*Contributed equally
Maiwei Yangfei (MWYF) is a compound Chinese herb that is safe and effective in the clinical setting in patients with pulmonary fibrosis (PF). The aim of the present study was to assess the role of a (MWYF) decoction in a bleomycin (BLM)-induced PF mouse model and to investigate the underlying functional mechanism. Chemical components within the MWYF decoction were analysed using liquid chromatography-mass spectrometry. A total of 50 C57BL/6 mice were randomly assigned to one of the following five groups with 10 mice per group: Control, model, low dose MWYF (20 g/kg), medium dose MWYF (40 g/kg) and high dose MWYF (60 g/kg). A mouse PF model was established by the tracheal instillation of BLM (5 mg/kg) prior to MWYF treatment, except for mice in the control group. After 21 days of treatment with MWYF, the mice were sacrificed and the body weights were recorded. In addition, pulmonary tissues and bronchial alveolar lavage fluid were collected. TNF-α, IL-6, IL-17, hydroxyproline, pyridinoline and collagen I levels were determined using ELISA. Vimentin, α-smooth muscle actin (α-SMA), fibronectin, TGF-β1, Smad3, TNF-α, IL-6, IL-17, collagen I and collagen III were determined using western blotting. Vimentin and α-SMA levels were also determined using immunofluorescence analysis. Collagens I and III were detected using immunohistochemical analysis and TGF-β1 and Smad3 levels were determined using reverse transcription-quantitative PCR. Following treatment with MWYF decoction, the body weight of the mice in the PF group increased, the degree of pulmonary alveolitis and PF was reduced, collagen levels were reduced and the expression levels of α-SMA, vimentin and fibronectin were decreased. Although both protein and mRNA expression levels of TGF-β1 and Smad3 were reduced, they remained higher than those observed in the control group. To conclude, MWYF decoction delayed the development of BLM-induced PF in mice, where the functional mechanism was likely associated with the TGF-β1/Smad3 signalling pathway.
Pulmonary fibrosis (PF) is a chronic, progressive and potentially lethal pulmonary interstitial disease that primarily occur in middle-aged and elderly individuals (
It has been previously hypothesized that PF is closely associated with alterations in alveolar epithelial cells and pulmonary fibroblasts (
At present, a therapeutic strategy that is fully effective for the treatment of PF remain unavailable. The clinically applied pirfenidone and nintedanib can only delay the deterioration of lung function in patients with slight-to-moderate PF (
In the present study, a single-use bleomycin (BLM) instillation method was used to establish a PF mouse model, which was subsequently treated using MWYF by intragastric administration. In the PF mice, the role of MWYF decoction was investigated, in addition to the possible mechanism of action.
The MWYF decoction was obtained from Jiangsu Province Hospital of Chinese Medicine. BLM was supplied by TCI (cat. no. B3972-10MG, Shanghai, China). Hydroxyproline (HYP; cat. no. F10614), pyridinoline (PYD; cat. no. F11442), collagen I (F5760; all from Shanghai Westang Bio-Tech Co., Ltd.), TNF-α (cat. no. 70-EK282/3-96), IL-6 (cat. no. 70-EK206/3-96) and IL-7 [cat. no. 70-EK207/2-96; all from MULTISCIENCES (LIANKE) BIOTECH, CO., LTD] were ELISA test kits. α-Smooth muscle actin (α-SMA; cat. no. 14395-1-AP), vimentin (cat. no. 10366-1-AP 14395-1-AP), fibronectin (cat. no. 15613-1-AP), collagen I (cat. no. 14695-1-AP), collagen III (cat. no. 22734-1-AP), TNF-α (cat. no. 17590-1-AP), IL-6 (cat. no. 21865-1-AP), IL-17 (cat. no. 13082-1-AP), TGF-β1 (cat. no. 21898-1-AP), Smad3 (cat. no. 25494-1-AP) and β-actin (cat. no. 20536-1-AP) antibodies were purchased from Wuhan Sanying.
