Sustainably released nanoparticle-based rhynchophylline limits pulmonary fibrosis by inhibiting the TEK-PI3K/AKT signaling pathway

Introduction

Pulmonary fibrosis (PF) is an ordinary response to various lung injuries, causing gas exchange disorder and leading to respiratory failure and asphyxiation death. Current pathogenesis of PF implicates a combination of age-related factors, genetic predisposition, a trigger and risk factors. A trigger which sets off repetitive epithelial cell injury/apoptosis/stress and subsequent abnormal repair responses and matrix accumulation leads to progressive fibrosis and loss of lung function (1,2). Risk factors include exposure to tobacco smoke, injury caused by refluxed gastric contents, virus infections, occupational/environmental exposures to both inorganic (metal, wood, and silica dusts) and inorganic dusts, and air pollution. Diffuse epithelial injury accompanied by aberrant wound healing responses lead to extensive fibroblastic foci that extend in a network-like fashion throughout the lung parenchyma (3). PF is the ultimate result of interstitial lung diseases (ILDs), which are characterized by diffuse inflammation of the lung interstitium in the early stage and collagen deposition caused by excessive proliferation of fibroblasts and excessive accumulation of extracellular matrix in the advanced stage (4-7). PF involves numerous fields. For instance, idiopathic pulmonary fibrosis (IPF), one of the most common types of ILD, is a progressive fibrosing disease with a median survival of 3 years (8,9). Other types of ILDs include chronic hypersensitivity pneumonitis (HP) (10), idiopathic non-specific interstitial pneumonia (11), systemic sclerosis (12), chronic sarcoidosis (13), rheumatoid arthritis (14), silicosis (15), and so on. With the development of treatment technology, percutaneous radiofrequency ablation (RFA) has been increasingly widely used in lung cancer therapy. Follow-up after RFA has shown that PF was observed in 36% of the scars and remained stable over time (16). Since the Corona Virus Disease 2019 (COVID-19) outbreak, a growing number of doctors have noticed that PF was a major complication of COVID-19 (17).

At present, PF treatment mainly relies on anti-fibrosis drugs; however, due to their inadequate selectivity, these drugs are not effective in preventing the progression of fibrosis (18). As a result, the development of new pharmaceuticals that can intervene in the PF process is necessary to provide multiple options, even combination therapies. An increasing number of studies have indicated that traditional Chinese medicine (TCM) is beneficial for PF treatment (19,20), which would offer more treatment options.

Rhynchophylline (Rhy) is a small molecular drug that is extracted and purified from Uncaria. It is located throughout the provinces of Guangxi, Guangdong, Fujian, Guizhou, and Sichuan in China. With the advantage of hypotoxicity, Rhy, which is extremely popular in TCM (21-23), has been utilized to treat high blood pressure, convulsions, vertigo, coma, and dizziness (24-27). Recently, a growing number of studies on Rhy have reported its functions in sleep regulation (28), neurotoxicity reduction (29), anti-inflammatory activity (30), asthma treatment (31), and so on. Song (32) found that Rhy reduced experimental intraperitoneal adhesion as well as Interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in serum and that TGF-β1 expression in the peritoneal fluid was also significantly decreased. In our previous study, we also observed that Rhy hindered tendon adhesion and stimulated the healing of injured tendon structures (33). Given that Rhy plays a role in adhesion formation inhibition, and considering the similar mechanisms between adhesion formation and PF, we naturally speculated that Rhy could retard the progression of PF.

The formation of PF is divided into two successive inflammatory and fibrotic phases; the 7-day time point is recognized as a separation between the two phases, and the 21-day time point is recognized as a fibrosis peak (34,35). However, the half-life period of Rhy is only approximately 12 hours (36). Also, long-term oral Rhy can cause systemic reactions such as liver and kidney injury. More importantly, Rhy could pass through the blood-brain barrier to influence the cardiovascular and central nervous systems (37-39). Additionally, the low water solubility of Rhy makes it difficult to dissolve in physiological saline and restricts its concentration. Therefore, given these limitations, a more suitable drug delivery system for Rhy is needed to solve these problems.

In the present study, we initially confirmed that the intraperitoneal injection (i.p.) of Rhy could exert an anti-PF function both in vivo and in vitro. However, considering the invasive nature of intraperitoneal injection drug delivery as well as the low water solubility, short half-life, and toxicity of Rhy solution (i.p) in the clinic, we designed and prepared a Rhy-loaded nanoparticle intratracheal spray for precise treatment and to avoid systemic reactions. Next, we tested its characteristics and endocytosis efficiency to identify whether the Rhy-loaded nanoparticles were suitable for animal experiments. Subsequently, the Rhy-loaded nanoparticles were applied to BLM-treated C57BL/6 mice, and its effects on anti-PF were observed via lung morphology, Masson’s trichrome and hematoxylin-eosin (HE) staining, lung index, hydroxyproline assay, and Western blot (WB). Then, transcriptome sequencing technology was utilized to analyze and determine the mechanism of Rhy. We present the following article in accordance with the ARRIVE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-675/rc).

