrn3 expression during lung fibrosis (Fig. 5H). Taken together, these findings indicate that ALB may be a potential strategy to efficiently ameliorate BLM-induced pulmonary fibrosis.

Fig. 5
figure 5

Administration of albendazole protects mice from BLM-induced pulmonary fibrosis. A BLM-induced mouse model of pulmonary fibrosis and the drug-delivery method used for ALB. B Representative images for H&E (top), Masson staining (middle) and Sirius red (bottom) in mice after BLM induction. Magnification × 200. C A bar graph showing the average score of fibrosis severity. D WB analysis of Fibronectin, Collagen 1, and α-SMA in BLM-treated mice after ALB and PFD exposure. RT-PCR assessment of Fn1 (E), Col1a1 (F), Acta2 (G) and Lrnn3 (H) in the homogenates from BLM-treated mice after ALB and PFD treatment. Mice, n = 4–5/group. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Administration of albendazole ameliorates pulmonary fibrosis in silicosis

To further validate our hypothesis that ALB effectively mitigate pulmonary fibrosis, we utilized an alternative mouse model of SiO2-induced silicosis, which is also a common subtype of pulmonary fibrotic disease [29]. Silicosis, characterized by alveolar destruction and irreversible pulmonary fibrosis, is an occupational lung disease that is still a significant public health concern [30]. Initially, silicosis was induced by intratracheally instilling silica suspensions for 28 days, followed by intragastric gavage of either vehicle or ALB for 14 days (Fig. 6A). Subsequently, the lung tissues were harvested and assessed for pathophysiological parameters of pulmonary fibrosis. Histological examination of the lung tissues revealed extensive lung fibrosis after exposure to silica, whereas treatment with ALB significantly alleviated pulmonary fibrosis (Fig. 6B, C). Furthermore, we performed WB analysis to evaluate the protein levels of myofibroblast markers, including Fibronectin, Collagen 1, and α-SMA, in the lungs of silica-exposed mice. Remarkably, the ALB-treated group exhibited a substantial reduction in the levels of these fibrotic biomarkers compared with the vehicle-treated group, further confirming the antifibrotic effects of ALB (Fig. 6D). These findings were further validated through RT-PCR analysis, which demonstrated consistent downregulation of fibrotic markers at the RNA level in ALB-treated mice (Fig. 6E–G). In addition to its effects on fibrotic markers, ALB treatment also restored the expression of Lrrn3, which had been suppressed by the silica suspensions (Fig. 6H). This observation suggests that ALB may have a positive regulatory effect on Lrrn3 expression, potentially contributing to its therapeutic efficacy in pulmonary fibrosis. Collectively, our data from the silicosis mouse model provide additional in vivo evidence supporting the therapeutic effects of ALB in ameliorating lung fibrosis.

Fig. 6
figure 6

Treatment with albendazole reverses the established silicosis induced by SiO2. A SiO2-induced mouse model of silicosis and the drug-delivery method used for ALB. B Representative images for H&E (top), Masson staining (middle) and Sirius red (bottom) in mice after SiO2 induction. Magnification × 200. C A bar graph showing the fibrosis foci. D WB analysis of Fibronectin, Collagen 1, and α-SMA in SiO2-treated mice after ALB and PFD exposure. RT-PCR assessment of Fn1 (E), Col1a1 (F), Acta2 (G) and Lrnn3 (H) in the homogenates from SiO2-treated mice after ALB and PFD treatment. Mice, n = 4–5/group. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Albendazole diminishes fibrosis-associated phenotypes in hPCLS following TGF-β1 stimulation

A major limitation of the mouse pulmonary fibrosis model induced by BLM or SiO2 is that it does not adequately mimic the pathogenetic process of pulmonary fibrosis in humans [31]. Therefore, to verify the therapeutic effects of ALB on human pulmonary fibrosis, an ex vivo approach utilizing hPCLS derived from human lung tissues was employed, which has emerged as a promising model system for studying pulmonary fibrosis [32].

