Tuberostemonine may alleviates proliferation of lung fibroblasts caused by pulmonary fibrosis

Abstract

Objectives: Tuberostemonine has several biological activity, the aim of study examined the impact of tuberostemonine on the proliferation of TGF-β1 induced cell model, and its ability to alleviate pulmonary fibrosis stimulated by bleomycin in mice. Methods: In vitro, we assessed the effect of tuberostemonine (350, 550 and 750 µM) on the proliferation of cells stimulated by TGF-β1 (10 μg/L), as well as on parameters such as α-SMA vitality, human fibronectin, collagen, and hydroxyproline levels in cells. In vivo, we analyzed inflammation, hydroxyproline, collagen activity and metabolomics in the lungs of mice. Additionally, a comprehensive investigation into the TGF-β/smad signaling pathway was undertaken, targeting lung tissue as well as HFL cells. Results: Within the confines of an in vitro setup, the tuberostemonine manifested a discerned IC50 of 1.9 mM. Furthermore, a significant reduction of over fifty percent was ascertained in the secretion levels of hydroxyproline, fibronectin, collagen type I, collagen type III and α-SMA. In vivo, tuberostemonine obviously improved the respiratory function percentage over 50% of animal model and decreased the hydroxyproline, lung inflammation and collagen deposition. A prominent decline in TGF-β/smad pathway functioning was identified within both the internal and external cellular contexts. Conclusions: Tuberostemonine is considered as a modulator to alleviate fibrosis and may become a new renovation for pulmonary fibrosis.

Introduction

Among interstitial lung diseases, pulmonary fibrosis stands out as a widespread occurrence, exhibiting a predilection for affecting males over females. ¹ The etiology of this condition is linked to environmental factors, such as exposure to dust and lung inflammatory conditions. These multifaceted issues pose challenges in effectively managing pulmonary fibrosis (PF). ² Patients with pulmonary fibrosis often experience varying degrees of breathing difficulties, frequently accompanied by other complications. ³ Those with severe pulmonary fibrosis may experience structural changes in the pulmonary alveoli and impaired gas exchange, which can contribute to their discomfort. ⁴ Treatment with immunosuppressive agents such as prednisolone in clinical settings may lead to an increase in patient mortality. ⁵ Common pathological changes seen in individuals with pulmonary fibrosis encompass widespread interstitial pneumonia, progressive formation of fibrotic scars, dysregulated proliferation of fibroblasts, excessive accumulation of extracellular matrix resulting from reciprocal stimulatory interactions within the lung microenvironment, anomalous expansion of myofibroblasts, sustained inflammatory injury and structural deterioration affecting the tissue surrounding alveolar cells within the pulmonary region. ⁶ The progression of alveolar epithelial impairment is of pivotal significance in initiating pulmonary fibrosis. ⁷ The regulatory role of transforming growth factor-β1 (TGF-β1) becomes evident in directing the recruitment of fibroblasts to injury sites and initiating their differentiation into myofibroblasts. ⁸ Furthermore, the continuous activation of monocytes and T cells sustains an inflammatory environment, which in turn triggers the transformation of pulmonary fibroblasts into myofibroblasts and accelerates the synthesis of the extracellular matrix.

Many natural products that possess both food-like characteristics and drug efficacy are considered effective anti-fibrotic compounds, such as Astragalus membranaceus var. mongholicus (Bunge) P. K. Hsiao, Rheum palmatum L, Salvia miltiorrhiza Bunge, Curcuma longa L and Rhodiola rosea L. The components contained in these herbs, such as curcumin, ⁹ astragaloside IV ¹⁰ and quercetin, ¹¹ can also improve lung function and relieve dyspnea. Stemona tuberosa Lour is a medicinal plant widely used in Asia for the treatment of respiratory ailments. Within its composition lies tuberostemonine, an active compound renowned for its antineoplastic, antitussive, and anti-inflammatory properties.^12,13 Nevertheless, the question regarding its efficacy in improving pulmonary fibrosis through modulation of the TGF/Smad signaling pathway remains unresolved. Therefore, this investigation aims to clarify the role of tuberostemonine from the whole plant of Stemona tuberosa Lour in treating PF via the TGF/Smad signaling pathway.