The MWYF decoction included 15 g
The characteristic components in the MWYF decoction were qualitatively analysed using liquid chromatography-mass spectrometry (LC-MS). Briefly, 50 mg freeze-dried samples were extracted using 800 µl 80% methanol and 10 µl internal standard (2.8 mg/ml, DL-o-chlorophenylalanine) and then vortexed for 30 sec. Next, the samples were ultrasonicated for 30 min at 40 kHz at 50˚C and all owed to stand for 1 h at 20˚C. The samples were centrifuged 1,800 x g and 4˚C for 15 min. The supernatant was collected in small vials and then used for evaluation by LC-MS (UltiMate™ 3000, Q Exactive™; Thermo Fisher Scientific, Inc.). LC-MS was performed using a C18 chromatographic column (Hypersil GOLD C18; 100x2.1 mm, 1.9 µm; Thermo Fisher Scientific, Inc.). Chromatographic isolation was performed at a column temperature of 40˚C with a 0.3 ml/min flow rate. The composition of the mobile phase A was water + 5% acetonitrile and 0.1% formic acid, while mobile phase B was composed of acetonitrile + 0.1% formic acid. The injection volume was 10 µl. The temperature of the autosampler was 4˚C.
A single dose intratracheal instillation of BLM was used to construct the PF mouse model. The mice were first anesthetized by an intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg) and fixed on the operating table (
A total of 50 SPF grade C57BL/6 male mice (weight, 20-25 g) were supplied by the Qinglongshan Animal Centre [Jiangning, Nanjing; licence no. SCXK (Su) 2017-0001; certification no. 201903602]. The mice were reared at the SPF grade test animal centre at the Nanjing University of Traditional Chinese Medicine at 22˚C and 46% relative humidity, with a 12-h light/dark cycle. The mice were provided with
Lung tissues were fixed in 4% polyoxymethylene (4˚C) for 24 h. The tissues were embedded in paraffin and cut into 4-µm thick sections. The slices were then subjected to H&E and Masson staining. The levels of alveolar inflammation were scored using Szapiel's method (
The optical density (OD) value was measured after ELISA was performed according to the manufacturer's protocols, before the final content of HYP and PYD was calculated using a standard curve.
TNF-α, IL-6 and IL-17 levels in BALF were determined using ELISA, according to the manufacturer's protocol. The colour was developed at room temperature, the OD was recorded and TNF-α, IL-6 and IL-17 levels in the BALF were calculated using a standard curve.
Lung tissue sections (5 µm) were deparaffinized with xylene and ethanol and washed with PBS. A few drops of 3% H2O2 were added to block endogenous peroxidase activity at 25˚C for 25 min in the dark. Antigen retrieval was performed by boiling in citrate buffer solution at 95˚C for 20 min. The tissues were then blocked with 3% BSA (cat. no. R22294; ShangHai YuanYe Biotechnology Co., Ltd.) at 25˚C for 30 min. Collagen I and collagen III antibodies were added to the samples at 4˚C for 12 h, which were placed in a wet box. After overnight incubation, the secondary antibody (Biotin-conjugated Affinipure Goat Anti-Rabbit; 1:500; cat. no. SA00004-2; ProteinTech Group, Inc.) was applied at 25˚C for 50 min, and the signal was revealed using 3,3'-diaminobenzidine reagent (cat. no. 8059S; Cell Signalling Technology, Inc.) followed by sealing of the sections with neutral resin. The expression of collagen I and collagen III were observed under a light microscope (XSP-C204; Chongqing Chongguang Industrial Co., Ltd.) at a magnification of x200. Results were quantified using ImageJ software (v.1.52; National Institutes of Health).
Lung tissue slices (5 µm) were incubated in citrate buffer (0.01 mol/l; pH 6.0) at 95˚C for 20 min. The sections were then sealed with 10% goat serum at 37˚C for 30 min. The primary antibody (α-SMA or vimentin; 1:200) was added and the sections were incubated overnight at 4˚C. The following day, the sections were washed with PBS and then incubated with the secondary antibody (Alexa Fluor 647; 1:500; cat. no. A0468; Beyotime Institute of Biotechnology) at 25˚C for 50 min in the dark. Finally, the cell nuclei were stained using DAPI (cat. no. C1002; Beyotime Institute of Biotechnology) at 37˚C for 10 min in the dark. The slides were observed under a fluorescence microscope (magnification, x200; Leica Microsystems GmbH).