Briefly, 400 µm thick hPCLS derived from donors were cultured stimulated by TGF-β1, and then subjected to either ALB or vehicle treatment. Histological evaluation of the ALB-treated hPCLS revealed a remarkable decrease in the accumulation of collagens after TGF-β1 stimulation, as demonstrated by Masson's trichrome and Sirius red staining (Fig. 7A). Furthermore, WB (Fig. 7B) and RT-PCR (Fig. 7C) analysis revealed that ALB treatment resulted in a decreased expression of fibrotic markers, including Fibronectin and Collagen 1, thus indicating a mitigation of fibrotic processes. Additionally, the number of Collagen 1+ cells in hPCLS alleviated after ALB treatment, suggesting a decline in the formation of myofibroblasts within the ALB-treated hPCLS (Fig. 7D). Importantly, we found an obvious enhancement in the expression of LRRN3 in myofibroblasts following ALB treatment (Fig. 7D). Above all, these data show that ALB exerts a therapeutic effect in hPCLS through enhancing the expression of LRRN3.

Fig. 7
figure 7

Albendazole attenuates fibrosis-associated phenotypes in hPCLS after TGF-β1 stimulation. A Masson staining (top) and Sirius red staining (bottom) of the fibrosis-associated phenotypes in TGF-β1-stimulated hPCLS after ALB treatment. B WB analysis of Fibronectin and Collagen 1 in hPCLS after TGF-β and ALB treatment. Left side: Representative image of WB results. Right side: Bar chart depicting WB data. C RT-PCR assessment of FN1 in the homogenates from hPCLS. D Immunofluorescence of LRRN3 and Collagen 1 in the hPCLS sections after TGF-β1 and ALB treatment. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Full size image

Discussion

IPF is a severe lung disease with limited therapeutic options to halt or reverse its progression. Although FDA-approved drugs like pirfenidone and nintedanib offer some benefit, they are insufficient to prevent the high mortality associated with IPF. Therefore, exploring novel therapeutic approaches is crucial [33, 34]. ALB, an FDA-approved broad-spectrum anthelmintic and antiprotozoal agent with low toxicity, is widely used in both humans and animals for several decades [35]. Recently, it has garnered attention as a potential candidate for cancer treatment due to its anti-tumor effects, including the inhibition of cancer cell division, interference with protein synthesis and the cytochrome P450 system, induction of apoptosis, and inhibition of angiogenesis [36,37,38]. Our results showed that ALB effectively reversed fibrosis in mouse models induced by BLM or SiO2 and demonstrated significant therapeutic effects on TGF-β1-induced fibrosis in hPCLS. Mechanistic investigations revealed that ALB remarkably attenuated TGF-β1 induced aerobic glycolysis in fibroblasts via activation of the LRRN3/PFKFB3 signaling pathway (Fig. 8), consistent with its known ability to reprogram glucose metabolism [13].

Fig. 8
figure 8

Schematic diagram for the mechanisms of albendazole in fibroblasts. TGF-β1 treatment facilitates fibroblasts undergoing DNA hypermethylation via TGFβR1/Smad3 signaling and abrogates the expression of LRRN3. Then, LRRN3 blunts PFKFB3 expression and enhances aerobic glycolysis, which subsequently induces the differentiation/proliferation/migration of fibroblasts. Administration of ALB reverses the downregulation of LRRN3 and ameliorates the established pulmonary fibrosis

Full size image

Research indicates that fibroblast-myofibroblast transition is a critical step in various pathological processes, including lung fibrosis, scar formation, cardiac fibrosis, and vascular thickening [39,40,41]. This makes targeting myofibroblasts a promising strategy for developing clinical treatments for multiple diseases. The phenotypic transition of fibroblasts is regulated by several key pathways, such as aging, autophagy, extracellular matrix, and metabolism [42,43,44,45]. Interestingly, our RNA-seq data revealed that ALB's effects were linked to the PI3K-Akt signaling pathway, which is closely associated with glycolysis [46]. Western blot analysis confirmed that ALB treatment abrogated this pathway (Supplementary Fig. 6C). Unlike previous studies, our results indicated that ALB could remarkably decreased PFKFB3 expression, but had no significant effect on other glycolytic enzymes like HK2, PKM2, and LDHA, which may due to the different cell types and disease conditions [47]. Moreover, we found that ALB regulates the expression of PFKFB3 through LRRN3. LRRN3, known for its role in neuronal development and disease [48], was downregulated in IPF patients and correlated with disease progression. Similarly, we have confirmed that LRRN3 expression levels are closely related to lung function, and ROC curve analysis indicates that it is a promising diagnostic marker. Moreover, knocking down LRRN3 reversed ALB's impact on glycolysis, which highlights LRRN3 as a potential factor against myofibroblast formation in IPF.

