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,<....

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....

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)