Materials and methods

Chemical reagents and antibodies

Sourced from DESITE Biotechnology Co., Ltd. (Chengdu, China), tuberostemonine (#DST210509-038) was obtained with an exceptional purity of over 98%. InvivoGen supplied bleomycin (#S1214) (Shanghai, China), while Continent Pharmaceutical Co., Ltd. provided pirfenidone (PFD) (#161003) (Beijing, China). Abcam (Shanghai, China) supplied the following antibodies: β-Actin antibody (#ab8226), anti-collagen I (Col I) antibody (#ab254113), anti-smad2 antibody (#ab33875), anti-TGF beta receptor II antibody (#ab259360), anti-collagen III (Col III) antibody (#ab7778), anti-smad7 antibody (#ab216428) and anti-smad3 antibody (#ab52903). CST (Shanghai, China) furnished the subsequent antibodies: phospho-smad2 (p-smad2) rabbit mAb (#18338), TGF-β antibody (#3711), α-SMA rabbit mAb (#19245), e-cadherin rabbit mAb (#3195), LOX rabbit mAb (#58135), vimentin rabbit mAb (#5741), and phospho-smad3 (p-smad3) rabbit mAb (#9520). Rabbit anti-phospho-smad7 (p-smad7) antibody (#bs-20024R), rabbit anti-MMP7 antibody (#bs-0423R) and rabbit anti-MMP9 antibody (#bs-20619R) were bought from Bioss (Beijing, China). RIPA buffer (#R0010), BSA blocking buffer (#SW3015), masson staining kit (#G1340), phosphate-buffered solution (#P1010), and sodium carboxymethyl cellulose (#IS9000) were obtained from Solarbio (Beijing, China). Heparin sodium (0.1%, #320121) was purchased from Bioisco (Lianyungang, China). The micro BCA protein assay kit (#CW2011), hailing from Cwbio (Beijing, China), was acquired, whereas the immunohistochemistry kit (#PV-9000) was secured from ZSGB-Bio (Beijing, China). The acquisition of the hydroxyproline assay kit (#A030-2-1) was accomplished through Jianchen Bioengineering Institute (Nanjing, China). Acquired from Beyotime (Shanghai, China), the following enzyme-linked immunosorbent assay kits were obtained: mouse IL-1β (#PI301), mouse monocyte chemoattractant protein 1 (MCP-1) (#PC125), mouse IL-2 (#PI575), mouse interferon-γ (#PI508), and mouse IL-6 (#PI326). NeoBioscience (Shenzhen, China) facilitated the purchase of the mouse macrophage inflammatory protein 2 (MIP-2) ELISA kit (#EMC122.96). Human fetal lung fibroblasts (HFL) (#06.0047) were sourced from EallBio (Beijing, China). Ham’s F-12K (Kaighn’s) medium (#2276929) and fetal bovine serum (#42F3261 K) were procured from Thermo Fisher Technology Co., Ltd. (Shanghai, China). Mouse tumor necrosis factor α (TNF-α) ELISA kit (#CSB-E04741m), mouse macrophage colony-stimulating factor (M-CSF) ELISA kit (#CSB-E04659 m), human fibronectin ELISA kit (CSB-E04551h), human collagen type III ELISA kit (#CSB-E04799h), human collagen type I ELISA kit (#CSB-E08082 h) were purchased from CUSABIO (Wuhan, China).

Cells culture

The human fetal lung fibroblasts (HFL) were cultured in Ham’s F-12K medium, supplemented with 10% fetal bovine serum. Modification of the medium and subsequent phosphate buffer saline (PBS) washes were performed upon reaching 80% cell confluence. Subsequently, trypsin was utilized for cell detachment, and digestion was terminated once they became rounded. The subsequent step involved placing the cells in a 5% CO₂ incubator at a temperature of 37°C for their continued cultivation.