Several studies have suggested that aerobic glycolysis contributes to the development of lung fibrosis by promoting the differentiation and proliferation of fibroblasts. Lu et al. and Xie et al. have shown that glycolysis inhibitors like 2-DG and 3-PO can reverse silicosis and IPF without significant side effects, supporting the therapeutic potential of targeting glycolysis in fibrotic diseases [27, 49]. Our findings align with these studies, as ALB was found to mitigate aerobic glycolysis and reduce fibroblast activation, further indicating that targeting this metabolic pathway could be an effective strategy for treating pulmonary fibrosis. ALB shows promise in treating IPF by targeting glycolysis as a widely used and low-cost FDA-approved drug. This repurposing could expedite its availability to patients, offering a new therapeutic option beyond current treatments like pirfenidone and nintedanib, which only slow disease progression [50,51,52].

However, our study primarily focused on the effects of ALB in glycolysis on fibroblasts, without extensively examining its impact on other lung cell types, such as epithelial and endothelial cells. Previous studies have demonstrated that glycolysis plays a critical role not only in fibroblast activation but also in the behavior of epithelial and endothelial cells in the context of pulmonary fibrosis. For example, studies have shown that inhibiting glycolysis in lung epithelial cells can reduce epithelial-mesenchymal transition (EMT), a key process in fibrosis progression [53]. Similarly, Huang et.al revealed activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells [54], suggesting that glycolysis in endothelial cell may be a key factor in the ongoing fibrosis. These findings suggest that the effects of ALB on these cell types should be explored to fully understand its therapeutic potential in IPF.

Our research has few limitations. Although we clarified that ALB reversed the reduction of LRRN3 induced by TGF-β1, the underlying mechanism remains unclear. A previous study indicated that DNA methylation could orchestrate LRRN3 expression [55]. Specifically, the methylation of cg03636183 (Chr19: position17000585) negatively associated with LRRN3 expression. In our previous study, fibroblasts underwent DNA hypermethylation after TGF-β1 stimulation [2]. Interestingly, the expression of LRRN3 was enhanced following SB431542 (a TGF-β receptor-1 inhibitor) and SIS3-HCl (a Smad3 phosphorylation inhibitor) treatment, suggesting that DNA methylation may be involved in regulating LRRN3 expression during fibroblast differentiation (Supplementary Fig. 7A). And the further methylation analysis revealed that there exist DNA methylation during LRRN3 regulation after TGF-β1 stimulation (Supplementary Fig. 7B and C). Consistently, inhibiting DNA methylation with 5-Aza-2'-deoxycytidine (a DNA methyltransferase inhibitor) facilitated LRRN3 expression (Supplementary Fig. 7D). We also made prediction diagram for the interaction between ALB and DNMT1, DNMT3a and DNMT3b, respectively (Supplementary Fig. 7E). The results showed intense interaction between ALB and these three methyltransferases. Thus, we speculate that the inductive effect of ALB on LRRN3 expression may involve a reduction in DNA methylation, which requires further investigation. Furthermore, although we demonstrated that PFKFB3 appeared to a downstream target of LRRN3, the underlying mechanism by which LRRN3 inhibits aerobic glycolysis and reduces fibroblast activation is still unknown, necessitating further exploration.

