Qingzao Jiufei Decoction in the Regulation of TGF-β/Smad Signaling in Idiopathic Pulmonary Fibrosis

Summary

We applied animal and cell experiments to verify the effect of Qingzao Jiufei Decoction (QJD) on idiopathic pulmonary fibrosis (IPF). The findings of this study showed that QJD delays and reverses IPF progression by inhibiting the TGF-β/Smad signaling pathway.

Abstract

This study aimed to investigate the therapeutic effects of Qingzao Jiufei Decoction (QJD) on idiopathic pulmonary fibrosis (IPF) and its regulatory mechanism involving the TGF-β/Smad signaling pathway.

Rats were divided into six groups: control, model, pifenidone (PFD), QJD Low Dose (QJD-L), QJD Medium Dose (QJD-M), and QJD High Dose (QJD-H). IPF was induced using bleomycin. Histopathological and ultrastructural changes in the lung tissue were analyzed. Levels of α-smooth muscle actin (α-SMA), collagen I (COL-1), fibronectin (FN1), TGF-β1, Smad2/3, and p-Smad2/3 proteins were measured. A TGF-β1-induced HFL-1 cell model was used to investigate the effects of QJD on the expression of α-SMA, COL-1, and FN1. Additionally, the TGF-β1 signaling agonist SRI-011381 was used to further determine the role of the TGF-β/Smad signaling pathway.

Results: In vivo, compared with the control group, rats in the model group showed significantly reduced dietary and water intake, along with an increased lung coefficient. Lung tissue showed infiltration of inflammatory cells, thickening of alveolar walls, and marked signs of fibrosis. Additionally, edematous alveolar type II epithelial cells with severe fibrosis were observed, characterized by swollen and deformed intracellular organelle structures and a reduced number of rough endoplasmic reticulum. Levels of α-SMA, COL-1, FN1, TGF-β1, and p-Smad2/3 were significantly elevated. Treatment with QJD or PFD markedly alleviated BLM-induced symptoms. In vitro, whereas TGF-β1 induction significantly upregulated the expression of FN1, COL-1, and α-SMA, these effects were significantly attenuated by QJD treatment. Activation of the TGF-β/Smad signaling pathway with the TGF-β1 agonist SRI-011381 increased FN1, COL-1, and α-SMA expression and aggravated pulmonary fibrosis. However, QJD reversed the fibrotic effects induced by SRI-011381, suggesting that QJD ameliorates pulmonary fibrosis by inhibiting TGF-β/Smad signaling pathway.

The findings of this study suggest that QJD attenuates and reverses the progression of pulmonary fibrosis by modulating the TGF-β/Smad signaling pathway.

Introduction

Idiopathic pulmonary fibrosis (IPF), a chronic interstitial lung disease of unknown etiology with an annual prevalence of (2-29)/100,000 and average survival time of 3.2 years, is characterized by cough, sputum, exertional dyspnea, and progressive exacerbation^1,2,3. Given the poor prognosis and short survival span, the primary therapeutic focus is to prevent and inhibit its further progression. Although current therapeutic approaches, such as nidazanib and pirfenidone, effectively delay disease progression, they do not reduce the mortality rate^4,5. Furthermore, their widespread use is limited by side effects and high cost⁶. Thus, new therapies able to treat IPF with fewer side effects are required.

IPF is categorized in traditional Chinese medicine (TCM) under "pulmonary atrophy" and "pulmonary obstruction", characterized by a complex pathogenesis involving both deficiency and excess. As a delicate and yin-deficient organ, the lung thrives on moisture and is vulnerable to dryness. When dryness predominates, it readily damages body fluids, impairing the lung's ability to maintain proper moisturizing function. This disrupts the transport of fluids within the lung lobules, leading to the withering and dysfunction of lung tissue, and ultimately the onset of disease⁷.