Cells viability assay

HFL cells were cultured using 96-well plates, with each well seeded at a density of 5 × 10³ cells. 24 h later, the experimental groups were established by the addition of different concentrations of tuberostemonine including 0, 80.25, 156.45, 298.85, 534.60, 760.89 and 890.75 µM. Upon undergoing a 48-h incubation at 37°C with 5% CO₂, the cells were treated with the addition of 10 μL cell counting kit-8 (CCK-8) in every well of each group. Subsequently, the samples underwent a 4h incubation period, after which the absorbance value was assessed at 450 nm. ¹⁴

Immunofluorescence assay

After disposing of the culture medium, the cultured cells were washed once with PBS. For each group, fixation was conducted with 4% paraformaldehyde for 30 min. The samples, having undergone immersion in 1% Triton X-100 for 10 min, were subsequently subjected to a series of three PBS washes. Subsequent to this, a blocking process was carried out, entailing the utilization of serum sourced from goats. The anti α-SMA antibody (1:100) was introduced to the cells and incubated at 4°C overnight. Subsequent to a triple wash using PBS for cells, the goat anti-rabbit IgG antibody (1:400) was mixed into cells and incubated at room temperature under dark conditions for 1h. The coverslip containing 4’, 6-diamidino-2- phenylindole (DAPI) covered the cells. The cell images were captured by a fluorescence microscope (Leica TCS SP8 Imaging System, Germany).

Animal and experimental design

A total of forty male C57BL/6J mice, adhering to specific pathogen-free conditions and weighing between 18 and 22 g, were procured from TianQin Biotechnology Co., Ltd (Changsha, China). Animal models were created based on a reference 15. An intratracheal injection method of 5 mg/kg bleomycin was administered to induce pulmonary fibrosis in animals. After the administration of the injection, subsequent to a randomized partitioning procedure, four distinct groups emerged: sham group (n = 10), model group (n = 10), model + 100 mg/kg tuberostenomine group (n = 10), and model + 100 mg/kg pirfenidone (PFD) group (n = 10). Over the course of 28 successive days, the mice assigned to these groups were administered either tuberostenomine or PFD. The Animal Ethics Committee of Guizhou University of Traditional Chinese Medicine granted authorization for the implementation of the experimental protocol (Reference number: 20210025). The experimental procedure adheres to the implementation guidelines stipulated by the National Measures for the Administration of Medical Experimental Animals.

Pulmonary function assay

The animals were positioned within an enclosed apparatus of whole-body plethysmography, facilitating the systematic monitoring of respiratory function in each group of animals (Shanghai TOW Intelligent Technology Co., Ltd). The parameters including inspiration time, tidal volume, expiration time, promoted expiratory flow 50 and pause of the animals were recorded for statistical analysis. ¹⁵ After completing the experiment, mice were euthanized by isoflurane overdose to ensure their humane death. Once euthanized, the entire pulmonary organs were promptly extracted and their weights documented.

Morphological and histological analysis

Each group’s animal lung specimens were subjected to fixation using a 4% paraformaldehyde solution for a duration of 24 h. A thorough rinsing with tap water followed, preceding a meticulously executed one-hour alcohol gradient dehydration process, beginning with lower concentrations and ascending to higher concentrations. The samples were then immersed in xylene for 20 min. Subsequently, the samples were enveloped within a paraffin block and expertly cut into sections measuring 4 µm in thickness. Following the sections’ drying, xylene was dewaxed, and the sections were subjected to dehydration with gradient alcohol series. The sections underwent subsequent staining employing hematoxylin-eosin (HE) and masson staining methodologies. Subsequently, an optical instrument (Olympus, Japan) was employed to assess the histopathological modifications in lung tissues, adhering to Szapiel’s mode of pathological evaluation.

Hydroxyproline assay

Lung tissue (30 mg) was harvested for each sample. In accordance with the prescribed methodology of the kit, the biological sample was situated within a designated test tube, into which 1 mL of hydrolysate was introduced. The ensuing step involved subjecting the tube to a robust boiling water bath for a precise span of 20 min. Once this thermal exposure was completed, a minimal volume of 10 µL indicator solution was judiciously administered, with careful attention to attaining a pH level in the vicinity of 6.0. Next, 10 mL of dH₂O was added, and 3 mL of diluted hydrolysate was sampled. Introduction of activated carbon (20 mg) was conducted into the diluted hydrolysate, succeeded by centrifugation at 3500 r/min lasting 10 min. Measurement of the supernatant’s absorbance value took place at 550 nm using instrumentation from Thermo Fisher Scientific, China. Similarly, the determination of hydroxyproline (HYP) content within the cell supernatant was conducted following an analogous method.