Conclusion

This research indicated that the administration of ALB, a broad-spectrum anthelmintic and antiprotozoal agent, dramatically reversed different types of pulmonary fibrosis. We further demonstrated that ALB attenuated aerobic glycolysis via activating the LRRN3/PFKFB3 signaling axis, consequently abrogating fibroblast-to-myofibroblast differentiation. Considering the well-established safety profile and low clinical cost of ALB, our study provides promising proof-of-concept for the potential application of ALB in treating pulmonary fibrosis within clinical settings. Subsequent studies will be crucial for evaluating the therapeutic efficacy of ALB in clinical trials.

Availability of data and materials

The data and materials utilized in this study can be made available by contacting the corresponding author upon a reasonable request.

References

  1. Podolanczuk AJ, Thomson CC, Remy-Jardin M, et al. Idiopathic pulmonary fibrosis: state of the art for 2023. Eur Respir J. 2023;61(4):78.

    Article  Google Scholar 

  2. Wang Y, Zhang L, Huang T, et al. The methyl-CpG-binding domain 2 facilitates pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation. Eur Respir J. 2022;60(3):65.

    Article  Google Scholar 

  3. Yao Y, Wang Y, Zhang Z, et al. Chop deficiency protects mice against bleomycin-induced pulmonary fibrosis by attenuating m2 macrophage production. Mol Ther. 2016;24(5):915–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Liu GY, Budinger GRS, Dematte JE. Advances in the management of idiopathic pulmonary fibrosis and progressive pulmonary fibrosis. BMJ. 2022;377: e066354.

    Article  PubMed  Google Scholar 

  5. Almuntashiri S, Alhumaid A, Zhu Y, et al. TIMP-1 and its potential diagnostic and prognostic value in pulmonary diseases. Chin Med J Pulm Crit Care Med. 2023;1(2):67–76.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Yao L, Xu Z, Davies DE, Jones MG, Wang Y. Dysregulated bidirectional epithelial-mesenchymal crosstalk: a core determinant of lung fibrosis progression. Chin Med J Pulm Crit Care Med. 2024;2(1):27–33.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Zheng Z, Peng F, Zhou Y. Biomarkers in idiopathic pulmonary fibrosis: Current insight and future direction. Chin Med J Pulm Crit Care Med. 2024;2(2):72–9.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Xie T, Wang Y, Deng N, et al. Single-cell deconvolution of fibroblast heterogeneity in mouse pulmonary fibrosis. Cell Rep. 2018;22(13):3625–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zeng C, Wang Q, Liu X, et al. Localized administration of Bcar3 siRNA via nano-self-assembly to treat idiopathic pulmonary fibrosis by disrupting macrophage-fibroblast crosstalk. Int J Nanomed. 2024;19:1827–42.

    Article  CAS  Google Scholar 

  10. Hu Y, Wang Q, Yu J, et al. Tartrate-resistant acid phosphatase 5 promotes pulmonary fibrosis by modulating β-catenin signaling. Nat Commun. 2022;13(1):114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jacob J, Steel A, Lin Z, Berger F, Zöeller K, Jarvi S. Clinical Efficacy and Safety of Albendazole and Other Benzimidazole Anthelmintics for Rat Lungworm Disease (Neuroangiostrongyliasis): A Systematic Analysis of Clinical Reports and Animal Studies. Clin Infect Dis. 2022;74(7):1293–302.

    Article  CAS  PubMed  Google Scholar 

  12. Tang Y, Liang J, Wu A, et al. Co-delivery of trichosanthin and albendazole by nano-self-assembly for overcoming tumor multidrug-resistance and metastasis. ACS Appl Mater Interfaces. 2017;9(32):26648–64.

    Article  CAS  PubMed  Google Scholar 

  13. Albendazole VP. J Antimicrob Chemother. 1998;41(2):145–7.

    Article  Google Scholar 

  14. Wang Y, Zhang L, Wu G-R, et al. MBD2 serves as a viable target against pulmonary fibrosis by inhibiting macrophage M2 program. Sci Adv. 2021;7(1):345.

    Article  Google Scholar 

  15. Yang M, Wang D, Gan S, et al. Triiodothyronine ameliorates silica-induced pulmonary inflammation and fibrosis in mice. Sci Total Environ. 2021;790: 148041.