Methods

Solution groups

For better apply to the clinic, 0.9% saline solution was used as the solvent. Because of the extremely low concentration for cell, 1 mg of Rhy (CAS 76-66-4; Allcells, Shanghai, China) was directly dissolved in 40 mL of 0.9% saline solution [Rhy solution (cell)] without medium according to our previous study. In mice, Rhy has an extremely low water solubility but it can be dissolved in ethanol. Although ethanol is toxic, a 10% ethanol solution is acceptable for C57BL/6 mice (40). After numerous attempts, we succeeded in dissolving Rhy into a harmless solution. 2.1 mg of Rhy was thoroughly dissolved in 200 µL 100% ethanol, and 1.8 mL of physiological saline was then slowly added to Rhy/ethanol solution to obtain the limpid Rhy solution (i.p). The concentration of the Rhy solution (i.p) must be lower than 1.05 mg/mL or Rhy would precipitate from the solution. Based on the ratio of ethanol to water, different concentrations of Rhy solution (i.p) could be obtained by changing the weight of Rhy. The Rhy-loaded nanoparticles were also mixed in a 0.9% saline solution (the Rhy-loaded nanoparticles solution).

Animal model of PF

Eight- to nine-week-old male C57BL/6 mice weighing about 25–30 g were purchased from the Experimental Animal Center of Nantong University [institutional license: SYXK(SU)-2012-0030]. Experiments involving animals were performed under a project license (No. 20140901-001) granted by Animal Ethics Committee of Nantong University, in compliance with Nantong University guidelines for the care and use of animals. A single dose of 50 µL BLM solution (CAS 9041-93-4; Macklin, Shanghai, China) at a concentration of 2.0 mg/mL (41) was loaded into a MicroSprayer aerosolizer (YAN 30012), which was provided by Yuyan Instruments Co., Ltd. (Shanghai, China) and intratracheally sprayed into mice that were anesthetized. After BLM treatment, a Rhy solution (i.p) at different concentrations was intraperitoneally injected in the mice every other day from day 2, or a single dose of 50 µL Rhy-loaded nanoparticles solution was loaded into a MicroSprayer aerosolizer and intratracheally sprayed into the mice that were anesthetized from day 2. On day 21 after BLM treatment, the mice were euthanized and their lung tissues were collected for protein and histological assays.

Culture of HFL1 cells

Human lung fibroblast (HFL1) cells, which were purchased from the Institute of Cell Research (Chinese Academy of Sciences, China), were cultured in a complete medium containing 87% Ham’s F-12K medium (Gibco, NY, USA), 10% fetal bovine serum (Gibco, NY, USA), 1% Glutamax (Gibco, NY, USA), 1% Non-essential Amino Acids (Gibco, NY, USA), and a 1% Sodium Pyruvate 100 mM solution (Gibco, NY, USA) or TGF-β1 (R&D; Minneapolis, MN, USA) in a 37 ℃ incubator containing 5% carbon dioxide (CO₂).

Cytotoxicity study

The cytotoxicity of Rhy and the unloaded nanoparticles in HFL1 cells was detected using a CCK-8 assay. In general, the cells were planted into 96-well plates (2×10³ cells/well). We added the Rhy solution (cell) at different concentrations (0, 50, 100, 200, 400 ng/mL) into each well for 12 hours after cell adherence. Then, the CCK-8 reagents (Dojindo, Kumamoto, Japan) were added and cultured for 2 hours in a 37 ℃ incubator containing 5% CO_2. The absorbance values at 450 nm were examined using an automatic spectrophotometer (Bio-Rad; Richmond, CA, USA). Cells treated with an equal volume of 0.9% saline (NC) were used as the 100% survival control. The experiment was repeated at least three times.

Nanoparticles characterization

The mean size of the nanoparticles was measured using a transmission electron microscope (TEM, JEM-2100, JEOL, Tokyo, Japan). The morphology of the nanoparticles, which were covered with platinum after freeze-drying, was observed by scanning electron microscopy (SEM, Hitachi, S-3400N, Japan).

Preparation of the Rhy-loaded nanoparticles

100 mg of polylactic-co-glycolic acid (PLGA) (Sigma-Aldrich, St. Louis, MO, USA) and 20 mg of Rhy were mixed in 1 mL of dichloromethane (DCM). 3 mL of a 7% (w/v) aqueous solution of poly(vinylalcohol) (PVA) (Sigma-Aldrich, St. Louis, MO, USA) was then added into the PLGA+Rhy/DCM solution for emulsification by sonication (Bandelin electronic, Berlin, Germany) for 1 minute to obtain the original emulsion. Immediately thereafter, the original emulsion was transferred to a 1% (w/v) aqueous PVA solution containing 2% isopropanol, with a volume of 50 mL. This was subsequently sonicated for 3 minutes to collect into the final emulsion and softly stirred overnight at room temperature to evaporate the methylene. Ultimately, the formed aggregation was washed and isolated via centrifugation (13,000 rpm, 5 minutes, 4 ℃) twice to obtain the Rhy-loaded nanoparticles. These were then redispersed in 0.9% saline. After evaporating the cleaning fluid, the residual Rhy was quantified to calculate the weight of Rhy in the Rhy-loaded nanoparticles. The drug wrapping efficiency of nanoparticles was about 10%. Coumarin-loaded nanoparticles were also prepared in the same way. We assumed that the amount of Rhy reaching the lungs accounted for 1/10 of the total amount of Rhy injected intraperitoneally. Considering the Rhy solution (i.p) (5, 10, 20 mg/kg) inhibited PF in mice, we believed that Rhy achieved the effect when the pulmonary dosing was less than 2 mg/kg. Ultimately, the Rhy-loaded nanoparticles were mixed in 0.9% saline solution (the Rhy-loaded nanoparticles solution) at a concentration of 0.33 mg/50 µL (nanoparticles) or 0.033 mg/50 µL (Rhy), which meant 75 µL Rhy-loaded nanoparticles solution contained 0.5 mg nanoparticles.