Enzyme-linked immunosorbent assay

Blood was collected from the orbit of the animals. Enzyme-linked immunosorbent assay (ELISA) kits were adopted to evaluate the concentrations of numerous inflammatory factors in plasma. Similar approach was employed to quantify human fibronectin (FN), Collagen I (Col I), Collagen III (Col III), and hydroxyproline contents in both cellular and tissue specimens.

Immunohistochemical assay

After fixation with 4% paraformaldehyde, all specimens underwent a process of dehydration, followed by embedding in paraffin and subsequent uniform sectioning at a thickness of 5 μm. The sample slices underwent baking, followed by deparaffinization in xylene and treatment with a gradient of ethanol. Subsequently, the slices were immersed in a 3% H₂O₂ solution for a duration of 5 min. Post three successive immersions in PBS, the slices were subjected to repair via citric acid buffer under high temperature and pressure. Following this, the slices underwent a thorough triple wash with PBS. The anti-Col I and Col III antibodies were introduced into the slices and stored at 4°C for 24 h. The antibodies in the PV9000 kit were added into slices. After reagent color development, the slices dehydration via a gradient of ethanol, permeation in xylene, sealing with neutral balsam, and were subsequently examined using a microscope.

Western blotting assay

The culture medium of HFL cells were discarded and rinsed twice with PBS. The radio-immunoprecipitation assay protein lysate, phenylmethylsulfonyl fluoride and protease inhibitors were added into each well. The supernatant was delicately moved to a new centrifuge tube, positioned for forthcoming application. The lung tissue of each group was minced and incubated at low temperature after the addition of lysate in the same way as the cell was used to extract total protein, and then the supernatant was centrifuged for reserve. The bicinchoninic acid assay was employed to ascertain the protein concentration in tissue or cell samples. At the end of protein denaturation, separating and concentrating gels were prepared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Within each loading well of the gel, an amalgamation of 50 μg total protein and 5 μL of marker was deposited, initiating electrophoresis under a voltage of 80 V for a duration of 40 min. Subsequently, the voltage was adjusted to 120 V and continued for 1 h. The membrane was transferred from 200 mA to 300 mA at constant flow for 40 min. The polyvinylidene fluoride membrane was situated within a solution consisting of 5% skim milk powder prepared in tris buffered saline tween, and the membrane underwent blocking at room temperature for a duration of 1 h. Subsequently, a primary antibody working solution was meticulously concocted, inclusive of anti-β-actin (1:8000), anti-TGF-β (1:1000), anti-TGF-β receptor II (1:1000), anti-ɑ-SMA (1:1000), anti-smad2 (1:1000), anti-smad3 (1:1000), anti-smad7 (1:1000), anti-p-smad2 (1:500), anti-p-smad3 (1:500), anti-p-smad7 (1:500), anti-MMP-7 (1:1000), anti-MMP-9 (1:1000), anti-e-cadherin (1:1000), anti-LOX (1:500), anti-vimentin (1:500) and goat anti-rabbit antibody (1:5000). This experiment was repeated three times with the enhanced chemiluminescence mixture prepared for imaging. After regenerating each membrane with the regenerating agent, antibody reintroduction and subsequent re-incubation were performed, repeating the test.

Metabolomics assay

High-resolution untargeted metabolomics analysis of lung samples from the control, model, and tuberostemonine groups was conducted. Samples were added to a pre-cooled solution of methanol/acetonitrile/water (2:2:1, v/v), and ultrasonicated at 4°C for 30 min. The mixture was centrifuged at 14000 g at 4°C for 20 min, and the supernatant was collected. Subsequently, 100 μL of an acetonitrile aqueous solution (1:1, v/v) was added to the supernatant, which was centrifuged at 14000 g at 4°C for another 15 min. Finally, the supernatant was sampled for analysis. Metabolites in the samples were detected by the UHPLC-Q-TOF/MS method (AB SCIEX, Agilent, USA).

Statistical analysis

The analysis of data resulting from a minimum of three distinct experimental instances was carried out using SPSS 23.0 software, and the generation of visual outputs was achieved via GraphPad Prism 9.0. Trial data was based on non-parametric analysis. In cases where p value > .05, the observed difference was deemed nonsignificant. Conversely, when the p value < .05, the difference was regarded significant.