    Article  CAS  PubMed  Google Scholar 

  16. Rao L-Z, Wang Y, Zhang L, et al. IL-24 deficiency protects mice against bleomycin-induced pulmonary fibrosis by repressing IL-4-induced M2 program in macrophages. Cell Death Differ. 2021;28(4):1270–83.

    Article  CAS  PubMed  Google Scholar 

  17. Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol. 1988;41(4):467–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ahangari F, Becker C, Foster DG, et al. Saracatinib, a Selective Src kinase inhibitor, blocks fibrotic responses in preclinical models of pulmonary fibrosis. Am J Respir Crit Care Med. 2022;206(12):1463–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang Q, Liu J, Hu Y, et al. Local administration of liposomal-based Srpx2 gene therapy reverses pulmonary fibrosis by blockading fibroblast-to-myofibroblast transition. Theranostics. 2021;11(14):7110–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Pan T, Zhou Q, Miao K, et al. Suppressing Sart1 to modulate macrophage polarization by siRNA-loaded liposomes: a promising therapeutic strategy for pulmonary fibrosis. Theranostics. 2021;11(3):1192–206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mou Y, Wu G-R, Wang Q, et al. Macrophage-targeted delivery of siRNA to silence Mecp2 gene expression attenuates pulmonary fibrosis. Bioeng Transl Med. 2022;7(2): e10280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Negreros M, Hagood JS, Espinoza CR, Balderas-Martínez YI, Selman M, Pardo A. Transforming growth factor beta 1 induces methylation changes in lung fibroblasts. PLoS ONE. 2019;14(10): e0223512.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tian Y, Morris TJ, Webster AP, et al. ChAMP: updated methylation analysis pipeline for Illumina BeadChips. Bioinformatics. 2017;33(24):3982–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang L, Wang Y, Wu G, et al. Blockade of JAK2 protects mice against hypoxia-induced pulmonary arterial hypertension by repressing pulmonary arterial smooth muscle cell proliferation. Cell Prolif. 2020;53(2): e12742.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Wang Q, Yu J, Hu Y, et al. Indirubin alleviates bleomycin-induced pulmonary fibrosis in mice by suppressing fibroblast to myofibroblast differentiation. Biomed Pharmacother. 2020;131: 110715.

    Article  CAS  PubMed  Google Scholar 

  26. Mei S, Xu Q, Hu Y, et al. Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis. Theranostics. 2022;12(14):6057–68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Xie N, Tan Z, Banerjee S, et al. Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis. Am J Respir Crit Care Med. 2015;192(12):1462–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Della Latta V, Cecchettini A, Del Ry S, Morales MA. Bleomycin in the setting of lung fibrosis induction: From biological mechanisms to counteractions. Pharmacol Res. 2015;97:122–30.

    Article  CAS  PubMed  Google Scholar 

  29. Handra C-M, Gurzu I-L, Chirila M, Ghita I. Silicosis: New Challenges from an Old Inflammatory and Fibrotic Disease. Front Biosci (Landmark Ed). 2023;28(5):96.

    Article  CAS  PubMed  Google Scholar 

  30. Leung CC, Yu ITS, Chen W. Silicosis. Lancet. 2012;379(9830):2008–18.

    Article  CAS  PubMed  Google Scholar 

  31. Williamson JD, Sadofsky LR, Hart SP. The pathogenesis of bleomycin-induced lung injury in animals and its applicability to human idiopathic pulmonary fibrosis. Exp Lung Res. 2015;41(2):57–73.

    Article  CAS  PubMed  Google Scholar 

  32. Alsafadi HN, Staab-Weijnitz CA, Lehmann M, et al. An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices. Am J Physiol Lung Cell Mol Physiol. 2017;312(6):L896–902.

    Article  PubMed  Google Scholar 

  33. Moss BJ, Ryter SW, Rosas IO. Pathogenic mechanisms underlying idiopathic pulmonary fibrosis. Annu Rev Pathol. 2022;17:515–46.

