Effects and mechanism of eleutheroside E on the growth of human hypertrophic scar fibroblasts

Objective To investigate the effects and mechanism of eleutheroside E on the growth of human hypertrophic scar fibroblasts (Fbs). Methods The experimental research method was used. The hypertrophic scar tissue was collected from 6 patients with hypertrophic scar (1 male and 5 females, aged 20 to 51 (37±8) years) admitted to General Hospital of Northern Theater Command, from October 2018 to March 2019. The third to seventh passages of human hypertrophic scar Fbs were cultured for later experiments. Cells were divided into normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group, and normal saline, eleutheroside E at the final molarity of 100, 200, and 400 μmol/L were added to cells in the corresponding groups. Cells were collected and divided into small interfering RNA (siRNA)-negative control alone group, siRNA-thrombospondin 1 (THBS1) alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group. Cells in siRNA-negative control alone group and siRNA-negative control+ 400 μmol/L eleutheroside E group were transfected with siRNA-negative control, cells in siRNA-THBS1 alone group and siRNA-THBS1+ 400 μmol/L eleutheroside E group were transfected with siRNA-THBS1. At 24 h after transfection, cells in siRNA-negative control alone group and siRNA-THBS1 alone group were added with normal saline, and cells in siRNA-negative control+ 400 μmol/L eleutheroside E group and siRNA-THBS1+ 400 μmol/L eleutheroside E group were added with eleutheroside E at the final molarity of 400 μmol/L. At 0 (immediately), 12, 24, 36, and 48 h after treatment, the cell proliferation activity (expressed as absorbance value) was detected by thiazolyl blue assay. Cells were divided into normal saline group, 200 μmol/L eleutheroside E group, 400 μmol/L eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group. The corresponding treatments in each group were the same as before. At 24 h after treatment, the apoptosis was observed by Hoechst 33258 staining. Cells were collected and divided into normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, 400 μmol/L eleutheroside E group, siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group. The corresponding treatments in each group were the same as before. At 24 h after treatment, the THBS1 protein level of cells was detected by Western blotting. The number of sample in each group was all 3 at each time point. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample

test, and Bonferroni correction. Results At 0 h after treatment, the absorbance values of cells in normal saline group, 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group were similar (

> 0.05). At 12, 24, 36, and 48 h after treatment, the absorbance values of cells in 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group were significantly lower than those of normal saline group (

= 7.64, 28.94, 13.69, 5.87, 6.96, 22.83, 14.75, 11.52, 21.09, 20.15, 29.52, 23.12,

< 0.05 or

< 0.01). At 0 h after treatment, the absorbance values of cells in siRNA-negative control alone group, siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group were similar (

> 0.05). At 12, 24, 36, and 48 h after treatment, the absorbance values of cells in siRNA-THBS1 alone group and siRNA-negative control+ 400 μmol/L eleutheroside E group were significantly lower than those in siRNA-negative control alone group (

= 7.14, 44.87, 20.67, 40.98, 9.26, 11.08, 15.33, 20.56,

< 0.05 or

< 0.01); the absorbance values of cells in siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group were similar (

> 0.05). Compared with that in normal saline group, the numbers of apoptotic cells in 200 μmol/L eleutheroside E group and 400 μmol/L eleutheroside E group were increased at 24 h after treatment. At 24 h after treatment, compared with that in siRNA-negative control alone group, the numbers of apoptotic cells in siRNA-THBS1 alone group and siRNA-negative control+ 400 μmol/L eleutheroside E group were increased, while the numbers of apoptotic cells in siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group were similar. At 24 h after treatment, the protein levels of THBS1 of cells in 100 μmol/L eleutheroside E group, 200 μmol/L eleutheroside E group, and 400 μmol/L eleutheroside E group (0.87±0.12, 0.38±0.07, 0.20±0.09) were significantly lower than 1.83±0.17 in normal saline group (

= 16.61, 16.17, 17.29,

< 0.01). At 24 h after treatment, the protein levels of THBS1 of cells in siRNA-THBS1 alone group and siRNA-negative control+ 400 μmol/L eleutheroside E group (0.61±0.07, 0.58±0.07) were significantly lower than 1.86±0.07 in siRNA-negative control alone group (

= 71.06, 83.80,

< 0.01), and the protein levels of THBS1 of cells siRNA-THBS1 alone group, siRNA-negative control+ 400 μmol/L eleutheroside E group, and siRNA-THBS1+ 400 μmol/L eleutheroside E group (0.63±0.11) were similar (

> 0.05). Conclusions Eleutheroside E can inhibit the growth of human hypertrophic scar Fbs by down-regulating the expression of THBS1.