Results

Tuberostemonine inhibits human fetal lung fibroblasts proliferation

After 48h incubation of human fetal lung fibroblasts, we got the optimal drug concentration. Derived from this ideal concentration, subsequent experiments encompassed the introduction of drug concentrations set at 350 µM, 550 µM, and 750 µM. The ascertained tuberostemonine concentration facilitated an extensive assessment of its suppressive impact on cellular activity. With the increasing of drug concentration, tuberostemonine could significantly inhibited cell growth (Figure 1A and B).

Figure 1.

Effect of tuberostemonine on growth and suppression ratio of human fetal lung fibroblasts. Cells were incubated with 10 μg/L TGF-β1 or different concentrations of tuberostemonine, and the inhibitory effect of tuberostemonine on cell proliferation was analyzed using CCK-8 protocol after a 48-hour incubation period. (A) Cell viability of human fetal lung fibroblasts. (B) The suppression ratio of human fetal lung fibroblasts. (^**p < .01 vs.TGF-β1 group, n = 3).

Tuberostemonine reduces expression of human fibronectin, human collagen type I, human collagen type III and hydroxyproline in human fetal lung fibroblasts

When fibroblasts showed continuous activation and proliferation, an abundant release of FN contributes to the forming of the cell scaffold framework. On basis of cell scaffold structure, Col I, Col III and HYP would by fibroblasts to continuous secrete. Upon 48 h of incubation, human fetal lung fibroblasts subjected to TGF-β1 induction showcased elevated production of FN, Col I, Col III, and HYP compared to the control group. Subsequent exposure to varying concentrations of tuberostemonine resulted in a progressive reduction in FN, Col I, Col III, and HYP levels (Figure 2A and B).

Figure 2.

Using ELISA to detect the effect of tuberostemonine on the expressions of FN, Col I, Col III, and HYP in cell culture supernatant(A)(B). (^##p < .01 vs Ctrl group; ^**p < .01 vs TGF-β1 group, n = 6).

Tuberostemonine lessens α-SMA expression in human fetal lung fibroblasts

α-smooth muscle actin (α-SMA) overexpression was a marker of continuous activation and proliferation in fibroblasts. After 48h of incubation, TGF-β1-induced human fetal lung fibroblasts displayed boosted synthesis of α-SMA protein in the cell supernatant in comparison to the control group. Following treatment with distinct concentrations of tuberostemonine, the α-SMA protein content exhibited a gradual decline (Figure 3A and B).

Figure 3.

Effect of tuberostemonine on expression of α-SMA protein in human fetal lung fibroblasts. 10 μg/L TGF-β1 and different concentrations of tuberostemonine were added in cells and incubated for 48h. (A)(B) The expression of α-SMA protein in 10 to 15 human fetal lung fibroblast cells. (^##p < .01 vs Ctrl group; ^**p < .01 vs TGF-β1 group, n = 5).

Tuberostemonine decreases the over-expression of TGF-β/smad signaling in cell

When TGF-β was added in cells, various of proteins in Smad signaling were changed, and smad 2/3 and its phosphorylation, vimentin and LOX were over-activated. The physiological activity of smad signaling was significantly inhibited after the addition of tuberostemonine. The results indicated a pronounced inhibitory outcome on TGF-β1-induced fibroblast proliferation, attributed to the modulation of smad 2/3 signaling via tuberostemonine (Figure 4A and B).

Figure 4.

(A)(B) The semi-quantitative analysis of the experimental results involves comparing the grayscale of the images from other groups with those from the control group. Expression levels of α-SMA, smad2, smad3, p-smad2, p-smad3, vimentin and LOX in HFL cells (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 3).

Effect of tuberostemonine or pirfenidone on respiratory dysfunction in mice

Based on the results of the previous cell experiment, further comparison was made with TGF inhibitor pirfenidone (PFD) of the same dosage to observe the biological mechanism of tuberostemonine in animals. It is clear that the introduction of bleomycin (BLM) into the trachea of mice results in the manifestation of fibrotic lesions typically within a range of 14-28 days. After 28 days of BLM induction or drug intervention, The respiratory function of the lungs revealed a substantial change in comparison to the control group, with markedly declined pulmonary Vt, significantly extended Ti and Te, significantly boosted Penh and significantly reduced EF50. In contrast to the model group, the administration of same application amount tuberostemonine and PFD could significantly alleviate lung dysfunction caused by BLM (Figure 5A–E).