Qingzao Jiefei Decoction (QJD) is a classic TCM formula used for treating lung disorders. It consists of Morus indica L. (Sang Ye), CaSO₄·2H₂O (Shi Gao), Glycyrrhiza uralensis Fisch. (or Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L.) (Gan Cao), Panax ginseng C.A.Mey. (Ren Shen), Sesamum indicum L. (Hu Ma Ren), Equus asinus L. (E Jiao), Ophiopogon japonicus (Linn. f.) Ker-Gawl. (Mai Dong), Prunus armeniaca L. var. ansu Maxim. (or P. sibirica L., or P. mandshurica (Maxim. ) Koehne, or P. armeniaca L.) (Xing Ren), and Eriobotrya japonica (Thunb.) Lindl. (Pi Pa Ye). It clears dryness, moisturizes the lungs, nourishes yin, and replenishes qi. Although QJD is widely used for the treatment of IPF with remarkable clinical efficacy⁸, the underlying mechanisms are still unclear. Therefore, previous studies lack in vivo and in vitro experiments, and this study aims to fill the gap.

Previous network pharmacology predictions suggest that QJD may influence the pathogenesis of IPF by regulating the TGF-β/Smad signaling pathway through its active components, quercetin and β-sitosterol⁹. Research indicates that QJD exerts a "moistening dryness" effect by alleviating pulmonary inflammation, reducing Mycoplasma pneumoniae (MP) toxin levels, and increasing levels of bioactive substances in lung tissue¹⁰. Zhao et al. reported that QJD treatment in IPF patients enhances anti-inflammatory activity and promotes lung tissue repair, while lowering serum MMP-7, osteopontin, and KL-6 levels, thereby improving exercise endurance, quality of life, and clinical outcomes for IPF patients¹¹.  Although other commonly used clinical formulas, such as Bu Fei Tang and Shen Long Decoction, have also been shown in preliminary studies to ameliorate the progression of pulmonary fibrosis in mice by regulating the TGF-β1/Smad signaling pathway, these formulas primarily focus on tonifying lung qi and promoting blood circulation to remove stasis^12,13. Considering that the TGF-β/Smad signaling pathway was previously shown being involved in the pathological process of pulmonary fibrosis¹⁴, we used the PFD-induced rat model and the TGF-β1-induced HFL-1 model to verify the involvement of the TGF-β/Smad pathway in the positive effect of QJD on IPF. To the best of our knowledge, this study is the first to demonstrate, through both in vivo and in vitro models, that QJD demonstrates anti-fibrotic effects by modulating the TGF-β/Smad signaling pathway, thereby revealing a potential molecular mechanism underlying its therapeutic action in IPF. The overall experimental workflow is illustrated in Figure 1.

Protocol

Animal experiments were conducted in accordance with the principles of the ARRIVE Guidelines. All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Ningxia Medical University (Approval No. IACUC-NYLAC-2022-151). Cell experiments using human embryonic lung fibroblasts (HFL-1) that are commercially available, thus ethical approval was waived.

Experimental preparation

Experimental animals
Sprague Dawley (SD) (n = 60, SPF grade, 6-8 weeks, 180-200 g, 50% male and 50% female) were purchased at the Animal Experimentation Center (Ningxia Medical University, License No. SCXK (Ningxia)2020-0001) and housed in the Animal Barrier Center of Ningxia Medical University at 23-26 °C, 40-60% humidity, and 12 h light and darkness cycles. The rats were allowed free access to food and water.

Cell culture

Human embryonic lung fibroblasts (HFL-1) were grown in Ham's F-12K culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 °C, 5% CO₂, and 95% air. For experiments, cells in the logarithmic phase were used.

Preparation of QJD

QJD is composed of Sang Ye (9 g), Shi Gao (8 g), Gan Cao (3 g), Ren Shen (2 g), Hu Ma Ren (3 g), E Jiao (3 g), Mai Dong (4 g), Xing Ren (2 g), Pi Pa Ye (3 g). All components were authenticated by Professor Gao Xiaojuan, School of Medicine, Ningxia Medical University.

