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Title: [Effects of endostatin pretreatment on fibrosis of human skin fibroblasts and the mechanisms].
Author: Ren HT, Li Y, Wang SD, Han CM.
Journal: Zhonghua Shao Shang Za Zhi; 2017 Nov 20; 33(11):694-698. PubMed ID: 29166712.
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-β(1) (TGF-β(1)), and endostatin+ TGF-β(1) 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-β(1) group were cultured with DMEM medium containing 200 ng/mL PDGF-BB and 10 ng/mL TGF-β(1) 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-β(1) 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-β(1) 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-β(1) 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-β(1) group [(5.41±0.20) pg/mL] was lower than that of TGF-β(1) 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-β(1) 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-β(1) group were significantly lower than those in PDGF-BB group and TGF-β(1) 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. 目的： 探讨内皮抑素预处理对人皮肤Fb纤维化的作用和相关机制。 方法： 将人皮肤Fb体外常规培养后，取第3～5代细胞进行实验。按照随机数字表法将细胞分为空白对照组、内皮抑素组、血小板源性生长因子BB(PDGF-BB)组、内皮抑素＋PDGF-BB组、TGF-β(1)组、内皮抑素＋TGF-β(1)组，每组3孔。空白对照组细胞仅用DMEM培养液培养24 h；内皮抑素组细胞用含5 μg/mL内皮抑素的DMEM培养液培养24 h；PDGF-BB组和TGF-β(1)组细胞分别用含200 ng/mL PDGF-BB、10 ng/mL TGF-β(1)的DMEM培养液培养24 h；内皮抑素＋PDGF-BB组细胞用含5 μg/mL内皮抑素的DMEM培养液预处理48 h后，再用含200 ng/mL PDGF-BB的DMEM培养液培养24 h；内皮抑素＋TGF-β(1)组细胞用含5 μg/mL内皮抑素的DMEM培养液预处理48 h后，再用含10 ng/mL TGF-β(1)的DMEM培养液培养24 h。每组收集3孔细胞的培养上清液，ELISA法检测Ⅰ型胶原含量；每组收集3孔细胞，采用蛋白质印迹法检测细胞α平滑肌肌动蛋白(α-SMA)、PDGF受体β(PDGFRβ)、磷酸化PDGFRβ(p-PDGFRβ)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的蛋白表达。对数据行单因素方差分析、SNK检验。 结果： (1)与空白对照组的(5.05±0.29)pg/mL比较，内皮抑素组细胞培养上清液中Ⅰ型胶原含量为(4.72±0.37)pg/mL，二者相近(P>0.05)；PDGF-BB组和TGF-β(1)组细胞培养上清液中Ⅰ型胶原含量分别为(8.60±0.57)、(9.20±0.64)pg/mL，均显著升高(P值均小于0.05)。内皮抑素＋PDGF-BB组细胞培养上清液中Ⅰ型胶原含量为(5.32±0.17)pg/mL，低于PDGF-BB组(P<0.05)；内皮抑素＋TGF-β(1)组细胞培养上清液中Ⅰ型胶原含量为(5.41±0.20)pg/mL，低于TGF-β(1)组(P<0.05)。(2)与空白对照组细胞α-SMA、PDGFRβ、p-PDGFRβ、p-ERK1/2蛋白表达量比较，内皮抑素组无明显差异(P值均大于0.05)，PDGF-BB组、TGF-β(1)组均明显增加(P值均小于0.01)。内皮抑素＋PDGF-BB组、内皮抑素＋TGF-β(1)组细胞α-SMA、PDGFRβ、p-PDGFRβ、p-ERK1/2蛋白表达量均分别低于PDGF-BB组、TGF-β(1)组(P值均小于0.05)。 结论： 内皮抑素预处理可抑制人Fb纤维化和向肌Fb转化，这可能与内皮抑素下调p-PDGFRβ、PDGFRβ和p-ERK蛋白表达有关。.

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