Figure 5.

(A–E) Tuberostemonine or PFD could be improved bleomycin-induced respiratory dysfunction in animal lung. (F)Tuberostemonine could be improved the organ coefficient of the lungs (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 10).

Tuberostemonine improves the morphological and histologic appearance of the lung

Upon completion of a 28-days BLM induction, the determination of organ coefficients for the lungs in the animal subjects was accomplished through visual assessment and rigorous statistical analysis. The model group showed congestion, larger tissue, and a significantly higher organ coefficient than the control group. Although the animals in the same amount of medication of tuberostemonine and PFD groups showed congestion in the lungs, their organ coefficient was found to be considerably lower than that observed in the model group (Figures 5F and 6A). Histopathologically, the control group exhibited preserved alveolar structures characterized by an absence of significant lymphocyte infiltration. The morphological integrity was well-maintained, accompanied by minimal collagen deposition. In the model group, there was large infiltration of lymphocytes, severe adhesion of alveolar and the surrounding tissues, a large number of red blood cells, high abnormal content of collagen within the lung, and loss of normal structure of gas exchange. A large amount of adhesion and a large number of erythrocytes within the lung tissue were also observed in the tuberostemonine and PFD groups. Collagen proliferation is obvious, but a certain amount of alveolar and surrounding structures still existing (Figure 6B–E).

Figure 6.

(A)Tuberostemonine or PFD has a good protective effect on bleomycin-induced pulmonary fibrosis in C57BL/6 mice. (B)(D) HE staining score for each group (100×). (C)(E) Masson staining score for each group (100×). (F) The content of hydroxyproline in animal lung tissue. (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 10).

Tuberostemonine prevents the up-regulation of the hydroxyproline content in the lung

Following 28 days of BLM-induced pulmonary fibrosis in animal subjects, the pulmonary HYP content witnessed a noteworthy rise within the model group, surpassing that observed in the control group. For equitably applying tuberostemonine and PFD in the same quantity, the HYP content in the lung reduced (Figure 6F).

Tuberostemonine prevents enhancement of inflammatory factors in the blood

By comparison to the control group, the model group manifested a boost in several inflammation-related cytokines in the surrounding blood, and the level of these inflammation-related factors decreased following the administration same dosage of tuberostemonine and PFD (Figure 7A and B).

Figure 7.

(A) (B) Employing ELISA to detect the secretion levels of IL-1β, IL-2, IL-6, TNF-a, IFN-y, M-CSF, MIP-2 and MCP-1 in animal plasma (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 10).

Tuberostemonine restrains the over-expression of TGF-β/smad signaling in lung

Fibrosis, featured by tissue scarring and stiffening, arises as a consequence of excessive extracellular matrix protein deposition facilitated by myofibroblasts within a prolonged inflammatory context. Deleterious stimuli such as toxins, autoimmune response, and mechanical stress are capable of inducing fibrotic cellular responses. TGF are the major effector of the fibrosis process. Subsequent to the induction of fibrosis in animal lung tissues by BLM or the introduction of TGF-β1 to human fetal lung fibroblast cells, an evident increase in the expression levels of TGF-β, TGF-β receptor II, α-SMA, smad2, p-smad2, smad3, p-smad3, MMP-7, MMP-9, LOX, and vimentin was observed. In contrast, the expression of e-cadherin, p-smad7, and smad7 went down. Following the application of matched dosages of tuberostemonine and PFD, a decline was evident in the expression levels of TGF-β, TGF-β receptor II, α-SMA, smad2, p-smad2, smad3, p-smad3, MMP-7, MMP-9, LOX, and vimentin. Simultaneously, an upswing was observed in the expression levels of e-cadherin, p-smad7, and smad7. TGF-β could activated biological effects via downstream smad signaling (Figure 8).

Figure 8.

