Eight-week-old male C57BL/6 mice weighing approximately 23 g were obtained from Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China). They were kept on a standard diet and provided with water under controlled environmental conditions: a 12-h light-dark cycle, humidity at 55 ± 5% and temperature at 23 ± 2°C. Following one-week adaptation time, 15 mice were randomly divided into three groups (n = 5 each) for conducting PF-induced PH models according to a previous research (23): the Control (CON) group receiving intratracheal injections of normal saline, the bleomycin (BLM)-administered group (BLM), and the BLM + SBC-115076 treated group (SBC), which received intratracheal injections of BLM (2 mg/kg, Selleck). Thirty minutes after the intratracheal injection, normal saline was intraperitoneally administered to mice in the CON and BLM groups, while those in the SBC group received SBC-115076 (10 mg/kg, Selleck). This intraperitoneal injection mode was repeated every other day for a duration of 3 weeks.

On day 21, all mice underwent anesthetized with 1% isoflurane. Under ventilator anesthesia, right ventricular systolic pressure (RVSP) was measured using a microtip Millar pressure transducer catheter (SPR-839) inserted into the RV via the right external jugular vein. The curve graph depicting RVSP was continuously recorded and subsequently analyzed with the PowerLab system and LabChart software. The right ventricular hypertrophy index (RVHI), also known as the Fulton index, was evaluated as the ratio of the RV weight to the combined weight of the left ventricle (LV) and septum (S) [RV/(LV + S) ratio]. Following the surgical procedures, all mice were euthanized by cervical dislocation, and bronchoalveolar lavage fluid (BALF) along with lung tissues were collected.

After RVSP measurement, BALF was collected by cannulating the trachea with a sterile catheter and instilling 1 mL of sterile phosphate-buffered saline (PBS) into the lungs, followed by gentle aspiration. This process was repeated three times, and the recovered lavage fluid was pooled for each animal. The collected BALF was then centrifuged at 1,500 rpm for 10 min at 4°C to remove cellular debris, and the supernatant was carefully separated and stored at −80°C until analysis. Levels of IL-6, TGF-β, and TNF-α in the BALF were quantified using commercially available ELISA kits (Elabscience, China) according to the manufacturer’s instructions, with absorbance measured at the appropriate wavelength using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Fisher Scientific, US).

The lung tissues, following fixation in 4% paraformaldehyde and subsequent embedding in paraffin, underwent sectioning into 5 μm slices. These sections were then subjected to staining using hematoxylin-eosin solution (H&E), Masson’s Trichrome stain, and Sirius Red stain kit (Servicebio, China).

The paraformaldehyde is dissolved in phosphate-buffered saline (PBS) to fix mice lung specimens at room temperature (RT) for 48 h, and subsequently embedded in paraffin. After embedding, tissues underwent sectioning into slices, deparaffinizing and rehydrating. To block endogenous peroxidase activity, 3% hydrogen peroxide was applied for 20 min at RT. Non-specific binding was then prevented by incubating the slides in 5% bovine serum albumin (BSA) for 2 h. Antibodies targeting alpha smooth muscle actin (αSMA) and collagen I (COL1A1) (Proteintech) were diluted in blocking solution and applied to the slides, which were incubated for 2 h at RT, followed by an additional hour of incubation with secondary antibodies at RT. Subsequently, diaminobenzidine (DAB) reagent and hematoxylin were used to counterstained the slides. The resulting images were captured using an ECLIPSE Ti2 inverted microscope (Nikon, Japan).

Mouse lung epithelial (MLE-12) cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). The cells were maintained in Procell medium supplemented with 10% fetal bovine serum (FBS, ABW, China) and 1% penicillin-streptomycin, and cultured at 37°C in a humidified atmosphere with 5% CO2. The cells were divided into four groups: (1) the CON group, which received no special treatment; (2) the TGF-β group, where cells were treated with recombinant TGF-β (200 ng/ml); (3) the SBC group, where cells were treated with TGF-β and SBC-115076 at concentrations of 5, 10, or 20 μM; and (4) the SKL2001 group, where cells were treated with TGF-β, SBC-115076 and SKL2001 (20 μM), an agonist of the Wnt/β-catenin pathway. Phosphate-buffered saline (PBS) was used to adjust the final volumes of all solutions. Recombinant TGF-β was purchased from PeproTech (New Jersey, USA), and SKL2001 was obtained from Selleck Chemicals (Texas, USA).

MLE-12 cells were seeded in 6-well plates. Sterile 1,000 μl pipette tips were used to create scratches in the cell monolayer, and any dislodged cells were removed by washing with PBS. Then serum-free medium was used to substituted the normal culture medium. Initial images were captured immediately following the scratch, and again 24 h later, using a light microscope. Cell migration was quantified by converting the images to grayscale and analyzing them with ImageJ software.

Immunofluorescence staining of MLE-12 cells was performed according to the protocol of the Immunol Fluorescence Staining Kit (Beyotime Biotechnology, China). MLE-12 cells were stained with αSMA specific rabbit polyclonal antibody and anti-COL1A1 rabbit antibody (Proteintech, China). After overnight incubation at 4°C, cells were incubated with anti-rabbit IgG conjugated secondary antibody and counterstained with DAPI (Beyotime Biotechnology, China). Fluorescent positive cells were detected using an ECLIPSE Ti2 inverted fluorescent microscope (Nikon, Japan).

MLE-12 cells and mouse lung tissues were lysed using lysis buffer (Thermo Fisher Scientific, USA) with phosphatase and protease inhibitors. Protein concentrations were measured using the Pierce BCA Protein Assay Kit. Equal amounts of protein (10–20 μg) were separated by SDS-PAGE (10–12%) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking, membranes were incubated with primary antibodies against β-actin (1:7000), E-cadherin (1:20000), N-cadherin (1:7000), Vimentin (1:20000), Snail (1:700), Wnt5A/B (1:2000), and β-catenin (1:10000) (Proteintech, China), followed by secondary antibody incubation (1:10000, Proteintech). Protein bands were visualized using ECL detection reagents and quantified with ImageJ software (V1.8.0.112).

