Preparation of purified proteins from fresh Pheretima and their inhibitory effect against pulmonary fibrosis in mice.
10.12122/j.issn.1673-4254.2022.04.20
- Author:
Shu Yu LI
1
;
Qi Xin YANG
1
;
An Na ZUO
1
;
Lin Hua TIAN
2
;
Jin Hai HUO
2
;
Yan Li MENG
2
;
Qing Fa TANG
1
;
Wei Ming WANG
1
Author Information
1. School of Traditional Chinese Medicine, Southern Medical University//Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics//Guangdong Provincial Engineering Laboratory of Traditional Chinese Medicine, Guangzhou 510515, China.
2. Institute of Chinese Medicine, Heilongjiang Provincial Academy of Chinese Medicine, Harbin 150036, China.
- Publication Type:Journal Article
- Keywords:
fresh Pheretima;
mass spectrometry;
myofibroblasts;
protein purification;
transforming growth factor -β1
- MeSH:
Animals;
Biological Products/pharmacology*;
Bleomycin/adverse effects*;
Cadherins/metabolism*;
Collagen Type I;
Lung/pathology*;
Male;
Mice;
Mice, Inbred C57BL;
Oligochaeta/chemistry*;
Pulmonary Fibrosis/drug therapy*;
Transforming Growth Factor beta1/metabolism*;
Vimentin/metabolism*
- From:
Journal of Southern Medical University
2022;42(4):618-624
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To develop a convenient method for rapid purification of fresh Pheretima proteins and assess the inhibitory effect of these proteins against pulmonary fibrosis.
METHODS:The crude extract of fresh Pheretima was obtained by freeze-drying method and then purified by size exclusion chromatography. The composition of the purified proteins was analyzed by mass spectrometry. MRC-5 cells were treated with 5 ng/mL TGF-β1 alone (model group) or in combination with SB431542 (2 μmol/L) or the purified proteins (13.125 μg/mL), and the cytotoxicity of purified proteins and their inhibitory effects on cell proliferation were detected with CCK8 assay. Flow cytometry was used to detect the changes in cell apoptosis, and the cellular expressions of α-SMA, Vimentin, E-cadherin, collagen I, Smad2/3 and P-Smad2/3 were detected using RT-PCR and Western blotting. In the animal experiment, adult male C57BL/6 mice were subjected to intratracheal instillation of bleomycin followed by treatment with the purified proteins (5 mg/mL) for 21 days, after which HE and Masson staining was used to observe the pathological changes in the lung tissue of the mice.
RESULTS:We successfully obtained purified proteins from fresh Pheretima protein by size exclusion chromatography. Treatment with the purified proteins significantly inhibited TGF-β1-induced proliferation of MRC-5 cells (P < 0.01), reduced the cellular expressions of α-SMA, Vimentin and collagen I (P < 0.001 or P < 0.01), increased the expression of E-cadherin (P < 0.01), and inhibited the expressions of Smad2/3 and P-Smad2/3 (P < 0.001 or P < 0.01). In male C57BL/6 mice models of bleomycin-induced pulmonary fibrosis, treatment with the purified proteins obviously reduced the number of inflammatory cells and fibrotic area in the lungs.
CONCLUSION:The purified proteins from fresh Pheretima obtained by size exclusion chromatography can inhibit pulmonary fibrosis in mice by regulating the TGF-β/ Smad pathway.