The herbs were soaked in 10 times their volume of water for 1 h, then boiled for 30 min to obtain the first extract. Then an additional 8 times volume of water was added to the residue and boiled for another 30 min to obtain the second extract. The two extracts were mixed and filtered through gauze, and concentrated to 1.12 g/mL.

Experimental procedures

In vivo experiments

Animal grouping and drug administration

After having a normal diet for 1 week of adaptive feeding, 60 rats were randomly assigned to 6 groups of 10 animals (control, model group, pifenidone group (PFD, 162 mg/kg), QJD-L, QJD-M, and QJD-H.

Except for the control group, all animals were anaesthetized by intraperitoneal injection of 2% pentobarbital sodium solution (40 mg/kg), fixed on a rat plate with the head upwards, and laryngoscopic intubation was performed. The rats received BLM (5 mg/kg), while control animals received an equal volume of saline. Then, the rats were held upright and rotated along the longitudinal axis for 3 min in order to diffuse the drug in the lungs¹⁵. After successful modeling, administration of the drug started on the following day. Considering the clinical QJD and PFD doses (37 g/day and 1800 mg/day, respectively) and based on the equivalent dose of the drug in humans and rats according to the body surface area, the PFD group received 162 mg/kg PFD while the QJD-L, QJD-M, and QJD-H groups received 1.65 g/kg, 3.3 g/kg, and 6.6 g/kg QJD, respectively. Control and model groups received saline for 4 weeks¹³.

Anesthesia and sampling of animals

After the last administration, the rats had only access to water for 12 h. At the time of sampling, rats were anaesthetized with 2% pentobarbital sodium solution (40 mg/kg) through intraperitoneal injection. Lungs were excised, washed with pre-cooled physiological saline to remove blood stains and residual connective tissues, weighed, and the lung coefficient was calculated (lung coefficient = lung wet weight (mg)/body mass (g) × 100%). The upper lobe was fixed in 4% paraformaldehyde for hematoxylin and eosin (HE), Masson staining, and immunofluorescence. The lower lobe of the left lung was cut and fixed in 2.5% glutaraldehyde for transmission electron microscopy observation. The right lung was stored at -80 °C for future analysis.

General condition of the animal

During the experimental period, the rats were observed daily for mental status, respiration condition, behavior, and coat luster. In case of death, the date and time of death were recorded.

Histopathological observations of the lungs

The collected lung tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded, and sectioned. HE and Masson staining were performed according to the kit instructions. The degree of alveolar inflammation was evaluated with the Szapiel scale, and the severity of fibrosis was assessed with the Ashcroft scale¹⁶.

Ultrastructural observation of lung tissue

The collected lung tissues were fixed in 2.5% glutaraldehyde for more than 4 h, washed with PBS, and fixed with 1% osmium tetroxide for 1.5 h. Then, they were dehydrated with ethanol and acetone gradients, followed by impregnation, embedding, trimming, sectioning, and staining. Finally, they were observed under a transmission electron microscope to observe the ultrastructural changes.

Immunofluorescence of lung tissue

The paraffin sections were dewaxed and subjected to antigen retrieval. Then, they were blocked at room temperature for 1 h, and the sections were incubated with the corresponding primary antibodies (α-SMA, COL 1, FN1, TGF-β1, Smad2/3, and p-Smad2/3; dilution: 1:200) overnight at 4 °C. Then, the sections were washed with PBS and incubated with the secondary antibody (Goat Anti-Rabbit IgG (H+L) Cy3 conjugate; dilution: 1: 1000) for 1 h at room temperature. Finally, the sections were washed with PBS, and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in the dark for 10 min at room temperature. Finally, the sections were observed under a fluorescence microscope, and images were captured.