Release of the Rhy-loaded nanoparticles

50 µL of the Rhy-loaded nanoparticles was added into an eppendorf (EP) tube with a specification of 1.5 mL. After 24 hours at room temperature, we collected the supernatant, replaced 50 µL of fresh 0.9% saline in the tube, and tested the absorbance value of the supernatant at 246 nm (in China) and 254 nm (42,43) using a molecular device (SpectraMax M5, MDC, CA, USA) every 24 hours. Meanwhile, the absorbance value of the Rhy solution (Rhy in saline) was also tested with different concentration gradients in 50 µL, which was the basis for drawing the curves.

Endocytosis efficiency in vitro

HFL1 cells were planted in 6-well plates (Corning Inc., Corning, NY, USA). When they reached 70–80% confluence, the fresh medium and coumarin-loaded nanoparticles solution was replaced in the 6-well plates for 48 hours. The cells in the plates were resuspended in phosphate buffer saline (PBS) to gauge the endocytosis efficiency using a FACSCalibur flow cytometer (BD FACSCalibur, BD Bioscience, San Jose, CA, USA). Moreover, the HFL1 cells were fixed with 4% paraformaldehyde, stained with 4,6-diamino-2-phenyl indole (DAPI) solution (Beyotime, Jiangsu, China), and then observed under a microscope (Leica DMR 3000; Leica Microsystem, Bensheim, Germany).

Endocytosis efficiency in vivo

Following intratracheal atomization instillation of the coumarin-loaded nanoparticles for 48 hours, we collected the lung tissues and fixed them with 4% paraformaldehyde for 24 hours. The tissues were then exposed to gradient alcohol, embedded in paraffin, cut into 4 mm sections, and then placed onto polylysine-coated slides. After deparaffinization in xylene and rehydration with graded alcohol, the sections were stained with DAPI solution at room temperature for 20 minutes. The immunofluorescence of the lung sections was then observed under a microscope (Leica DMR 3000; Leica Microsystem, Bensheim, Germany).

WB analysis

Tissues and cells were collected and lysed in Radio Immunoprecipitation Assay Lysis buffer (RIPA) containing phosphatase inhibitors and protease inhibitors (P1261, Solarbio, Beijing, China). The supernatant was harvested after centrifugation (12,000 rpm at 4 ℃ for 20 minutes). Samples with the same protein concentration were separated by Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and the total protein of samples was transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk in TBST [100 mm Sodium chloride (NaCl), 0.1% Tween-20 and 50 mm Tris-HCl, pH 7.6] for 2 hours at room temperature, the membranes were rinsed with TBST and incubated for a specific time with the following antibodies at 4 ℃: anti-FN antibody (1:1,000, Proteintech Cat# 15613-1-AP, RRID:AB_2105691), anti-α-SMA antibody (1:2,000, Proteintech Cat# 55135-1-AP, RRID:AB_10949628), anti-type I collagen antibody (1:1,000, Proteintech Cat# 14695-1-AP, RRID:AB_2082037), anti-TEK antibody (1:1,000, Cat#YT6112; Immunoway), anti-p-AKT antibody (1:1,000, ImmunoWay Cat# YP0006, RRID:AB_2814755), anti-AKT antibody (1:1,000, Proteintech Cat# 10176-2-AP, RRID:AB_2224574), or anti-β-actin antibody (1:1,000, Proteintech Cat# 66009-1-Ig, RRID:AB_2687938). After being rinsed with TBST every 5 minutes (three times), the membranes were further incubated with Goat anti-Mouse (1:10,000, Proteintech Cat# SA00001-1, RRID:AB_2722565) or Goat anti-Rabbit (1:10,000, Proteintech Cat# SA00001-2, RRID:AB_2722564) at 4 ℃ overnight. After washing every 15 minutes (three times), the membranes were scanned and imaged using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The intensity of each band was quantitatively determined by the ImageJ analysis system (Wayne Rasband, National Institutes of Health, USA).

Masson’s trichrome staining and HE staining

The left lung lobes of mice were fixed with 4% paraformaldehyde for 24 hours. Then, the lungs exposed to gradient alcohol were embedded in paraffin and cut into 4 mm sections onto polylysine-coated slides. The sections were then deparaffinized in xylene and rehydrated with graded alcohol. Next, we performed staining using a Masson stain kit (Solarbio, Beijing, China) and a HE stain kit (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. The lung sections were then observed under a microscope (Leica DMR 3000; Leica Microsystem, Bensheim, Germany).

Hydroxyproline quantification

A hydroxyproline assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was used to detect the hydroxyproline content in the lungs according to the manufacturer’s instructions.

