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中华临床医师杂志(电子版) ›› 2021, Vol. 15 ›› Issue (09) : 691 -698. doi: 10.3877/cma.j.issn.1674-0785.2021.09.009

基础研究

生姜提取物6-姜酚对人皮肤成纤维细胞增殖、迁移的影响及其机制研究
侯春1, 王肃生1,†(), 梁刚1, 张志华1, 冀航1, 王平平1, 施然1, 李伯辉1   
  1. 1. 510120　广州，广州医科大学附属第一医院整形外科
  • 收稿日期:2021-06-29 出版日期:2021-09-15
  • 通信作者: 王肃生
  • 基金资助:
    广东省中医药局科研项目(20201209)

Effect of 6-gingerol on proliferation and migration of human skin fibroblasts and underlying mechanism

Chun Hou1, Susheng Wang1,†(), Gang Liang1, Zhihua Zhang1, Hang Ji1, Pingping Wang1, Ran Shi1, Bohui Li1   

  1. 1. Department of Plastic and Cosmetic Surgery, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, China
  • Received:2021-06-29 Published:2021-09-15
  • Corresponding author: Susheng Wang
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引用本文：

侯春, 王肃生, 梁刚, 张志华, 冀航, 王平平, 施然, 李伯辉. 生姜提取物6-姜酚对人皮肤成纤维细胞增殖、迁移的影响及其机制研究[J]. 中华临床医师杂志(电子版), 2021, 15(09): 691-698.

Chun Hou, Susheng Wang, Gang Liang, Zhihua Zhang, Hang Ji, Pingping Wang, Ran Shi, Bohui Li. Effect of 6-gingerol on proliferation and migration of human skin fibroblasts and underlying mechanism[J]. Chinese Journal of Clinicians(Electronic Edition), 2021, 15(09): 691-698.

目的

研究不同浓度生姜提取物6-姜酚对人皮肤成纤维细胞增殖、迁移能力的影响，并探讨相关机制。

方法

采用MTT活力试验检测不同浓度6-姜酚（0、25、50、75、100、150、200 mg/L）对皮肤成纤维细胞活性的影响，设空白对照组（人成纤维细胞）、阳性药物组（重组表皮生长因子20 mg/L）、不同浓度（100、50、25 mg/L）6-姜酚处理组。采用流式细胞术检测细胞增殖情况，Transwell实验检测细胞迁移能力，实时荧光定量及蛋白免疫印迹实验检测细胞血小板源性生长因子（PDGF）、血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、基质金属蛋白酶2（MMP2）、基质金属蛋白酶9（MMP9）mRNA和蛋白的表达情况。采用方差分析进行多组样本的差异性比较。

结果

MTT及流式细胞实验结果显示，与空白对照组相比，在50~200 mg/L的范围内随着6-姜酚浓度的增大，皮肤成纤维细胞活性及增殖能力逐渐增加，差异有统计学意义（P＜0.05）。Transwell结果显示，与空白对照组相比，100、50 mg/L 6-姜酚处理组的穿膜细胞数明显增多（P均＜0.01），而25 mg/L 6-姜酚处理组的迁移细胞数量与空白对照组比较差异无统计学意义（P＞0.05）。与空白对照组相比，100、50 mg/L 6-姜酚处理组的细胞羟脯氨酸分泌表达量明显升高（P均＜0.01），而25 mg/L 6-姜酚处理组的细胞羟脯氨酸分泌表达量与空白对照组比较，差异无统计学意义（P＞0.05）。实时定量聚合酶链反应结果显示，不同浓度6-姜酚作用于皮肤成纤维细胞24 h后，100、50 mg/L 6-姜酚处理组PDGF、VEGF、MMP2、MMP9 mRNA表达水平较空白对照组显著升高，差异均有统计学意义（P＜0.05）；100 mg/L 6-姜酚处理组TGF-β1 mRNA表达水平较空白对照组显著升高，差异均有统计学意义（P＜0.05）。蛋白免疫印迹结果显示，各浓度6-姜酚处理组PDGF、VEGF、MMP9蛋白表达水平均较空白对照组显著升高，差异均有统计学意义（P＜0.05）；100、50 mg/L 6-姜酚处理组TGF-β1、MMP2蛋白表达水平较空白对照组显著升高，差异有统计学意义（P＜0.05）。

结论

6-姜酚能够增强成纤维细胞的增殖和迁移能力，具有促进创面修复的潜能，机制可能与6-姜酚能够促进生长因子及金属蛋白酶的表达相关。

关键词: 6-姜酚, 人皮肤成纤维细胞, 生长因子, 基质金属蛋白酶
Objective

To investigate the effect of different concentrations of ginger extract 6-gingerol on the proliferation and migration of human skin fibroblasts and explore the possible mechanisms involved.

Methods

MTT assay was used to detect the effect of different concentrations of 6-gingerol (0, 25, 50, 75, 100, 150 and 200 mg/L) on the viability of skin fibroblasts. The cells were then divided into a blank control group (untreated human fibroblasts), a positive drug group (recombinant epidermal growth factor 20 mg/L), and 6-gingerol treatment groups (100, 50, and 25 mg/L). Flow cytometry was used to detect cell proliferation. Transwell assay was used to detect cell migration ability. Real-time PCR and Western blot were used to detect the mRNA and protein expression of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9), respectively. Analysis of variance was used to compare the differences among multiple groups of samples.