Western blot (WB) analysis of lung tissue

Approximately 10 mg of frozen lung tissue was minced and lysed in 100 µL of strong radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors. The tissue was thoroughly homogenized using a homogenizing rod and incubated on ice for 30 min. The lysates were then centrifuged at 10,625 g for 15 min at 4°C, then the supernatant was collected. Protein concentration in the supernatant was determined using the bicinchoninic acid (BCA) assay, and the samples were normalized accordingly. After electrophoresis, membrane transfer, and blocking, the membranes were incubated with primary antibodies (α-SMA, COL-1, FN1, TGF-β1, Smad2/3, and p-Smad2/3; dilution: 1:1000) at 4 °C overnight and then with secondary antibodies (Goat Anti-Rabbit IgG (H+L) HRP; dilution: 1: 10,000) at room temperature for 2 h. Subsequently, luminescent-mediated imaging was performed on a gel imaging system. Gray-scale scanning analysis was conducted using ImageJ software. All experiments were independently repeated three times.

In vitro tests

CCK- 8 analysis

HFL-1 cells grown in log phase were taken and inoculated in 96-well plates at 1×10⁴ cells per well. The next day, the cells were treated with different concentrations of TGF-β1 (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 µg/L) for 12, 24, and 36 h. Then, 10 µL of CCK-8 solution was added, the plates were incubated at 37 °C for 2 h, and optical density (OD) was detected at 450 nm. The optimal TGF-β1 concentration and intervention time were selected for subsequent experiments.

Next, HFL-1 cells were inoculated in 96-well plates at 1 × 10⁴ cells per well, QJD was added with or without TGF-β1, and incubated for 24 h at 37 °C. Then, 10 µL of CCK-8 solution was added and incubated at 37 °C for 2 h. The OD was detected at 450 nm, and cell viability was estimated as cell proliferation viability (%) = ([OD experimental group - OD blank]/[OD control - OD blank]) × 100.

Cell grouping

Cells were grouped as Control (conventional culture), Model (10 µg/L TGF-β1), QJD (model group with 50 mg/L QJD), SRI-011381 (model group with 10 mg/L SRI-011381), and QJD + SRI-011381 (model group with 10 mg/L SRI-011381 and 50 mg/L QJD). Cells in each group were treated as outlined above and cultured for 24 h.

WB analysis

Collected cells were lysed in 100 µL of strong RIPA lysis buffer containing protease and phosphatase inhibitors. The lysate was thoroughly mixed and incubated on ice for 30 min. Samples were then centrifuged at 10,625 g for 15 min at 4 °C, and the supernatant was collected. Protein concentration in the supernatant was determined using the BCA assay, and samples were normalized accordingly. After electrophoresis, membrane transfer, blocking, membranes were incubated with primary antibodies (α-SMA, COL-1, FN1, TGF-β1, Smad2/3, and p-Smad2/3; dilution: 1:1000) at 4 °C for overnight, and secondary antibodies (Goat Anti-Rabbit IgG (H+L) HRP; dilution: 1: 10,000) at room temperature for 2 h. Subsequently, a luminescent solution was added, and imaging was performed on a gel imaging system. Gray-scale scanning analysis was conducted using ImageJ software. All experiments were independently repeated three times.

RT-qPCR analysis

Total RNA was extracted from the collected cells using Trizol lysis buffer (1 mL per dish). The lysate was thoroughly mixed and incubated at room temperature for 5 min. Then 200 µL chloroform was added, vortexed vigorously for 15 s, and incubated for 3 min at room temperature. Samples were then centrifuged at 10,625 g for 15 min at 4 °C, and the aqueous phase was collected. An equal volume of isopropanol was added, mixed thoroughly, and incubated for 10 min at room temperature. The mixture was centrifuged at 10,625 g for 10 min at 4 °C, and the supernatant was discarded. A white RNA pellet was visible at the bottom of the tube. The pellet was washed with 1 mL of pre-chilled 75% ethanol by gently inverting the EP tube, then centrifuged at 4150 g for 5 min at 4 °C. The supernatant was discarded, and the pellet was air-dried for 5 min at room temperature. The RNA was then dissolved in 20-50 µL of RNase-free water and stored at -80 °C. Total RNA was purified using a commercial RNA extraction kit. The concentrations were normalized prior to reverse transcription, and RT-qPCR was performed. Primers used in this study are listed in Table 1.

