Wen Yang Ding Chuan Tang Attenuates Asthmatic Airway Remodeling by Inhibiting M2 Macrophage-Driven Fibroblast Activation

Summary

This article describes a study on the regulation of pulmonary fibroblast fibrosis by Wenyang Dingchuan Decoction through inhibition of M2 macrophage polarization.

Abstract

To study the effects of Wen Yang Ding Chuan Tang (WYDCT) on fibroblast function, airway remodeling in asthma, and the underlying mechanism. IL-4 was used to induce M2 polarization of RAW264.7 macrophages, and the polarized macrophages were then treated with WYDCT to investigate its effect on mouse lung fibroblasts. Polarized RAW264.7 cells were subjected to different treatments and then co-cultured with fibroblasts. M1 and M2 marker levels, cell morphology, and IL-6 concentration were assessed. STAT3 protein level was analyzed. Fibroblast viability, proliferation, colony formation, PCNA expression, and the expression of fibronectin, COL1A1, and α-SMA were analyzed using various assays, including real-time PCR, Western blot, CCK-8, Edu, immunofluorescence, and colony formation assays. WYDCT reversed the IL-4-induced morphological changes in RAW264.7 cells, reduced the levels of M2 markers (Arg-1, CD206) and IL-6, and decreased STAT3 expression in RAW264.7 cells, while supplementation with IL-6 restored the effect of WYDCT on M2 markers. Furthermore, WYDCT inhibited fibroblast viability and proliferation, reduced PCNA expression, and decreased fibrosis markers (fibronectin, COL1A1, α-SMA), suggesting that WYDCT inhibits the viability, proliferation, and fibrosis of lung fibroblasts by suppressing macrophage M2 polarization, in which the IL-6/STAT3 signaling pathway may play a key role.

Introduction

Asthma, also known as bronchial asthma, is a persistent inflammatory condition of the respiratory passages¹. Frequently linked to allergen exposure, cold temperatures, and irritants of both physical and chemical nature, this condition is characterized by infiltration of inflammatory cells, reversible constriction of airways, mucus secretion, and bronchospasm². Currently, there are at least 300 million asthma patients worldwide, and the prevalence is increasing year by year³. In 2015, 400,000 people died from asthma, making it a serious global health issue^4,

Protocol

This study was conducted following international, national, and institutional guidelines for humane animal treatment and in compliance with relevant legislation. Specifically, all procedures involving mice were approved by the Animal Ethical Care Committee of The Third Affiliated Hospital of Qiqihar Medical College (AECC-2022-009). Mice were housed under standard laboratory conditions with access to food and water ad libitum, and all efforts were made to minimize suffering. Humane endpoints were established and monitored throughout the study.

1. Preparation of WYDCT extract

The 12 herbs of Wen Yang....

Results

The effects of WYDCT on RAW264.7 cells M2 polarization of macrophages

We observed RAW264.7 cell morphology using a light microscope. As shown in Figure 1A, IL-4 stimulation caused cells to elongate, resulting in a more elongated, spindle-like shape compared to untreated cells. Additionally, the IL-4-stimulated cells exhibited increased cytoplasmic granularity and showed enhanced adherence to the culture substrate. These morphological changes sugge.......

Discussion

The objective of this study was to explore the impact of WYDCT on fibroblast activity and airway remodeling in asthma, as well as the underlying mechanisms. The current findings demonstrate that WYDCT can inhibit M2 polarization of RAW264.7 macrophages, which subsequently affects the viability, proliferation, and fibrosis of mouse lung fibroblasts. Moreover, the IL-6/STAT3 signaling pathway played a key role in mediating these effects.

Macrophage polarization is a crucial process in the pathog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Apollo 643 Hybridization Reagent	Guangzhou RiboBio Co., Ltd.	C10371-2
Arginase-1 (E4U1I) Mouse mAb	Cell Signaling Technology, Inc.	43933T
BCA protein concentration determination kit	Beyotime Biotechnology Co., Ltd.	P0012
BeyoBlot HRP-Conjugated Mouse Anti-Rabbit Native IgG (H+L)	Beyotime Biotechnology Co., Ltd.	A0210
Carbon dioxide incubator	Shanghai Lishen Scientific Equipment Co., Ltd.	HF240
CD206/MRC1 (E6T5J) XP® Rabbit mAb	Cell Signaling Technology, Inc.	24595T
Cell Counting Kit-8	Beyotime Biotechnology Co., Ltd.	C0037
COL1A1 (E8F4L) XP Rabbit mAb	Cell Signaling Technology, Inc.	72026T
Dimethyl Sulfoxide	Beyotime Biotechnology Co., Ltd.	ST038-100ml
DMEM high sugar medium	Grand Island Biological Company	11965092
EDU	Beyotime Biotechnology Co., Ltd.	ST067-250mg
FcγRIII/CD16 (F6K6G) Rabbit mAb	Cell Signaling Technology, Inc.	71345T
fetal calf serum	Grand Island Biological Company	A5256701
Fibronectin/FN1 Antibody	Cell Signaling Technology, Inc	63779S
FITC-Conjugated Goat Anti-Mouse IgG (H+L)	Beyotime Biotechnology Co., Ltd.	A0568
Fluorescence quantitative PCR instrument	Yarui Biotechnology Co., Ltd.	MA-6000
Hoechst 33342 Live Cell Staining Solution (100X)	Beyotime Biotechnology Co., Ltd.	C1029
iNOS (E1W4J) Rabbit mAb	Cell Signaling Technology, Inc.	70706SF
inverted microscope	Guangzhou Mingmei Optoelectronics Technology Co., Ltd.	MI52-N
LRRK2 T1348N mut RAW 264.7	ATCC	SC-6005
mmunostaining Permeabilization Buffer (with Triton X-100)	Beyotime Biotechnology Co., Ltd.	P0096-100ml
Mouse IL-6 ELISA Kit	Beyotime Biotechnology Co., Ltd.	PI326
Mouse Lung Fibroblast Cells	Wuhan Procell Life Technology Co., Ltd.	CP-M006
PCNA (PC10) Mouse mAb	Cell Signaling Technology, Inc.	2586T
Penicillin-streptomycin	Grand Island Biological Company	15140148
Recombinant Murine IL-4	Beyotime Biotechnology Co., Ltd.	P5916-5μg
Recombinant Murine IL-6	Beyotime Biotechnology Co., Ltd.	P5925-10μg
Redu enzyme label instrument	Rayto Life Sciences	RT-6100
Stat3 (124H6) Mouse mAb	Cell Signaling Technology, Inc.	9139T
superclean bench	Jinan BioBase Biotech Co., Ltd.	BBS-SDC
SynGAP-α2 (E4Y6I) Rabbit mAb	Cell Signaling Technology, Inc.	56927T
Thermo Fisher Scientific RIPA Lysis and Extraction Buffer	ThermoFisher	89901
Trizol (Total RNA extraction reagent)	Beyotime Biotechnology Co., Ltd.	R0016
β-Actin Antibody	Cell Signaling Technology, Inc.	4967S