Transcriptome sequencing

The transcriptome sequencing data (mRNA) of the lung tissues in three groups [negative control (NC), BLM, BLM+Rhy] were analyzed using the Illumina platform by Personal Biotechnology Co. Ltd. (Shanghai, China). Differently expressed genes were screened by the absolute value of the log2 fold change >1 and P<0.05. Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway and Gene Ontology (GO) analyses were also performed to confirm the biological functions of Rhy. Protein-protein interactions (PPIs) were established using the STRING website (https://cn.string-db.org/) and visualized in CYTOSCAPE (version 3.7.3). The most essential hub genes were screened by the Hub plug-in component in CYTOSCAPE.

Statistical analysis

GraphPad 8 software was employed for statistical analysis. All statistics were reported as the mean ± standard deviation (SD). The analysis of variance was assessed using the Student’s t-test. P<0.05 was considered statistically significant.

Results

Rhy mitigates the BLM-induced PF and TGF-β1-induced fibrotic characteristics of pulmonary fibroblasts

To verify our hypothesis, we roughly evaluated Rhy’s efficacy in PF. For better application in the clinic, the in vitro anti-fibrotic effect of Rhy was assessed using HFL1 cells, and the toxicity of Rhy in HFL1 cells was detected by the CCK-8 assay. The HFL1 cells showed no remarkable cytotoxicity compared with the physiological saline and no treatment groups with the concentration of Rhy solution (cell) increased from 50 to 200 ng/mL after 12 hours of exposure. Nonetheless, when the concentration of Rhy reached 400 and 800 ng/mL, the viability of HFL1 decreased significantly (Figure 1A). As a fibrotic disease-promoting factor, TGF-β1 drives fibrotic disease progression (44). TGF-β1 has been used as one of the in vitro assay models to investigate new intervention targets for PF (45). In the recent research, FN, Col I, and α-SMA expression of HFL1 cells treated with TGF-β1 increased, which represented fibrotic characteristics (46). After treatment with TGF-β1 (5 ng/mL) and immediately adding the different concentrations of Rhy solution (cell) for 48 hours, the cells were harvested. Next, the cells were lysed and the protein expressions of FN, Col I, and α-SMA, which are fibrosis markers, were tested by WB. TGF-β1 markedly increased the expressions of FN, Col I, and α-SMA expression. Rhy decreased the TGF-β1-induced abnormal expression of FN, Col I, and α-SMA in a dose-dependent manner, and the 200 ng/mL group achieved the best effect (Figure 1B,1C). The in vivo anti-fibrotic effect of Rhy was estimated by C57BL/6 mice. BLM can cause pulmonary injury, inflammation, and subsequent fibrosis after being instilled in the lung (47). Jiang (48) found that a Rhy intraperitoneal injection (10 or 20 mg/kg) could improve Aβ1-42-induced cognitive impairment. To achieve the above concentration, we prepared a limpid, not opaque Rhy solution (i.p) as described in the Methods. As the phase in which Rhy may act on PF was unknown, its 12-hour half-life, coupled with high mortality of daily intraperitoneal injections, we intraperitoneally injected 0.5 mL of Rhy solution (i.p) of different concentrations every other day from day 2 after 50 µL of BLM (2.0 mg/mL) (49) intratracheal spray to day 20 and collected the samples on day 21 (Figure 1D). Masson staining revealed that Rhy reduced collagen deposition and the fibrotic area, thinned alveolar walls, rebuilt the alveolar structure in lesion regions, and increased the alveolar air area in a dose-dependent manner (Figure 1E). With the increase in Rhy concentration, the survival rate of mice also increased (Figure 1F). These results roughly showed that Rhy could inhibit PF in vivo and in vitro.

Figure 1 The role of Rhy in PF in vivo and in vitro. (A) Cytotoxicity study of Rhy and nanoparticles by CCK-8. (B) HFL1 cells stimulated with TGF-β1 (5 ng/mL) were treated with Rhy (0, 50, 100, 200, and 400 ng/mL) for 48 hours. Representative WB assay and protein expression of FN, Col I, and α-SMA in six groups. (C) Histograms after statistical analysis of each protein in six groups. (D) Schematic diagram of the experimental procedure in the lung fibrosis model. (E) Masson’s trichrome stain of lung sections from 0, 5, 10, and 20 mg/kg-treated mice for 10 days at day 21 after BLM. Scale bars: 200 µm. (F) The survival rate of mice in 0, 5, 10, and 20 mg/kg Rhy groups. (*, P<0.05; ***, P<0.001). CCK-8, Cell Counting Kit-8; Rhy, rhynchophylline; PF, pulmonary fibrosis; FN, fibronectin; WB, Western blot.

Characterization of the Rhy-loaded nanoparticles

Although Rhy did play a role in PF inhibition, it was restricted by the multiple invasive drug delivery, low water solubility, short half-life, and toxicity of the Rhy solution (i.p) in the clinic. To solve these limitations and precisely treat patients to avoid systemic reactions, the Rhy-loaded nanoparticles were successfully designed and prepared as described in the Methods according to our previous study (50).

The hypotoxicity of nanoparticles is shown in Figure 1C. As previously described in methods, Rhy-loaded nanoparticles were designed and prepared (Figure 2A). The size distribution of Rhy-loaded nanoparticles was scanned by dynamic light scattering (DLS) (Figure 2B). We observed that the mean diameter of the Rhy-loaded nanoparticles was about 143±44 nm. By magnifying, the morphology of Rhy-loaded nanoparticles was observed by SEM (Figure 2C). Also, the surface of the Rhy-loaded nanoparticles was very smooth. Next, we examined the in vitro release of Rhy embedded in nanoparticles, which presented a sustainable release profile in physiological saline. Over 21 days, the totally released Rhy accounted for 75.63% at 254 nm and 77.67% at 246 nm (Figure 2D).