Briefly, 20 mg lung tissue was added to RIPA Lysis Buffer (cat. no. P0013B; Beyotime Institute of Biotechnology). The tissues were homogenised and total protein was extracted. The protein content was measured using a bicinchoninic acid assay. The protein sample was subjected to 10% SDS-PAGE with 30 µg/well and then transferred onto a PVDF membrane, before and the membrane was blocked at room temperature for 2 h with 5% skimmed milk and washed with TBS with 100.1% Tween-20 (TBST) three times, 10 min per wash. The primary antibody was prepared according to the manufacturer's protocol and incubated at 4˚C for 12-18 h. The dilution of the α-SMA antibody used was 1:2,000, whilst the dilution of fibronectin, vimentin, collagen I, collagen III, TGF-β1 and Smad3 antibodies was 1:1,000. The membranes were washed again with TBST three times for 10 min. Subsequently, the membrane was incubated with an HRP-conjugated secondary antibody (cat. no. 3999S; Cell Signalling Technology, Inc.) at room temperature for 1 h. Next, the membranes were washed with TBST three times for 10 min. Signals were visualized using chemiluminescence reagent (cat. no. 170-5061; Bio-Rad Laboratories, Inc.). Densitometry analysis was performed using Image Lab software (v.5.1; Bio-Rad Laboratories, Inc.).
Pulmonary tissues were homogenised, and total RNA was extracted using TRIzol® reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.). The concentration of RNA was calculated, and cDNA was synthesised using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix kit (cat. no. 11141ES60; Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. Hieff® qPCR SYBR Green Master Mix kit (cat. no. 11201ES03; Shanghai Yeasen Biotechnology Co., Ltd.) was used to assay the relative expression levels of TGF-β1 and Smad3 in the pulmonary tissues. The thermocycling conditions used were as follows: 95˚C for 5 min; followed by 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 20 sec and elongation at 72˚C for 20 sec. The average expression of target genes and GAPDH was calculated using the Cq values. In each group, the relative amount of gene expression was calculated using the 2-ΔΔCq value of the model group (
All results are based on triplicate experiments and presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.). Inflammation and fibrosis scores were compared using Kruskal-Wallis test, followed by the Dunn post hoc test. One- or two-way analysis of variance followed by Tukey's test was used to determine statistical significance. P<0.05 was considered to indicate a statistically significant difference.
MWYF consists of 12 Chinese herbal medicines, indicating a complex composition. Amino acids, flavonoids, phenols, terpenes and organic acids were identified in the MWYF decoction and LC-MS was used for the analysis of these chemical ingredients. Under optimised LC-MS conditions, the primary components of MWYF were isolated and detected. The typical components are shown in
To assess the effects of MWYF fibrosis
Pulmonary alveolitis and PF were scored based on the H&E and Masson staining images to evaluate the degree of inflammation and fibrosis. Using H&E staining, the structure of the mouse lung tissues in the control group was clear, the morphology of pulmonary alveoli was normal, the walls of the pulmonary alveoli were thin and no inflammatory cell infiltration was observed in the interstitial lung (
In the control group, Masson staining demonstrated that the structure of pulmonary alveoli tissues was regular and arranged in an orderly manner, with only a small number of fine light blue fibres observed (
The inflammation score was calculated to be significantly higher in the model group compared with that in the normal control group (
The typical pathological change during PF is characterised by the deposition of collagen in the pulmonary fibres, particularly collagen I and collagen III (
HYP is the primary component of collagen, where its content is an effective indicator for evaluating collagen deposition in lung tissues (
In PF, the main pathological changes observed are the proliferation of pulmonary fibroblasts and the differentiation of fibroblasts into myofibroblasts (
Cytokines serve key roles in the occurrence and development of PF (
TGF-β1 serves a key role in the development of PF (
Pulmonary interstitial fibrosis is a connective tissue disease that is primarily characterized by dry cough and dyspnoea with an unknown pathogenesis (
There are four types of collagens in lung tissues, where collagen I and collagen III are the most abundant (
α-SMA is a marker of myofibroblast activation (
The inflammatory response in the pulmonary alveoli is one of the primary causes of inflammatory cytokine release (
TGF-β1/Smad3 is considered to be a classical signalling pathway associated with PF (
In conclusion,
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
XZ and QW designed the study. YX, WP and DH performed the experiments. FF, ZW and CG performed data analysis. YX and XZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was approved by the Ethics Commission for Animal Tests of the Nanjing University of Chinese Medicine (approval no. 012009012015; Nanjing, China).
