Effects and mechanism of eleutheroside E on the growth of human hypertrophic scar fibroblasts
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摘要:
目的 探讨刺五加皂苷E对人增生性瘢痕成纤维细胞（Fb）生长的影响及作用机制。 方法 采用实验研究方法。收集北部战区总医院2018年10月—2019年3月收治的6例增生性瘢痕患者［男1例、女5例，年龄20~51（37±8）岁］的增生性瘢痕组织，培养第3~7代人增生性瘢痕Fb用于后续实验。取细胞，分为生理盐水组、100 μmol/L刺五加皂苷E组、200 μmol/L刺五加皂苷E组和400 μmol/L刺五加皂苷E组，分别加入生理盐水，终物质的量浓度为100、200、400 μmol/L的刺五加皂苷E。取细胞，分为单纯小干扰RNA（siRNA）-阴性对照组、单纯siRNA-血小板反应蛋白1（THBS1）组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组和siRNA-THBS1+400 μmol/L刺五加皂苷E组，单纯siRNA-阴性对照组和siRNA-阴性对照+400 μmol/L刺五加皂苷E组细胞转染siRNA-阴性对照，单纯siRNA-THBS1组和siRNA-THBS1+400 μmol/L刺五加皂苷E组细胞转染siRNA-THBS1，转染24 h后单纯siRNA-阴性对照组和单纯siRNA-THBS1组细胞加入生理盐水，siRNA-阴性对照+400 μmol/L刺五加皂苷E组和siRNA-THBS1+400 μmol/L刺五加皂苷E组细胞加入终物质的量浓度为400 μmol/L的刺五加皂苷E。于处理后0（即刻）、12、24、36、48 h，采用噻唑蓝法检测细胞增殖活性（以吸光度值表示）。取细胞分为生理盐水组、200 μmol/L刺五加皂苷E组、400 μmol/L刺五加皂苷E组，另取细胞分为单纯siRNA-阴性对照组、单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组、siRNA-THBS1+400 μmol/L刺五加皂苷E组，相应处理同前，于处理后24 h，行Hoechst 33258染色观察细胞凋亡情况。取细胞分为生理盐水组、100 μmol/L刺五加皂苷E组、200 μmol/L刺五加皂苷E组、400 μmol/L刺五加皂苷E组，另取细胞分为单纯siRNA-阴性对照组、单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组、siRNA-THBS1+400 μmol/L刺五加皂苷E组，相应处理同前，于处理后24 h，采用蛋白质印迹法检测细胞THBS1蛋白水平。以上实验每组各时间点样本数均为3。对数据行析因设计方差分析、单因素方差分析、独立样本
t 检验、Bonferroni校正。 结果 处理后0 h，生理盐水组、100 μmol/L刺五加皂苷E组、200 μmol/L刺五加皂苷E组和400 μmol/L刺五加皂苷E组细胞吸光度值相近（
P >0.05）。处理后12、24、36、48 h，100 μmol/L刺五加皂苷E组、200 μmol/L刺五加皂苷E组、400 μmol/L刺五加皂苷E组细胞吸光度值均明显低于生理盐水组（
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或
P <0.01）。处理后0 h，单纯siRNA-阴性对照组、单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组、siRNA-THBS1+400 μmol/L刺五加皂苷E组细胞吸光度值相近（
P >0.05）；处理后12、24、36、48 h，单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组细胞吸光度值明显低于单纯siRNA-阴性对照组（
t =7.14、44.87、20.67、40.98，9.26、11.08、15.33、20.56，
P< 0.05或
P< 0.01），单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组细胞吸光度值与siRNA-THBS1+400 μmol/L刺五加皂苷E组相近（
P >0.05）。处理后24 h，与生理盐水组比较，200 μmol/L刺五加皂苷E组和400 μmol/L刺五加皂苷E组凋亡细胞数量增加。处理后24 h，与单纯siRNA-阴性对照组比较，单纯siRNA-THBS1组和siRNA-阴性对照+400 μmol/L刺五加皂苷E组凋亡细胞数量增加；单纯siRNA-THBS1组、siRNA-阴性对照+400 μmol/L刺五加皂苷E组和siRNA-THBS1+400 μmol/L刺五加皂苷E组凋亡细胞数量相近。处理后24 h，100 μmol/L刺五加皂苷E组、200 μmol/L刺五加皂苷E组、400 μmol/L刺五加皂苷E组细胞THBS1蛋白水平（0.87±0.12、0.38±0.07、0.20±0.09）均明显低于生理盐水组（1.83±0.17，
t ＝16.61、16.17、17.29，
P <0.01）。处理后24 h，单纯siRNA-THBS1组和siRNA-阴性对照+400 μmol/L刺五加皂苷E组细胞THBS1蛋白水平（0.61±0.07、0.58±0.07）均明显低于单纯siRNA-阴性对照组（1.86±0.07，
t ＝71.06、83.80，
P <0.01），单纯siRNA-THBS1组和siRNA-阴性对照+400 μmol/L刺五加皂苷E组细胞THBS1蛋白水平与siRNA-THBS1+400 μmol/L刺五加皂苷E组（0.63±0.11）相近（
P >0.05）。 结论 刺五加皂苷E能够通过下调人增生性瘢痕Fb中THBS1蛋白表达，发挥抑制人增生性瘢痕Fb生长的作用。
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 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
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 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 or
P < 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 (
P > 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 (
t = 7.14, 44.87, 20.67, 40.98, 9.26, 11.08, 15.33, 20.56,
P < 0.05 or
P < 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 (
P > 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 (
t = 16.61, 16.17, 17.29,
P < 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 (
t = 71.06, 83.80,
P < 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 (
P > 0.05). Conclusions Eleutheroside E can inhibit the growth of human hypertrophic scar Fbs by down-regulating the expression of THBS1.
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Key words:
- Cicatrix /
- Cell proliferation /
- Apoptosis /
- Fibroblasts /
- Saponins
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