Statistical analysis

Data were represented as mean ± standard deviation (x̅±s). Excel and GraphPad Prism 8 were used for analysis and figure generation. Student's t-test was used between two groups, and one-way ANOVA was used for comparison between multiple groups. A P<0.05 was considered statistically significant.

Results

In vivo experiments

Effects of QJD on the general condition of IPF rats

Rats in the control group had normal food and water intake, were in good spirits, had calm and smooth breathing, had lustrous fur, and moved about normally. Compared to the control group, rats in the model group had reduced food and water intake, were listless, had shortness of breath, rough and yellowish fur, were lethargic, and prefer...

Discussion

IPF is a chronic interstitial lung disease characterized by high mortality and complex course. In China, the prevalence of IPF has been steadily increasing, largely due to the factors such as population aging, environmental pollution, and a large smoking population²⁰. The inflammatory response triggered by early lung injury is considered one of the major contributing factors in the development of pulmonary fibrosis²¹. Dysregulated repair of AECs induces EMT, stimulating pul...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Apricot kernel	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2301003
Bleomycin (BLM)	Shanghai Yuanye Biotechnology Co., Ltd	11056-06-7	A glycopeptide antibiotic?used to induce IPF animal models
COL-1	Affinity Biosciences	AF7001	Primary antibody (RRID: AB_86351)
Fetal bovine serum	Beijing Rongxing Biotechnology Company Limited	10099141C	Serum (RRID: AB_2354062)
FN1	Affinity Biosciences	AF5335	Primary antibody (RRID: AB_10616058)
Fried linseed (Hu Ma Ren)	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2401260
GAPDH	Affinity Biosciences	AF7021	Primary antibody (RRID: AB_2721282)
Ginseng	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2405123
Goat anti-Rabbit	Affinity Biosciences	S0001	secondary antibody (RRID: AB_10774538)
Gypsum	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2405236
Hematoxylin-eosin staining (HE)	Wuhan Servicebio Biotechnology Co	G1076	Test kit
Human embryonic lung fibroblasts (HFL-1)	Wuhan Pricella Biotechnology	CL-0106	Cell
Licorice	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2312322
Loquat leaf	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2405027
Masson staining kit	Wuhan Servicebio Biotechnology Co	G1006	Test kit
Mulberry leaf	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2406235
Ophiopogon japonicus	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	2403378
Pirfenidone (PFD)	Beijing Contini Pharmaceutical Co., Ltd	H20133376	A new pyridone compound with broad-spectrum anti fibrotic effects
p-Smad2/3	Affinity Biosciences	AF3367	Primary antibody
Real Colla Corii Asini (Ejiao)	Ningxia Mingde Chinese Herbal Pieces Co., Ltd	20240103
Recombinant human TGF-β1	Peprotech	100-21	A growth factor that induces fibroblast growth in rodents (RRID: AB_2910510)
Smad2/3	Affinity Biosciences	AF6367	Primary antibody (RRID: AB_398162)
Sodium pentobarbital	Merck	11715	Anesthetic
SRI-011381	MedChemExpress	HY-100347	An activator of the TGF-β signalling pathway
TGF-β1	Affinity Biosciences	AF1027	Primary antibody (RRID: AB_2737614)
total RNA extraction kit	Wuhan Servicebio Biotechnology Co	G3607	Test kit
whole protein extraction kit	Wuhan Servicebio Biotechnology Co	G2002	Test kit
α-SMA	Affinity Biosciences	AF1032	Primary antibody (RRID: AB_2847835)