Effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms
10.3760/cma.j.issn.1009-2587.2017.11.007
- VernacularTitle: 内皮抑素预处理对人皮肤成纤维细胞纤维化的作用和机制
- Author:
Haitao REN
1
;
Yuan LI
;
Shengdong WANG
;
Chunmao HAN
Author Information
1. Department of Burns, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China
- Publication Type:Journal Article
- Keywords:
Endostatins;
Fibroblasts;
Platelet-derived growth factor;
Signal pathways
- From:
Chinese Journal of Burns
2017;33(11):694-698
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms.
Methods:Human skin fibroblasts were routinely cultured in vitro, and then the cells of passage 3 to 5 were used in the following experiments. The cells were divided into blank control, endostatin, platelet-derived growth factor-BB (PDGF-BB), endostatin+ PDGF-BB, transforming growth factor-β1 (TGF-β1), and endostatin+ TGF-β1 groups according to the random number table, with 3 wells in each group. Cells in blank control group were cultured with DMEM medium for 24 h. Cells in endostatin group were cultured with DMEM medium containing 5 μg/mL endostatin for 24 h. Cells in PDGF-BB group and TGF-β1 group were cultured with DMEM medium containing 200 ng/mL PDGF-BB and 10 ng/mL TGF-β1 for 24 h, respectively. Cells in endostatin+ PDGF-BB group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 200 ng/mL PDGF-BB for 24 h. Cells in endostatin+ TGF-β1 group were pretreated with DMEM medium containing 5 μg/mL endostatin for 48 h and then cultured with DMEM medium containing 10 ng/mL TGF-β1 for 24 h. The content of type Ⅰ collagen in the cell culture supernatant of three wells in each group was determined by enzyme-linked immunosorbent assay. The protein expression levels of α-smooth muscle actin (α-SMA), PDGF receptor β (PDGFRβ), phosphorylated PDGFRβ (p-PDGFRβ), and phosphorylated extracellular signal-regulated protein kinases 1/2 (p-ERK1/2) of three wells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and SNK test.
Results:(1) Compared with (5.05±0.29) pg/mL in blank control group, content of type Ⅰ collagen in the cell culture supernatant of endostatin group [(4.72±0.37) pg/mL] was close to it (P>0.05), content of type Ⅰ collagen in the cell culture supernatant of PDGF-BB group and TGF-β1 group [(8.60±0.57) and (9.20±0.64) pg/mL, respectively] was higher (with P values below 0.05). Content of type Ⅰ collagen in the cell culture supernatant of endostatin+ PDGF-BB group [(5.32±0.17) pg/mL] was lower than that of PDGF-BB group (P<0.05), and content of type Ⅰ collagen in the cell culture supernatant of endostatin+ TGF-β1 group [(5.41±0.20) pg/mL] was lower than that of TGF-β1 group (P<0.05). (2) Compared with those in blank control group, protein expression levels of α-SMA, PDGFRβ, p-PDGFRβ, and p-ERK1/2 of cells in endostatin group showed no obvious differences (with P values above 0.05), while those in PDGF-BB and TGF-β1 group were significantly higher (with P values below 0.01). Protein expression levels of α-SMA, PDGFRβ, p-PDGFRβ, and p-ERK1/2 of cells in endostatin+ PDGF-BB group and endostatin+ TGF-β1 group were significantly lower than those in PDGF-BB group and TGF-β1 group, respectively (with P values below 0.05).
Conclusions:Pretreatment of endostatin can inhibit the fibrosis of human skin fibroblast and its transformation into myofibroblast, which may be related to the down-regulation of protein expression of p-PDGFRβ, PDGFRβ, and p-ERK.