Figure 2 Characteristic detection of the Rhy-loaded nanoparticles. (A) Schematic diagram of the synthesis process of the Rhy-loaded nanoparticles. (B) Size distribution. (C) SEM. (D) The release of Rhy (%) from the Rhy-loaded nanoparticles in physiological saline at 254 and 246 nm during 21 days. DCM, dichloromethane; PLGA, polylactic-co-glycolic acid; PVA, poly(vinylalcohol); Rhy, rhynchophylline; SEM, scanning electron microscopy.

Endocytosis efficiency of nanoparticles

To validate the drug loading and transportation capacity of the nanoparticles, we prepared coumarin-loaded nanoparticles for more visual estimation. After administering the coumarin-loaded nanoparticles intratracheal spray for 48 hours in vivo, we collected the lung tissues and performed the immunofluorescence staining of the lung sections. As green fluorophores, we could see that the coumarin-loaded nanoparticles had diffused in the lung and were transfected into the cytoplasm and not the nucleus (Figure 3A).

Figure 3 Drug loading and efficiency of nanoparticles. (A) Immunofluorescence staining of lung sections from NC and coumarin-loaded nanoparticles-treated mice for 72 h. Scale bars: 200 µm. (B) Immunofluorescence staining of NC and coumarin-loaded nanoparticles-treated HFL1 cells for 48 h. Scale bars: 200 µm. (C) Endocytosis efficiency of NC and coumarin-loaded nanoparticles-treated HFL1 cells tested by flow cytometry analysis. DAPI, 4,6-diamino-2-phenyl indole; FITC, fluorescein isothiocyanate; NC, negative control.

In vitro, after adding the coumarin-loaded nanoparticles into the HFL1 cells for 48 hours, we noticed that the nanoparticles had transfected into the cytoplasm and not the nucleus (Figure 3B). Meanwhile, flow cytometry analysis was also conducted to analyze the percentage of cells with green fluorescence. The endocytosis efficiency of HFL1 cells reached 79.89% (Figure 3C). These results indicated that the nanoparticle drug delivery system could carry drugs into cells and had higher endocytosis efficiency.

Effects of the Rhy-loaded nanoparticles on PF therapy

We intratracheally sprayed 50 mL of Rhy-loaded nanoparticles solution on day 2 after 50 µL of BLM (2.0 mg/mL) intratracheal spray and collected the samples on day 21 (Figure 4A). Due to its high mortality (Figure 1F), we ultimately dissolved the BLM in physiological saline at a concentration of 1.0 mg/mL to conduct the animal experiments. As shown in Figure 4B, the physiological saline and unloaded nanoparticles treatment group (NC) displayed no differences with healthy mice. The lung morphology of the BLM and unloaded nanoparticles treatment group (BLM) revealed severe inflammatory edema and collagen deposition, while that of the BLM and Rhy-loaded nanoparticles treatment group (BLM+Rhy) manifested less inflammatory edema and collagen deposition (Figure 4B). It could be observed in the Masson’s trichrome and HE stains that BLM-induced mice demonstrated conspicuous PF, with collagen deposition, thickened alveoli walls, broad fibrotic area, and neutrophil aggregation in the interstitium, compared with the NC group. In contrast, collagen deposition, neutrophil aggregation, and fibrotic areas were notably reduced in the BLM+Rhy group (Figure 4C).

Figure 4 The role of the Rhy-loaded nanoparticles in PF in vitro. (A) Schematic diagram of the experimental procedure in lung fibrosis model. (B) Representative pictures of lung morphology in NC, BLM, BLM+Rhy groups. (C) Masson’s trichrome staining and HE staining of lung sections from mice in NC, BLM, BLM+Rhy groups. Scale bars: 200 µm. (D) Lung Index. (E) Quantification of pulmonary Hydroxyproline content. (F) Representative western blot assay and protein expression of FN, Col I, and α-SMA of lung tissues from mice in NC, BLM, BLM+TGF-β1 groups. (G) Histograms after statistical analysis of each protein in 3 groups. H&E, hematoxylin-eosin; PF, pulmonary fibrosis; FN, fibronectin; NC, negative control; BLM, bleomycin; Rhy, rhynchophylline; TGF, transforming growth factor.

The lung index is calculated by dividing the lung weight divided by the body weight and is an important index that reflects the degree of lung injury. The lung index value of the BLM group was significantly higher than that of the NC group (P<0.0001). Although the lung index value of the BLM+Rhy group was significantly lower than that of the BLM group (P=0.0159), it was still higher than that of the NC group (P=0.0003) (Figure 4D).

The hydroxyproline assay, which is a frequently-used method to quantify fibrosis, is applied for the preclinical evaluation of potential treatments for PF. Consistent with the lung index changes, the hydroxyproline content in the BLM+RHY group was markedly reduced compared to the BLM group (P=0.0468) but increased relative to the NC group (P=0.0367) (Figure 4E).