    Article  CAS  PubMed  Google Scholar 

  34. Spagnolo P, Kropski JA, Jones MG, et al. Idiopathic pulmonary fibrosis: disease mechanisms and drug development. Pharmacol Ther. 2021;222: 107798.

    Article  CAS  PubMed  Google Scholar 

  35. Movahedi F, Li L, Gu W, Xu ZP. Nanoformulations of albendazole as effective anticancer and antiparasite agents. Nanomedicine (Lond). 2017;12(20):2555–74.

    Article  CAS  PubMed  Google Scholar 

  36. Zhu L, Kuang X, Zhang G, et al. Albendazole induces immunotherapy response by facilitating ubiquitin-mediated PD-L1 degradation. J Immunother Cancer. 2022;10(5):7.

    Article  Google Scholar 

  37. Yi H, Liang L, Wang H, et al. Albendazole inhibits NF-κB signaling pathway to overcome tumor stemness and bortezomib resistance in multiple myeloma. Cancer Lett. 2021;520:307–20.

    Article  CAS  PubMed  Google Scholar 

  38. Noura M, Morita K, Kiyose H, et al. Albendazole induces the terminal differentiation of acute myeloid leukaemia cells to monocytes by stimulating the Krüppel-like factor 4-dihydropyrimidinase-like 2A (KLF4-DPYSL2A) axis. Br J Haematol. 2021;194(3):598–603.

    Article  CAS  PubMed  Google Scholar 

  39. Czubryt MP. Cardiac fibroblast to myofibroblast phenotype conversion-an unexploited therapeutic target. J Cardiovasc Dev Dis. 2019;6(3):6.

    Google Scholar 

  40. Liu C-T, Hsu S-C, Hsieh H-L, et al. Inhibition of β-catenin signaling attenuates arteriovenous fistula thickening in mice by suppressing myofibroblasts. Mol Med. 2022;28(1):7.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Morelli MB, Shu J, Sardu C, Matarese A, Santulli G. Cardiosomal microRNAs are essential in post-infarction myofibroblast phenoconversion. Int J Mol Sci. 2019;21(1):56.

    Article  Google Scholar 

  42. Pot SA, Lin Z, Shiu J, Benn MC, Vogel V. Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte-fibroblast/myofibroblast transition. Sci Rep. 2023;13(1):11350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Luo H, Zhao L, Ou Z, Li T, Liu Y, Yu Z. Novel lncRNA LNC_000113 drives the activation of pulmonary adventitial fibroblasts through modulating PTEN/Akt/FoxO1 pathway. J Cardiovasc Dev Dis. 2023;10(6):345.

    Google Scholar 

  44. Peng D, Li J, Li Y, et al. MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis. Pharmacol Res. 2024;200: 107070.

    Article  CAS  PubMed  Google Scholar 

  45. Xu R, Wu M, Wang Y, et al. Mesenchymal stem cells reversibly de-differentiate myofibroblasts to fibroblast-like cells by inhibiting the TGF-β-SMAD2/3 pathway. Mol Med. 2023;29(1):59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Su WY, Tian LY, Guo LP, Huang LQ, Gao WY. PI3K signaling-regulated metabolic reprogramming: From mechanism to application. Biochim Biophys Acta Rev Cancer. 2023;1878(5): 188952.

    Article  CAS  PubMed  Google Scholar 

  47. Zhou F, Du J, Wang J. Albendazole inhibits HIF-1α-dependent glycolysis and VEGF expression in non-small cell lung cancer cells. Mol Cell Biochem. 2017;428(1–2):171–8.

    Article  CAS  PubMed  Google Scholar 

  48. Guo X, Hu W, Gao Z, Fan Y, Wu Q, Li W. Identification of PLOD3 and LRRN3 as potential biomarkers for Parkinson’s disease based on integrative analysis. NPJ Parkinsons Dis. 2023;9(1):82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lu Y, Mu M, RenChen X, et al. 2-Deoxy-D-glucose ameliorates inflammation and fibrosis in a silicosis mouse model by inhibiting hypoxia-inducible factor-1α in alveolar macrophages. Ecotoxicol Environ Saf. 2024;269: 115767.