Results

MTT assay and flow cytometry showed that compared with the blank control group, as the concentration of 6-gingerol increased in the range of 50-200 mg/L, the activity and proliferation ability of skin fibroblasts increased gradually, with statistically significant differences (P＜0.05). Transwell assay showed that compared with the blank control group, the numbers of migrated cells in the 100 and 50 mg/L 6-gingerol treatment groups were significantly increased (P＜0.01 for both), while that in the 25 mg/L 6-gingerol treatment group had no statistically significant change (P＞0.05). Compared with the blank control group, the secretion of cellular hydroxyproline in the 100 and 50 mg/L 6-gingerol treatment groups was significantly increased (P＜0.01 for both), while in the 25 mg/L 6-gingerol treatment group there was no statistically significant difference (P＞0.05). Real-time PCR results showed that after treatment of skin fibroblasts with different concentrations of 6-gingerol for 24 h, the expression levels of PDGF, VEGF, MMP2, and MMP9 mRNA in the 100 and 50 mg/L 6-gingerol treatment groups, and TGF-β1 mRNA in the 100 mg/L 6-gingerol treatment group were significantly higher than those of the blank control group (P＜0.05 for all). Western blot analysis showed that the protein expression levels of PDGF, VEGF, and MMP9 in the 6-gingerol treatment group at various concentrations and TGF-β1 and MMP2 in the 100 and 50 mg/L 6-gingerol treatment groups were significantly higher than those in the blank control group (P＜0.05 for all).

Conclusion

The 6-gingerol can enhance the proliferation and migration of fibroblasts and has the potential to promote wound repair via mechanism that may be related to promoting the expression of growth factors and metalloproteinases.

Key words: 6-gingerol, Human skin fibroblasts, Growth factors, Matrix metalloproteinases
表1 实时定量聚合酶链反应引物序列
基因名称 正义链（5'-3'） 反义链（5'-3'） 预期扩增长度（bp）
PDGF GATACCTCGCCCATGTTCTG GATACCTCGCCCATGTTCTG 107
VEGF CCGGAATTCGGCGTGCAGCTGGT CCGCTCGAGGCTGCTCACGGTCA 217
TGF-β1 CCAAGCTTATGCCGCCCTCCGGGC GCGTCGACCAGCTGCACTTGCAGGAG 126
MMP2 TGATCTTGACCAGAATACCATCGA GGCTTGCGAGGGAAGAAGTT 184
MMP9 CCTGGAGACCTGAGAACCAATC CCACCCGAGTGTAACCATAGC 188
GAPDH CAAGGTCATCCATGACAACTTTG GTCCACCACCCTGTTGCTGTAG 287
图1 不同浓度6-姜酚对人皮肤成纤维细胞活性的影响注：与NC比较，*P＜0.05，**P＜0.01；NC为空白对照组；PC为阳性药物组
图2 不同浓度6-姜酚对人皮肤成纤维细胞增殖能力的影响注：与NC比较，*P＜0.05，**P＜0.01；NC为空白对照组；PC为阳性药物组
图3 不同浓度6-姜酚对人皮肤成纤维细胞迁移能力的影响（×400）。图a为空白对照组（NC）；图b为阳性药物组（PC）；图c为100 mg/L 6-姜酚处理组（GIN-H）；图d为50 mg/L 6-姜酚处理组（GIN-M）；图e为25 mg/L 6-姜酚处理组（GIN-L）；图f为各组比较统计图，随着6-姜酚浓度增加，穿膜细胞数量增加注：与NC比较，**P＜0.01，***P＜0.001
图4 不同浓度6-姜酚对人皮肤成纤维细胞羟脯氨酸分泌的影响注：与NC相比，**P＜0.01，***P＜0.001；NC为空白对照组；PC为阳性药物组；GIN-H为100 mg/L 6-姜酚处理组；GIN-M为50 mg/L 6-姜酚处理组；GIN-L为25 mg/L 6-姜酚处理组
图5 不同浓度6-姜酚对人皮肤成纤维细胞中生长因子及基质金属蛋白酶mRNA表达的影响注：与NC相比，*P＜0.05，**P＜0.01，***P＜0.001；PDGF为血小板源性生长因子；VEGF为血管内皮生长因子；TGF-β1为转化生长因子-β1；MMP2为基质金属蛋白酶2；MMP9为基质金属蛋白酶9；GAPDH为甘油醛-3-磷酸脱氢酶；NC为空白对照组；PC为阳性药物组；GIN-H为100 mg/L 6-姜酚处理组；GIN-M为50 mg/L 6-姜酚处理组；GIN-L为25 mg/L 6-姜酚处理组
图6 不同浓度6-姜酚对人皮肤成纤维细胞中生长因子及基质金属蛋白酶蛋白表达的影响。蛋白条带图中1~5分别对应NC、PC、GIN-H、GIN-M、GIN-L组注：与NC相比，**P＜0.01，***P＜0.001；PDGF为血小板源性生长因子；VEGF为血管内皮生长因子；TGF-β1为转化生长因子-β1；MMP2为基质金属蛋白酶2；MMP9为基质金属蛋白酶9；NC为空白对照组；PC为阳性药物组；GIN-H为100 mg/L 6-姜酚处理组；GIN-M为50 mg/L 6-姜酚处理组；GIN-L为25 mg/L 6-姜酚处理组
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