Not applicable.
The authors declare that they have no competing interests.
Elementary particle flow graph chromatogram of the Maiwei Yangfei decoction. Monitoring in (A) positive ion mode and (B) negative ion mode. In the chromatograms, succinic acid, ephedrine, ferulic acid, esperidin, rosmarinic acid, lobetyolin, baicalin, glycyrrhizic acid, schizandrin, asarinin, ruscogenin and shionone were detected.
Effects of MWYF on mice body weight in the different treatment groups. (A) Scheme of the experimental setup. After bleomycin exposure, mice received MWYF daily and were sacrificed on day 21. (B) Body weight measurement after BLM injection and various treatment regimens over the 21-day period. (C) Effects of MWYF on the terminal body weight in mice with BLM-induced pulmonary fibrosis. All data are expressed as mean ± SD (n=10), ###P<0.001 vs. Vehicle control. MWYF, Maiwei Yangfei decoction; BLM, bleomycin; Vehicle control, normal group; Model, model group.
Effects of MWYF on the histopathological changes in lung tissues of mice with BLM-induced pulmonary fibrosis. (A) Lung sections were stained with H&E or Masson Trichrome. Magnification, x200. (B) Effects of MWYF on the alveolitis score. (C) Effects of MWYF on the fibrosis score. All data are expressed as mean ± SD (n=10), ##P<0.001 and ###P<0.01 vs. Vehicle control. *P<0.05 and **P<0.01 vs. Model. MWYF, Maiwei Yangfei decoction; Vehicle control, normal group; Model, model group.
Effects of MWYF on collagen content in mice with BLM-induced pulmonary fibrosis. (A) Expression of collagen I and collagen III in the lung tissues was measured by immunohistochemistry. Magnification, x200. Quantitative results of immunohistochemical staining for (B) collagen I and (C) collagen III. Expression of (D) collagen I, (E) HYP and (F) PYD in the lung tissues was detected by ELISA. (G) Expression of collagen I and collagen III were measured by western blotting, (H) which was quantified. All data are expressed as mean ± SD (n=10). #P<0.05, ##P<0.01 and ###P<0.001 vs. Vehicle; *P<0.05, **P<0.01 and ***P<0.001 vs. Model. MWYF, Maiwei Yangfei decoction; Vehicle control, normal group; Model, model group; HYP, hydroxyproline; PYD, pyridinoline.
Effects of MWYF on the expression of marker proteins for fibrosis in pulmonary tissues of mice with BLM-induced pulmonary fibrosis. The red fluorescence represents the expression of α-SMA and vimentin, whereas the blue fluorescence represents cell nuclei stained with DAPI. (A) Expression of α-SMA and vimentin in the lung tissues were measured by immunofluorescence. Magnification, x200. (B) Quantitative results of immunofluorescence analysis of (B) α-SMA and (C) vimentin. (D) Expression of fibronectin, vimentin and α-SMA proteins were measured by western blotting. Quantitative results of (E) fibronectin, (F) vimentin and (G) α-SMA protein expression. All data are expressed as mean ± SD (n=10). ##P<0.001 and ###P<0.01 vs. Vehicle control. *P<0.05, **P<0.01 and ***P<0.001 vs. Model. MWYF, Maiwei Yangfei decoction; Vehicle control, normal group; Model, model group; α-SMA, α-smooth muscle actin.