(A)(B) The semi-quantitative analysis of the experimental results involves comparing the grayscale of the images from other groups with those from the control group. Expression levels of TGF-β, TGF-β Receptor II, α-SMA, smad2, smad3, smad7, p-smad2, p-smad3, p-smad7, MMP-7, MMP-9 and e-cadherin in animal lung. (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 5).

Tuberostemonine decreases the collagen proliferation in lung

The immunohistochemical images underwent a semi-quantitative analysis based on both staining area and intensity. Comparatively, the model group exhibited considerably heightened expression levels of Col I and Col III in relation to the control group. Nonetheless, administration of equivalent doses of tuberostemonine and PFD induced a substantial reduction in these levels (Figure 9A–C).

Figure 9.

Tuberostemonine could be reduced collagen formation in animal lung of C57BL/6 mice. (A)(B) Immunohistochemical staining for each group. (C) IHC score. The score was based on the intensity of staining and the proportion of positive cells (^##p < .01 vs Ctrl group; ^**p < .01 vs Model group, n = 6).

Tuberostemonine regulates metabolites in the lung

At the 28th day mark, following the onset of pulmonary fibrosis induced by BLM, internal metabolites were harvested from lung tissues of the control group, model group, and tuberostemonine-treated group. These metabolites underwent rigorous differential analysis. The extraction of peaks from all experimental samples and QC samples was carried out for PCA analysis (Figure 10A and B). The experimental findings revealed a tight clustering of the quality control (QC) samples in both the positive and negative ion modes, signifying the robust the good reproducibility of the experiment. By using an in-house database (Shanghai Applied Protein Technology, China) search library, metabolites in the biological samples were structurally identified by matching their retention time with those mentioned in the local database (within <25 ppm), secondary fragmentation spectrogram, collision energy, and the identification outcomes were subject to scrutiny and subsequently affirmed. The identification rating is above Level 2. After the identification of 485 metabolites, by using p < .05 and OPLS-DA VIP > 1 as the screening criteria for significantly different metabolites, 70 common metabolites were taken from the control group, the model group, and the tuberostemonine group to map the hierarchical clustering of significantly different metabolites in a heatmap. The analysis screened out 4 differential metabolites with a large contribution to distinguishing the three groups. The levels of L-citrulline, L-glutamate and L-asparagine were markedly upregulated, while the pyridoxal level was prominently downregulated in the model group in comparison to the control group. The contents of these differential metabolites were reversed after the administration of tuberostemonine (Figure 10C).

Figure 10.

(A)(B) PCA analysis of positive ion model and negative ion model in samples. The figure shows that each group of samples has a good dispersion without overlapping, indicating the reliability of the data. (C) Heatmap of potential biomarkers in different group. The rectangle represents the level of metabolite expression, with red indicating up-regulation and green indicating down-regulation.

Discussion

Histopathologically, areas of fibrosis, structural deformations, microscopic honeycombing, and scattered fibroblast foci were observed in close proximity to the visually normal lung tissue. In order to accurately identify targets for pulmonary fibrosis (PF) therapy, a comprehensive understanding of disease pathogenesis, genetic risk factors, and biomarkers of disease progression is imperative. Building upon this foundation, both traditional and innovative drugs and methodologies can be employed to pinpoint therapeutic targets for this progressive ailment. Tuberostemonine, one of chemical composition in herbs, was effective for treating acute and chronic cough. Tuberostemonine could be used as a quality control standard for herb. ¹⁶ We know PFD is a clinical drug or inhibitor of TGF-β and can inhibit TGF-β’s pro-proliferative effect on fibroblasts. The TGF-β protein, which holds significance in the advancement of lung fibrosis, emerges as a central target in the realm of drug research devoted to lung fibrosis treatment. The functional importance of this protein is demonstrated through its integral role in governing cell growth, differentiation, migration, embryonic development, apoptosis, organogenesis, immune regulation, cell fate decision, stress response and stem cell function. ¹⁷ In this study, the inhibition of TGF-β1-induced proliferation in human fetal lung fibroblast cells was confirmed through an initial in vitro experiment. The activation of TGF-β/Smad signaling in human fetal lung fibroblast cells was triggered by the amplified proliferation resulting from TGF-β1 stimulation. Various doses of tuberostemonine exhibited significant inhibitory effects on cell proliferation in human fetal lung fibroblasts. In the in vivo experiment, following the detection of the TGF-β/Smad signaling protein in mice with BLM-induced pulmonary fibrosis, the expression levels of α-SMA, TGF-β1, Col I, Col III and other marker proteins were up-regulated. This phenomenon was not observed in the tuberostemonine and PFD groups.