Next, we tested the PF-associated protein by WB. The results suggested that the Rhy-loaded nanoparticles diminished the BLM-induced abnormal overexpression of FN (P=0.048), Col I (P=0.017), and α-SMA (P=0.013) (Figure 4F,4G).

Taken together, these results showed that the Rhy-loaded nanoparticles exerted an anti-PF function. It could solve the concentration problems caused by low water solubility, short half-life, as well as the irritating smell of the solution, because Rhy-loaded nanoparticles dissolved in any volume of physiological saline and sustainably released Rhy. A disposable intratracheal spray achieved the desired purposes of a single non-invasive injury and precise treatment to avoid systemic reactions.

Analysis of the transcriptional sequencing

After determining the anti-PF effect of Rhy in C57BL/6 mice, we attempted to identify the key genes changed by Rhy through lung tissue transcriptome sequencing to explore the molecular mechanism of Rhy. All sequencing data is uploaded in NCBI (www.ncbi.nlm.nih.gov) under the bioproject PRJNA846551 and biosamples (SAMN28890083-SAMN28890091). The PCA analysis on the intact transcriptome of lung tissues in three groups indicated that the different groups could be clearly distinguished (Figure 5A). From the sample correlation results, it could be seen that the three repetitions in each group were highly correlated (Figure 5B). The ranges of correlation coefficients in the NC vs. BLM, BLM vs. BLM+Rhy, and NC vs. BLM+Rhy groups were 0.87–0.97, 0.89–0.99, and 0.87–0.98, respectively. The heat map confirmed the different mRNA expression profiles in all samples (Figure 5C), including 509 up-regulated and 428 down-regulated genes between NC and BLM, 568 up-regulated and 1,239 down-regulated genes between BLM and BLM+Rhy, 1,101 up-regulated and 997 down-regulated genes between NC and BLM+Rhy (Figure 5D), NC vs. BLM, BLM vs. BLM+Rhy, and NC vs. BLM+Rhy (Figure S1), respectively. As shown in the Venn diagram, the overlapping genes, which were upregulated or downregulated in the NC vs. BLM and BLM vs. BLM+Rhy groups but not in the NC vs. BLM+Rhy group (237 genes), were selected for further screening (Figure 5E).

Figure 5 Overall results of sequencing of all samples. (A) The PCA conducted on total transcriptome in all samples. (B) Correlation analysis of patterns of gene expression. (C) The heatmap of differentially expressed genes in all samples. (D) Up-regulated and down-regulated genes in differentially expressed genes in each group. (E) Venn diagram of differentially expressed genes. PCA, principal component analysis; BLM, bleomycin; Rhy, rhynchophylline; NC, negative control.

A volcano plot displayed 568 up-regulated and 1,239 down-regulated genes between BLM and BLM+Rhy (Figure 6A). The other groups are shown in Figure S2. In our previous study, Rhy was relevant to the extracellular matrix, and the fibrosis characteristics changes mainly occurred in the interstitium. The GO terms of the BLM vs. BLM+Rhy group revealed that the extracellular matrix and extracellular region were also frequently implicated (Figure 6B). KEGG analysis of the BLM vs. BLM+Rhy group manifested that ECM-receptor interaction was a significant KEGG pathway (Figure 6C). The GO terms and KEGG analysis of all groups are depicted in Figures S3,S4. The STRING website was used to establish the PPIs of differentially expressed mRNAs of the NC vs. BLM, BLM vs. BLM+Rhy, and NC vs. BLM+Rhy groups (Figure S5) as well as 237 differentially overlapping genes (Figure 6D). In addition, from among these 237 genes, 10 genes including CDH5, IGF1, ENG, TEK, FLT1, BDNF, COL3A1, COL5A1, ELN, and EDN1 were identified as the most essential hub genes (Figure 6E).

Figure 6 Representative gene enrichment analysis of the differentially expressed genes in some groups. (A) Volcano plot of BLM vs. BLM+Rhy group. (B) GO terms of BLM vs. BLM+Rhy group. (C) KEGG analysis of BLM vs. BLM+Rhy group. (D) PPI of 237 differentially overlapping genes in NC vs. BLM group and BLM vs. BLM+Rhy group but not NC vs. BLM+Rhy group. (E) Top 10 modules from PPI of 237 genes. CC, cellular component; MF, molecular function; BP, biological progress; KEGG, Kyoto Encyclopedia of Gene and Genomes; BLM, bleomycin; Rhy, rhynchophylline; GO, Gene Onology; PPI, protein-protein interaction; NC, negative control; FDR, false discovery rate.

The mechanism of Rhy in anti-PF

To further track the downstream targets and pathways of Rhy, KEGG analysis of the NC vs. BLM group showed PI3K/AKT was also a significant KEGG pathway (Figure S3), the Venn diagram was conducted to more precisely identify the overlapping genes. It was demonstrated that TEK and BDNF were the precisely overlapping genes, which existed in the KEGG/PI3K-AKT signaling, clust 5 (Figure 7A, Figure S6), 237 genes, and GO/extracellular region groups (Figure 7B). BDNF was reported to promote neuroprotection and neuroregeneration in neurological diseases (51,52). Although TEK exerted anti-inflammatory effects in the vasculature system (53), it was also reported that TEK overexpression could cause more clinically severe arthritis in mice (54) and that TEK was abundantly expressed in macrophages in the lungs of asthmatic mice (55). The above-mentioned studies demonstrated that TEK was bound with inflammation, which was more related to PF. Hence, we instigated TEK-PI3K/AKT as the downstream target and pathway of Rhy.

