Reducing Fibroblast Activation and Fibrosis in Systemic Sclerosis through Adropin

Fibrotic diseases of the skin and other organs have become significant socioeconomic challenges in modern society, and current treatment options are limited. This background for this study seeks to address the problem of fibrosis in Systemic Sclerosis (SSc), with the research team focusing on a peptide hormone called adropin. Adropin is encoded by the energy homeostasis associated gene (ENHO), and early studies have indicated its important role in metabolic and vascular homeostasis, though its role in the fibrotic pathological process remains unclear.

This paper, written by researchers Minrui Liang, Nicholas Dickel, Andrea-Hermina Györfi from University Hospital Düsseldorf, University of Erlangen-Nuremberg, Fuzhou Huashan Hospital, and other institutions, was published in the journal Science Translational Medicine on March 27, 2024.

Research Workflow

Research Procedure and Steps

The research includes multi-step experiments using machine learning techniques combined with in vivo and in vitro functional experiments to explore the regulatory effects of adropin on fibrosis in systemic sclerosis. Specifically, the steps are as follows: 1. Identification via Machine Learning: - Analyzed publicly available prospective early systemic sclerosis registry data using Elastic Net (EN) regularization, Support Vector Machine (SVM), and Gradient Boosting Machine (GBM) models to screen for disease classification marker genes. Results indicated that the ENHO gene ranked highly in multiple models, validating its potential as a regulatory factor.

1. Functional Validation Experiments:

   • Used RNA sequencing (RNA-seq) technology to screen gene expression data from patients with early diffuse cutaneous systemic sclerosis (dcSSc) in vitro, showing significant downregulation of the ENHO gene across different patient cohorts’ skin samples.
   • Further validated ENHO protein expression levels through immunofluorescence staining of skin samples from healthy volunteers and patients with systemic sclerosis, showing significantly reduced ENHO expression in the patients’ skin.

2. Exploration of Signaling Pathways:

   • Studied the effects of various fibrotic stimuli on ENHO expression in fibroblasts from healthy volunteers’ skin, finding that Transforming Growth Factor-β (TGF-β) downregulated ENHO expression via the JNK-dependent pathway.

3. In Vivo and In Vitro Verification of Adropin’s Anti-fibrotic Effects:

   • Investigated the effects of the Adropin34–76 peptide on TGF-β-induced fibroblast activation and fibrotic tissue remodeling.
   • Experiments in murine models (such as bleomycin-induced lung fibrosis and chronic graft versus host disease models) and ex vivo precision-cut human skin samples showed that the Adropin34–76 peptide had significant effects in alleviating fibrosis.

Main Results

1. Gene Expression Analysis:

   • ENHO gene and protein expression levels were significantly downregulated in the skin of systemic sclerosis patients as analyzed by various machine learning models and RNA-seq.
   • Comparing RNA-seq data with normal controls, ENHO mRNA abundance was significantly reduced in SSc patients’ skin and negatively correlated with the mRNA expression of type I collagen (COL1A1).

2. Functional Validation:

   • In vitro experiments showed that Adropin34–76 peptide intervention significantly reduced TGF-β-induced fibroblast activation and ECM release, including reduced mRNA abundance of ACTA2, COL1A1, connective tissue growth factor (CTGF), and plasminogen activator inhibitor 1 (PAI1), as well as decreased protein abundance of α-SMA and type I collagen.
   • 3D full-thickness skin equivalent models and murine fibrosis models demonstrated that Adropin34–76 effectively alleviated fibrotic lesions, reducing skin thickness and collagen deposition.

3. Signal Pathway Research:

   • Studies confirmed that TGF-β downregulates ENHO expression via a JNK-dependent pathway. JNK1 had a minor effect on this process, while specific knockdown of JNK2 could prevent TGF-β-induced downregulation of ENHO expression.
   • RNA-seq and subsequent GSEA analysis showed that Adropin34–76 inhibits Hedgehog/Gli signaling, blocking the upregulation of Gli1 and Gli2, and ultimately preventing fibroblasts from transforming into myofibroblasts.

Conclusion and Significance

Research results demonstrate that Adropin serves as a potential anti-fibrotic regulatory factor. Its restoration can reduce TGF-β-induced fibroblast activation and fibrotic tissue remodeling. This discovery may open a new therapeutic pathway to address systemic sclerosis and other fibrotic diseases in clinical practice. These findings offer important theoretical basis for the development of new treatment strategies and possess potential application value.

Highlights of the Study

1. Combination of Machine Learning and Functional Validation: For the first time, the study effectively screened and validated potential anti-fibrotic factors using a combination of machine learning and experimental validation.
2. Multi-Model Validation: The importance of the ENHO gene in systemic sclerosis was validated using multiple models and independent patient cohorts.
3. Mechanistic Exploration: The study revealed that ENHO gene expression is regulated by the TGF-β/JNK2 pathway and its mechanism in anti-fibrosis.
4. Potential Clinical Application: The anti-fibrotic effect exhibited by the Adropin34–76 peptide provides scientific basis for new therapeutic methods.

These findings lay an important foundation for further research and development of Adropin as a fibrosis treatment, contributing significantly to scientific understanding and clinical application.