Effects of MWYF on the levels of inflammatory factors in mice with bleomycin-induced pulmonary fibrosis. (A) Western blot analysis of the protein expression of TNF-α, IL-6 and IL-17 in the lung tissues. (B) Quantitative analysis of TNF-α, IL-6, and IL-17 protein expression. Levels of (C) TNF-α, (D) IL-6 and (E) IL-17 in the BALF were detected by ELISA. All data are expressed as mean ± SD (n=10). ##P<0.001 and ###P<0.01 vs. Vehicle control. *P<0.05, **P<0.01 and ***P<0.001 vs. Model. MWYF, Maiwei Yangfei decoction; Vehicle control, normal group; Model, model group; BALF, bronchial alveolar lavage fluid.
Effects of MWYF on the expression of TGF-β1 and Smad3 in the pulmonary tissue of mice with BLM-induced pulmonary fibrosis. (A) The expression of TGF-β1 and Smad3 in the lung tissues were measured by western blotting. (B) Quantitative analysis of TGF-β1 and Smad3 expression. Reverse transcription-quantitative PCR was used to measure the relative expression of (C) Smad3 and (D) TGF-β1. All data are expressed as mean ± SD (n=10). ##P<0.001 and ###P<0.01 vs. Vehicle control group. *P<0.05, **P<0.01 and ***P<0.001 vs. Model group. MWYF, Maiwei Yangfei decoction; Vehicle control, normal group; Model, model group.
Mechanism research diagram. MWYF can alleviate lung inflammation, reduce collagen deposition and decrease the expression level of PF marker proteins, and it may be potent in ameliorating PF via the TGF-β1/Smad3 signaling pathway. MWYF, Maiwei Yangfei decoction. LC-MS, liquid chromatography-mass spectrometry; RT-qPCR, reverse transcription-quantitative PCR; PF, pulmonary fibrosis.
Composition of Maiwei Yangfei decoction.
Compound number | Component | Formula | Adduct | Exact Mass | Retention time (min) | Mass/charge ratio | Parts per million | Chinese name |
---|---|---|---|---|---|---|---|---|
1 | Succinic acid | C4H6O4 | [M-H]- | 117.01823 | 1.20 | 191.01891 | -3.548 | Ban xia |
2 | Ephedrine | C10H15NO | [M+H]+ | 166.12264 | 2.75 | 447.09247 | 0.417 | Ma huang |
3 | Ferulic acid | C10H10O4 | [M-H]- | 193.04953 | 3.59 | 193.04967 | 0.698 | Dang gui |
4 | Hesperidin | C28H34O15 | [M+H]+ | 611.19704 | 3.70 | 611.19733 | 0.464 | Chen pi |
5 | Rosmarinic acid | C18H16O8 | [M-H]- | 359.07614 | 3.78 | 359.07700 | 3.470 | Zi su zi |
6 | Lobetyolin | C20H28O8 | [M+Na]+ | 419.16763 | 4.00 | 419.16718 | -0.969 | Dang shen |
7 | Baicalin | C21H18O11 | [M+H]+ | 447.09218 | 4.02 | 447.09247 | 0.631 | Huang qin |
8 | Glycyrrhizic acid | C42H62O16 | [M+H]+ | 823.41106 | 5.48 | 823.41187 | 0.981 | Gan cao |
9 | Schisandrin | C24H32O7 | [M+H]+ | 433.22207 | 6.41 | 433.22208 | 0.000 | Wu wei zi |
10 | Asarinin | C20H18O6 | [M-H]- | 353.10196 | 6.75 | 353.10315 | 3.357 | Xi xin |
11 | Ruscogenin | C27H42O4 | [M+H]+ | 431.31558 | 7.40 | 431.20694 | 0.774 | Mai dong |
12 | Shionone | C30H50O | [M+H]+ | 427.39344 | 13.45 | 427.39380 | 0.836 | Zi wan |