Figure 7 Downstream target and pathway of Rhy. (A) Expression patterns of genes in clust 5. (B) Venn diagram of differentially expressed genes in PI3K-AKT signaling of KEGG in the BLM vs. the BLM+Rhy group, clust 5, 237 genes, and extracellular region of GO in the BLM vs. BLM+Rhy group. (C) HFL1 cells stimulated with TGF-β1 (5 ng/mL) were treated with Rhy (200 ng/mL) for 12 hours. Representative Western blot assay and protein expression of TEK, p-AKT, and AKT in the NC, TGF-β1-treated, and TGF-β1+Rhy-treated groups. (D) Histograms after statistical analysis of each protein in the three groups. (E) HFL1 cells stimulated with TGF-β1 (5 ng/mL) were treated with Rhy (200 ng/mL) for 48 hours. Representative Western blot assay and protein expressions of TEK, p-AKT, and AKT in the NC, TGF-β1-treated, and TGF-β1+Rhy-treated groups. (F) Histograms after statistical analysis of each protein in the three groups. Rhy, rhynchophylline; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Gene and Genomes; BLM, bleomycin; TGF, transforming growth factor; NC, negative control.

To confirm this hypothesis, we applied WB to explore the relative molecular protein expressions. After adding TGF-β1 or TGF-β1+Rhy for 12 hours, we noticed that there was no difference in TEK expression between the NC and TGF-β1 groups; however, Rhy remarkably reduced TEK expression (P=0.017). Simultaneously, Rhy reduced the TGF-β1-induced elevated expression of p-AKT (P=0.029, P=0.006). There were no significant changes in AKT expression (Figure 7C,7D). After adding TGF-β1 or TGF-β1+Rhy for 48 hours, we continually detected the expressions of these genes. The results indicated that TEK expression in the TGF-β1 group was conspicuously higher than that in the NC group (P=0.003); meanwhile compared with the TGF-β1 group, TEK expression in the TGF-β1+Rhy group was minimized (P=0.044). Concurrently, there was no significant difference in the p-AKT and AKT expressions among the three groups (Figure 7E,7F). Together, these results highlighted that Rhy prevented PF by inhibiting the TEK-PI3K/AKT signaling pathway.

In summary, these findings suggest that after the Rhy-loaded nanoparticles solution penetrates the lungs via intratracheal spray, it sustainably restricts PF via the suppression of FN, Col I, and α-SMA expression in lung fibroblasts by inhibiting the TEK-PI3K/AKT signaling pathway (Figure 8).

Figure 8 Schematic diagram of the Rhy loaded nanoparticles intratracheal spray in the fibrotic lung. PLGA, polylactic-coglycolic acid; PVA, poly(vinylalcohol); DCM, dichloromethane; Rhy, rhynchophylline.

Discussion

PF is a fatal and almost incurable lung disease with the characteristics of functional replacement of extracellular matrix and fibroblast proliferation (56,57). IPF is the most common type of ILD, which will eventually cause PF (8,9); given the COVID-19 outbreak as well as the increasing popularity of RFA in lung cancer treatment, PF has become the major complication of both diseases (16,17).

TCM has accumulated the experience and theoretical knowledge of ancient Chinese people in combating diseases. It has gradually formed and developed into a medical theoretical system over more than 2,000 years of medical practice (58). The tremendous potential abilities of TCM are demonstrated in health improvement, disease prevention, and treatment of conditions such as cancers, cardiovascular diseases, and autoimmune diseases (59-61). TCM is also the focus of drug research. Numerous drugs including artemisinin, digitoxin, and quinine have shown advantages in disease treatment. Tu was awarded the Nobel Prize for physiology and medicine, which further bolsters the popularity of TCM (62). Nowadays, a growing number of studies have indicated that TCM is effective for PF treatment (19,20), which would provide more treatment options.

Rhy is a small molecular TCM drug, which is commonly applied in the clinic to cure cardiovascular disease. Given its increased attention among researchers, Rhy has been reported to function in certain fields (28-31); however, its effect on PF remains unclear. In addition, Song (32) and Yang (33) reported that Rhy plays a role in the inhibition of adhesion formation. Considering the similar mechanisms between adhesion formation and PF, we wondered whether Rhy could retard the progression of PF.

At present, BLM is most frequently used to induce experimental models of PF for potential therapy, as the histopathological varieties of model lung and human PF are extremely similar (34). Consequently, we utilized BLM-induced C57BL/6 mice as our experimental models. Through intraperitoneal injection of Rhy, we found (by Masson and HE staining) that Rhy reduced collagen deposition and the fibrotic area, thinned alveolar walls, rebuilt the alveolar structure in lesion regions, and increased the alveolar air area in a dose-dependent manner, which roughly identified the effect of Rhy on anti-PF in vivo. Also, the survival rate of mice increased with the increase in Rhy concentration. Considering that Rhy will be ultimately used to treat human patients, we discovered that a Rhy concentration of less than 200 ng/mL for HFL1 is safe and hypotoxic. As a promoting factor of fibrotic diseases, TGF-β1 stimulation induces IL-13 macrophages (63,64) and A549 (65) to express fibroblast specificity and HFL1 (66-69) activation. WB revealed that Rhy decreased the TGF-β1-induced abnormally high expression of FN, Col I, and α-SMA in a dose-dependent manner.