    Article  CAS  PubMed  Google Scholar 

  50. Noble PW, Albera C, Bradford WZ, et al. Pirfenidone for idiopathic pulmonary fibrosis: analysis of pooled data from three multinational phase 3 trials. Eur Respir J. 2016;47(1):243–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Richeldi L, du Bois RM, Raghu G, et al. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med. 2014;370(22):2071–82.

    Article  PubMed  Google Scholar 

  52. Sharma RK, Singh K, Saxena KK. The effect of levamisole and albendazole on some enzymes of Ascaridia galli and Heterakis gallinae. Vet Parasitol. 1989;30(3):213–22.

    Article  CAS  PubMed  Google Scholar 

  53. Sun Z, Ji Z, He W, Duan R, Qu J, Yu G. Lactate accumulation induced by Akt2-PDK1 signaling promotes pulmonary fibrosis. FASEB J. 2024;38(2): e23426.

    Article  CAS  PubMed  Google Scholar 

  54. Raslan AA, Pham TX, Lee J, et al. Lung injury-induced activated endothelial cell states persist in aging-associated progressive fibrosis. Nat Commun. 2024;15(1):5449.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Maas SCE, Mens MMJ, Kühnel B, et al. Smoking-related changes in DNA methylation and gene expression are associated with cardio-metabolic traits. Clin Epigenetics. 2020;12(1):157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We express our sincere gratitude to the individuals who generously donated their pulmonary tissue for the purpose of this study.

Funding

The present study was supported by the National Natural Science Foundation of China (82170078, 81900063), Huazhong University of Science and Technology Fundamental Research Program, the Tongji Hospital (HUST) Foundation for Excellent Young Scientists, the Tongji Hospital (HUST) Foundation for key project (2022A15), Hubei Provincial Medical Youth Elite Talent Program (SCZ202412), and Wuhan Science and Technology Bureau "Dawn Light Plan Talent Project” (2022020801020450).

Author information

Author notes

  1. Chenxi Zeng and Huihui Yue contributed equally to this work.

Authors and Affiliations

  1. Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Ave, Wuhan, 430030, China

    Chenxi Zeng, Congjian Wang, Xuetao Ju, Tianlai Wang, Xiangning Fu & Jun Yu

  2. Department of Pulmonary and Critical Care Medicine, NHC Key Laboratory of Respiratory Diseases, Tongji Hospital, Tongji Medical College and State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China

    Huihui Yue, Qing Zhou, Huilan Zhang & Yi Wang

  3. Department of Clinical Laboratory, School of Medicine, Shanghai East Hospital, Tongji University, Shanghai, China

    Long He

Authors
  1. Chenxi Zeng
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Huihui Yue
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Congjian Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Xuetao Ju
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Tianlai Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Xiangning Fu
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Qing Zhou
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Huilan Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Long He
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Jun Yu
    View author publications

    You can also search for this author inPubMed Google Scholar

  11. Yi Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

The experimental procedures were conducted by Chenxi Zeng and Huihui Yue. Congjian Wang, Xuetao Ju and Tianlai Wang diligently analyzed the gathered data, employing appropriate statistical techniques and methods. Xiangning Fu, Huilan Zhang and Long He provided human lung tissues and clinical data. Qing Zhou and Yi Wang designed the experiments and supported the preparation of the manuscript. Jun Yu and Yi Wang led the investigation and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Long He, Jun Yu or Yi Wang.

Ethics declarations

Ethics approval and consent to participate

The study was conducted in compliance with the Declaration of Helsinki and was approved by the Human Assurance Committee of Tongji Hospital (TJ-IRB20210942). All experimental procedures were approved by the Animal Care and Use Committee of Tongji Hospital (TJH-202210029).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests. All authors have carefully reviewed and complied with the journal’s authorship agreement.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zeng, C., Yue, H., Wang, C. et al. Albendazole ameliorates aerobic glycolysis in myofibroblasts to reverse pulmonary fibrosis. J Transl Med 22, 910 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05655-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05655-0

Keywords

Journal of Translational Medicine

ISSN: 1479-5876

Contact us