Tianlongkechuanling Inhibits Pulmonary Fibrosis Through Down-Regulation of Arginase-Ornithine Pathway.

Lili Zhang,Lu Wang,Yuanyuan Shi,Chunguo Wang,Peng Wang,Zhimin Zhang,Sihao Qu,Qinghe Yu,Yadong Li,Binbin Zhang,Yirui Diao

doi:10.3389/fphar.2021.661129

Abstract

Background: Pulmonary Fibrosis (PF) is an interstitial lung disease characterized by excessive accumulation of extracellular matrix in the lungs, which disrupts the structure and gas exchange of the alveoli. There are only two approved therapies for PF, nintedanib (Nib) and pirfenidone. Therefore, the use of Chinese medicine for PF is attracting attention. Tianlongkechuanling (TL) is an effective Chinese formula that has been applied clinically to alleviate PF, which can enhance lung function and quality of life. Purpose: The potential effects and specific mechanisms of TL have not been fully explored, yet. In the present study, proteomics was performed to explore the therapeutic protein targets of TL on Bleomycin (BLM)-induced Pulmonary Fibrosis. Method: BLM-induced PF mice models were established. Hematoxylineosin staining and Masson staining were used to analyze histopathological changes and collagen deposition. To screen the differential proteins expression between the Control, BLM, BLM + TL and BLM + Nib (BLM + nintedanib) groups, quantitative proteomics was performed using tandem mass tag (TMT) labeling with nanoLC-MS/MS [nano liquid chromatographymass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The protein–protein interactions (PPI) were established by STRING. Expressions of α-smooth muscle actin (α-SMA), Collagen I (Col1a1), Fibronectin (Fn1) and enzymes in arginase-ornithine pathway were detected by Western blot or RT-PCR. Result: TL treatments significantly ameliorated BLM-induced collagen deposition in lung tissues. Moreover, TL can inhibit the protein expressions of α-SMA and the mRNA expressions of Col1a1 and Fn1. Using TMT technology, we observed 253 differentially expressed proteins related to PPI networks and involved different KEGG pathways. Arginase-ornithine pathway is highly significant. The expression of arginase1 (Arg1), carbamoyltransferase (OTC), carbamoy-phosphate synthase (CPS1), argininosuccinate synthase (ASS1), ornithine aminotransferase (OAT) argininosuccinate lyase (ASL) and inducible nitric oxide synthase (iNOS) was significantly decreased after TL treatments. Conclusion: Administration of TL in BLM-induced mice resulted in decreasing pulmonary fibrosis. Our findings propose that the down regulation of arginase-ornithine pathway expression with the reduction of arginase biosynthesis is a central mechanism and potential treatment for pulmonary fibrosis with the prevention of TL.

Highlights

Pulmonary Fibrosis (PF) is a chronic, progressive fibrotic interstitial lung disease, characterized by the excessive accumulation of extracellular matrix and fibrotic tissue in the lungs (Lederer and Martinez, 2018; Sgalla et al, 2018)
Comparing the information collected from the literature and standards with the data obtained by the HPLC-LTQ-Orbitrap MS method mass spectrometry, a total of 57 compounds were identified from the TL extract, including Gallic acid, p-Cymene, Pinocembrin, Rutin, Skullcapflavone I, Chlorogenic acid, and other ingredients (Figure 1, Supplementary Table S1)
Fibrosis showed apparent symptomatic relief in BLM + TL group and BLM + Nib group compared to the BLM group (Figures 2D,E). α-Smooth muscle actin is a marker of myofibroblasts that plays an important role in the development of the fibrotic lesion (Ding et al, 2019)

Summary

Introduction

Pulmonary Fibrosis (PF) is a chronic, progressive fibrotic interstitial lung disease, characterized by the excessive accumulation of extracellular matrix and fibrotic tissue in the lungs (Lederer and Martinez, 2018; Sgalla et al, 2018). It leads to decreased lung compliance, disrupted gas exchange, and respiratory failure and death (Raghu et al, 2011; Richeldi et al, 2017). There are only two approved therapies for PF, nintedanib (Nib) and pirfenidone, which can reduce the rate of progression of fibrosis (Taniguchi et al, 2010; Wongkarnjana et al, 2019) They have some uncertainties, side effects and poor prognosis. Proteomics was performed to explore the therapeutic protein targets of TL on Bleomycin (BLM)-induced Pulmonary Fibrosis

Methods

Results

Conclusion