Given its characteristics of low water solubility and short half-life, the clinical application of Rhy will be limited according to the concentration, multiple administrations, and systemic reactions. To address these limitations, we designed and prepared Rhy-loaded nanoparticles. The total sustainably released Rhy dissolved in the physiological saline over 21 days accounted for about 60%.

To clarify whether nanoparticles were able to carry Rhy into cells or remained in the trachea, we prepared coumarin-loaded nanoparticles, which could emit green fluorescence when exposed to blue light or ultraviolet light. We noticed that the nanoparticles had transfected into the cytoplasm (not nuclei) of cells both in vivo and in vitro. The endocytosis efficiency test for HFL1 reached 79.89%. Taken together, these results indicated that the drug delivery system of nanoparticles could carry drugs into the cells and had higher endocytosis efficiency.

Next, we evaluated the effects of the Rhy-loaded nanoparticles on PF therapy in the C57BL/6 mice. Owing to the high mortality, we chose a concentration of 1.0 mg/mL BLM instead of 2.0 mg/mL. The mice were then treated as planned. The lung morphology of the BLM+Rhy group displayed less inflammatory edema and collagen deposition compared to the BLM group. Through Masson’s trichrome and HE staining, it was observed that the Rhy-loaded nanoparticles relieved collagen deposition, neutrophil aggregation, and fibrotic areas in the BLM+Rhy group. The lung index value of the BLM+Rhy group was significantly lower than that of the BLM group but was still higher than that of the NC group. Also, the hydroxyproline content in the RHY+BLM group was markedly lower than that in the BLM group but was higher than that in the NC group. Moreover, the Rhy-loaded nanoparticles also alleviated the BLM-induced abnormal overexpression of FN, Col I, and α-SMA. These results exhibited that the Rhy-loaded nanoparticles exerted an anti-PF function and could overcome the limitations of Rhy in clinical application.

Lung tissue transcriptome sequencing was applied to explore the molecular mechanism of Rhy. By analyzing the transcriptional sequencing results, we found that TEK and PI3K-AKT signaling were downstream targets and pathways of Rhy, which may have resulted from our Rhy intervention in the early inflammation phase of the fibrosis process. p-Akt changes from 2 hours after TGF-β1 stimulation, with the most obvious trend occurring at 12 hours and the most meaningless trend occurring at 48 hours (70). Accordingly, we examined p-AKT protein expression at 12 hours and TEK expression at 48 hours. After 12 hours of TGF-β1 or TGF-β1+Rhy administration, Rhy subtracted the TGF-β1-induced elevated expression of p-AKT. After 48 hours of TGF-β1 or TGF-β1+Rhy addition, it was noted that TEK expression in the TGF-β1 group was conspicuously higher than that in the NC group but was lower in the TGF-β1+Rhy group compared to the TGF-β1 group. Interestingly, via Western blotting, we observed that after 12 hours of TGF-β1 or TGF-β1+Rhy administration, there was no difference in TEK expression between the TGF-β1 and NC groups but was remarkably decreased in the TGF-β1+Rhy group. This illustrated that TGF-β1 could not increase TEK expression in a short time but Rhy could decrease the expressions of TEK and p-AKT immediately. After 48 hours, TGF-β1 gradually promoted TEK, and with Rhy degradation, the ability of Rhy to inhibit TEK and p-AKT expressions gradually weakened. Thus, it could be seen that with Rhy degradation, the expressions of TEK, p-AKT, and AKT were dynamic, which might explain why the trend of mRNA differs from that of protein in clust 5. It was also possible that TEK, like some proteins, exhibited other forms such as phosphorylation, which increased the degradation of phosphorylation and led to the decrease of total protein.

In this research, although we confirmed that Rhy suppresses the progression of PF by inhibiting the TEK-PI3K-AKT signaling pathway, only fibroblasts were involved. However, the effects of Rhy on other fibrosis-related cells such as epithelial cells, macrophages, neutrophils, and so on remain unclear. In the future, we intend to explore other downstream targets and pathways of Rhy on other cells or fields in-depth to ensure its safety and effectiveness in humans. With the increasingly severe situation of COVID-19 and the widespread application of RFA, PF is becoming more common than before. We hope that some researchers will investigate the effect of Rhy on fibrotic complications caused by these two diseases.

Conclusions

Rhy restricts PF progression by depressing the TEK-PI3K/AKT signaling pathway. Hence, the locally sustainable release of Rhy in nanoparticles is a highly effective therapy to limit PF and should be developed.

Acknowledgments

Funding: This work was supported by National Natural Science Foundation of China (No. NNSCF-81770266), Clinical Medical Research Center of Cardiothoracic Diseases in Nantong (No. HS2019001), Innovation Team of Cardiothoracic Disease in Affiliated Hospital of Nantong University (No. TECT-A04), China and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX20-2632), and Nantong Key Laboratory of Translational Medicine of Cardiothoracic Diseases.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-675/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-675/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-675/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. Experiments involving animals were performed under a project license (No. 20140901-001) granted by Animal Ethics Committee of Nantong University, in compliance with Nantong University guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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