Differences between two groups were assessed using Student’s t-test with Welch correction. For multiple comparisons, one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was employed. Data from a minimum of three independent experiments were analyzed using GraphPad Prism software and are presented as means ± SD. Statistical significance was defined as a p-value < 0.05.

We performed light microscopy measurements of lumen stenosis and pulmonary artery thickness to evaluate the effects of PCSK9 inhibition on pulmonary arterial reconstruction in mice induced with BLM. Histological analysis of the pulmonary arteries demonstrated a reduction in lumen area ratio, which was subsequently increased following PCSK9 inhibitor treatment. Conversely, an opposite trend was found in the pulmonary arteries using another index named the wall thickness percentage (WT%) (Figures 1A–C). These findings suggest a significant reversal of pulmonary arterial remodeling induced by PF-associated PH upon PCSK9 inhibitor administration. Notably, the observed remodeling in the pulmonary arteries was primarily confined to fibrotic lung regions, suggesting that this improvement is related to fibrosis alleviation.

Additionally, the right ventricle hypertrophy index (RVHI), also known as Fulton’s index, a parameter indicative of right ventricle hypertrophy and reconstruction, was evaluated. BLM-induced mice presented a substantial increase in RVHI compared to the control group, which was notably mitigated following PCSK9 inhibitor treatment (Figures 1D, E). We also assessed RVSP by closed-chest method to gauge hemodynamics of right ventricle. The results showed that RVSP was significantly elevated in the BLM-induced mouse model but exhibited a notable reduction post-SBC treatment compared to the control group (Figures 1F, G). These outcomes demonstrate beneficial effect of the PCSK9 inhibitor on RV remodeling and cardiopulmonary function in the PH mice model induced with BLM.

Histological fibrosis and alveolitis severity in the BLM-induced PH model were further evaluated by staining lung sections with H&E, Masson’s Trichrome, and Sirius Red reagents. As depicted in Figure 2A, histopathological images illustrated that sham control mice displayed normal lung architecture without collagen depositions or inflammatory cell infiltrations. BLM induction, in contrast, led to abnormal lung morphology where alveolar walls were thickened and both collagens and inflammatory cells were accumulated. Remarkably, SBC treatment mitigated these pathohistological abnormalities in lung tissues.

Furthermore, immunohistochemistry staining for αSMA and COL1A1 was conducted to comprehensively evaluate fibrotic conditions in BLM-induced lungs. As illustrated in Figures 2B–D, the average optical density (AOD) of COL1A1 and αSMA in BLM-induced lung sections significantly increased, while this metric was notably decreased in the SBC group. Additionally, as presented in Figures 2E–G, ELISA assays revealed a significant rise in the levels of inflammatory factors (IL-6, TGF-β, and TNF-α) in the BLM group’s lung tissues, which were notably suppressed after SBC treatment. These findings collectively suggest that PCSK9 inhibitor treatment ameliorated the lung architecture in BLM-induced PH by reducing collagen deposition, inflammation infiltration, and inflammatory cytokine release.

Protein expression analysis of biological markers associated with EMT process and Wnt/β-catenin signaling was conducted from mice lungs as well. Western blotting analysis revealed a significant increase in the synthesis levels of mesenchymal cell marker proteins including N-cadherin, Vimentin, Snail, Wnt5A/B, and β-catenin. Meanwhile, epithelial marker protein such as E-cadherin expression levels were concurrently reduced in the BLM group. Conversely, treatment with SBC exhibited the opposite trend. These findings suggest that PCSK9 inhibitor significantly attenuated the EMT process by repressing the Wnt/β-catenin signaling pathway (Figures 3A–G).

To further investigate the role of PCSK9 in pulmonary fibrosis (PF), MLE-12 cells were cultured in vitro and treated with SBC-115076. The scratch wound healing assay (Figures 4A–C) demonstrated that TGF-β significantly enhanced the migratory capacity of MLE-12 cells. Treatment with SBC-115076 at concentrations of 5, 10, and 20 μM progressively reduced cellular migration in a dose-dependent manner. Consequently, 20 μM was selected as the optimal concentration for SBC-115076 in subsequent in vitro experiments to achieve the best therapeutic effect. Furthermore, immunofluorescent staining Figures 4D–F, and Western blot analysis (Figures 4G–I) of fibrosis biomarkers, including α-SMA and COL1A1, revealed that while TGF-β markedly increased their expression, the PCSK9 inhibitor effectively mitigated pulmonary fibrotic progression in MLE-12 cells.

In experiments involving MLE-12 cells, notable trends in EMT-related protein expression were observed among the CON, TGF-β, SBC, and SKL2001 groups, as assessed by Western blot analysis. The results showed that TGF-β treatment significantly increased the expression of EMT markers, including N-cadherin, vimentin, and Snail. Similarly, proteins associated with the Wnt/β-catenin pathway, such as Wnt5A/B and β-catenin, displayed similar upregulation. In contrast, treatment with the PCSK9 inhibitor led to a marked increase in E-cadherin expression, countering the EMT-promoting effects of TGF-β. However, SKL2001, a known agonist of the Wnt/β-catenin pathway, reversed the effects of PCSK9 inhibition on EMT-related protein expression. Collectively, these findings indicate that the PCSK9 inhibitor ameliorates cellular fibrosis in MLE-12 cells by suppressing the EMT process via the Wnt/β-catenin signaling cascade (Figures 5A–G).