The extensive functional versatility of TGF-β signaling is associated with a range of human ailments, including cancer, fibrotic diseases, and inherited disorders. ¹⁸ During the development of pulmonary fibrosis (PF), TGF-β exerts a potent activating and catalytic effect on neutrophils, monocytes, macrophages, T cells, and B cells, leading to an inflammatory response within the lung tissue and promoting the conversion of pulmonary fibroblasts into myofibroblasts. ¹⁹ The formed myofibroblasts have the dual structure of fibroblasts and smooth muscle cells, which is an important feature in forming PF lesions. The α-SMA protein, a cytoskeletal protein, is one of the important markers of fibroblast proliferation. ²⁰ Past investigations have documented that the level of the α-SMA protein is consistent with the progression of lung fibrosis, where α-SMA protein expression is enhanced and vice versa. ²¹ Col I and col III function as structural proteins within the extracellular matrix, playing pivotal roles in its composition. ²² PF is a more difficult disease to treat than other respiratory diseases. Because effective gas exchange depends on the effective diffusion of carbon dioxide and oxygen molecules through the alveolar epithelium, inflammation and fibrosis can lead to impaired gas exchange, lung function and lung volume reduction, hypoxemia, breathing difficulties and cough, eventually triggering respiratory failure.^23,24 In accordance with a wealth of preexisting theoretical studies, the induction of lung fibrosis via BLM in mice led to significant reductions in lung tidal volume, inspiratory time, and expiratory time, coupled with an evident rise in airway resistance. In the course of treatment utilizing same dosage tuberostemonine and PFD, the lung tidal volume was enhanced and the respiratory time was shortened. The effect of tuberostemonine on lung function was the same as PFD. The HE staining and masson staining revealed that PF induced lymphocyte infiltration, an increase in collagen content, pathological changes in lung tissue structure, and replacement of the normal alveolar structure with a large number of abnormally proliferated collagen tissues. Following the treatment with same dosage tuberostemonine and PFD, lymphocyte infiltration and the overall collagen content decreased, the remaining alveolar structures were preserved, and the alveolar gas exchange and lung function improved. As a nonessential amino acid, HYP is the main amino acid of collagen synthesis. Within the model group, a marked escalation in HYP content was evident, while the application of tuberostemonine and PFD demonstrated a considerable decline in HYP content. Same weight tuberostemonine and PFD had an antagonistic effect on collagen formation and bleomycin-induced pulmonary fibrosis in mice. Many metabolites were differentially expressed in the lung tissue, with high levels of metabolites related to amino acid metabolism, such as glutamate, citrulline, and asparagine. These results confirmed that amino acid metabolism is altered during the development of PF. Tuberostemonine can regulate these metabolites to resist BLM-induced PF. ²⁵ Although the inhibitory effect of tuberostemonine on pulmonary fibrosis in animals and cells has been confirmed, further basic research is required to determine its efficacy in humans. It should be noted that pulmonary fibrosis in humans has multiple causes and differs from the induction of animal models. Additionally, the method used to induce the model relies heavily on the side effects of bleomycin in the lung, which significantly deviates from the actual human situation. In our next steps, we will explore and enhance basic research from various perspectives such as toxicology and pharmaceutics for future clinical translation.

Conclusions

Our findings illustrated that tuberostemonine possesses a remarkable ability to forestall the pathological modifications taking place in the lungs of mice subjected to BLM-induced pulmonary fibrosis. Moreover, it proficiently interferes with the excessive proliferation of human fetal lung fibroblast induced by TGF-β1, while simultaneously exerting regulatory control over the expression of proteins implicated in the TGF-β/smad signaling pathway. It also works to regulate the differential expression of metabolites related to amino acid metabolism and lipid metabolism and thus, play a role in controlling lung fibrosis. In this investigation, we confirmed the advantageous influence of tuberostemonine on pulmonary fibrosis, and this study serves as a provider of fundamental data reference for informing the potential application of the tuberostemonine.