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

Abstract

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 the Department of Burns and Plastic Surgery of General Hospital of Northern Theater Command, from October 2018 to March 2019. The third to seventh generations of human hypertrophic scar Fbs were cultured for the follow-up 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 10 μL 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 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 10 μL normal saline, 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, respectively. At 0 (immediately), 12, 24, 36 and 48 h after treatment, the cell proliferation activity was detected by thiazolyl blue assay. Cells were divided into normal saline group, 200 μmol/L Eleutheroside E group, and 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 were the same as before. At 24 h after treatment, the apoptosis was observed by Hoechst 33258 staining. 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, 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 were the same as before. At 24 h after treatment, the protein expression level of THBS1 of cells was detected by Western blotting. The number of sample in each group at each time point was all 3. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, independent sample t 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 (P>0.05). At 12, 24, 36, and 48 h after treatment, the cell 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 (t=7.64, 28.94, 13.69, 5.87, 6.96, 22.83, 14.75, 11.52, 21.09, 20.15, 29.52, 23.12, P 0.05). At 12, 24, 36 and 48 h after treatment, the cell 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 (t=7.14, 44.87, 20.67, 40.98, 9.26, 11.08, 15.33, 20.56, P 0.05). Compared with that in normal saline group, the number 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 number 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 expression 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 (t=16.61, 16.17, 17.29, P 0.05). Conclusions: Eleutheroside E can inhibit the growth of human hypertrophic scar Fbs by down regulating the expression of THBS1.