切换至 "中华医学电子期刊资源库"
高级检索
中华临床医师杂志(电子版)

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
全文 ( ) 下载PDF ( ) 导出引用
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/OL]. 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/OL]. 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
1
Hu F, Yan Y, Wang CW, et al. Effect and mechanism of ganoderma lucidum polysaccharides on human fibroblasts and skin wound healing in mice [J]. Chin J Integr Med, 2019, 25(3): 1-7.
2
Darby IA, Hewitson TD. Fibroblast differentiation in wound healing and fibrosis [J]. Int Rev Cytol, 2007, 257: 143-179.
3
Wilkinson HN, Hardman MJ. Wound healing: cellular mechanisms and pathological outcomes [J]. Open Biol, 2020, 10(9): 1-14.
4
Bhaskar A, Kumari A, Singh M, et al. [6]-Gingerol exhibits potent anti-mycobacterial and immunomodulatory activity against tuberculosis [J]. Int Immunopharmacol, 2020, 87: 106809.
5
Bhagavathula N, Warner RL, Dasilva M, et al. A combination of curcumin and ginger extract improves abrasion wound healing in corticosteroid-impaired hairless rat skin [J]. Wound Repair Regen, 2009, 17(3): 360-366.
6
Zick SM, , Djuric Z, Ruffin MT, et al. Pharmacokinetics of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol and conjugate metabolites in healthy human subjects [J]. Cancer Epidemiol Biomarkers Prev, 2008, 17(8): 1930-1936.
7
Baliga MS, Haniadka R, Pereira MM, et al. Update on the chemopreventive effects of ginger and its phytochemicals [J]. Crit Rev Food Sci Nutr, 2011, 51(6): 499-523.
8
Lee C, Park GH, Kim CY, et al. [6]-Gingerol attenuates β-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system [J]. Food Chem Toxicol, 2011, 49(6): 1261-1269.
9
Li XH, McGrath KCY, Tran Van H, et al. Attenuation of proinflammatory responses by S-[6]-gingerol via inhibition of ROS/NF-Kappa B/COX2 activation in HuH7 cells [J]. Evid Based Complement Alternat Med, 2013, 2013: 1-8.
10
Prasad S, Tyagi AK. Ginger and its constituents: role in prevention and treatment of gastrointestinal cancer [J]. Gastroenterol Res Pract, 2015, 2015: 142979.
11
Zhang MZ, Xu CL, Liu DD, et al. Oral Delivery of nanoparticles loaded with ginger active compound, 6-shogaol, attenuates ulcerative colitis and promotes wound healing in a murine model of ulcerative colitis [J]. J Crohns Colitis, 2018, 12(2): 217-229.
12
Bhagavathula N, Warner RL, DaSilva M, et al. A combination of curcumin and ginger extract improves abrasion wound healing in corticosteroid-impaired hairless rat skin [J]. Wound Repair Regen, 2009, 17(3): 360-366.
13
Sinno H, Prakash S. Complements and the wound healing cascade: an updated review [J]. Plast Surg Int, 2013, 2013: 1-7.
14
Rousselle P, Montmasson M, Garnier C. Extracellular matrix contribution to skin wound re-epithelialization [J]. Matrix Biol, 2019, 75-76: 12-26.
15
Martin P, Nunan R .Cellular and molecular mechanisms of repair in acute and chronic wound healing [J]. Br J Dermatol, 2015, 173(2): 370-378.
16
Bielefeld KA, Amini-Nik S, Alman BA .Cutaneous wound healing: recruiting developmental pathways for regeneration [J]. Cell Mol Life Sci, 2013, 70(12): 2059-2081.
17
Wang PH, Huang BS, Horng HC, et al. Wound healing [J]. J Chin Med Assoc, 2018, 81(2): 94-101.
18
Greenhalgh DG, Sprugel KH, Murray MJ, et al. PDGF and FGF stimulate wound healing in the genetically diabetic mouse [J]. Am J Pathol, 1990, 136(6): 1235-1246.
19
Demaria M, Ohtani N, Youssef SA, et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA [J]. Dev Cell, 2014, 31(6): 722-733.
20
Li HH, Fu XB, Zhang L, et al. Research of PDGF-BB gel on the wound healing of diabetic rats and its pharmacodynamics [J]. J Surg Res, 2008, 145(1): 41-48.
21
Lobmann R, Zemlin C, Motzkau M, et al. Expression of matrix metalloproteinases and growth factors in diabetic foot wounds treated with a protease absorbent dressing [J]. J Diabetes Complications, 2006, 20(5): 329-335.
22
Cheng JW, Cheng SW, Wei RL, et al. Anti-vascular endothelial growth factor for control of wound healing in glaucoma surgery [J]. Cochrane Database Syst Rev, 2016(1): 1-39.
23
Angelo LS, Kurzrock R. Vascular endothelial growth factor and its relationship to inflammatory mediators [J]. Clin Cancer Res, 2007, 13(10): 2825-2830.
24
Zhou KH, Ma Y, Brogan MS. Chronic and non-healing wounds: the story of vascular endothelial growth factor [J]. Med Hypotheses, 2015, 85(4): 399-404.
25
Leonov YI, Shkumat MS, Klymenko PP, et al. Effect of insulin-like growth factor transgene on wound healing in mice with streptozotocin-induced diabetes [J]. Cytology Genet, 2015, 49(1): 19-26.
26
Roberts AB. Transforming growth factor-beta: activity and efficacy in animal models of wound healing [J]. Wound Repair Regen, 1995, 3(4): 408-418.
27
Ehanire T, Ren LC, Bond J, et al. Angiotensin II stimulates canonical TGF-β signaling pathway through angiotensin type 1 receptor to induce granulation tissue contraction [J]. J Mol Med (Berl), 2015, 93(3): 289-302.
28
Alves CC, Torrinhas RS, Giorgi R, et al. TGF-β1 expression in wound healing is acutely affected by experimental malnutrition and early enteral feeding [J]. Int Wound J, 2014, 11(5): 533-539.
29
Hozzein WN, Badr G, Ghamdi AA, et al. Topical application of propolis enhances cutaneous wound healing by promoting TGF-beta/Smad-mediated collagen production in a streptozotocin-induced type Ⅰ diabetic mouse model [J]. Cell Physiol Biochem, 2015, 37(3): 940-954.
30
Jude EB, Blakytny R, Bulmer J, et al. Transforming growth factor-beta 1, 2, 3 and receptor type Ⅰ and Ⅱ in diabetic foot ulcers [J]. Diabet Med, 2002, 19(6): 440-447.
31
Heublein H, Bader A, Giri S. Preclinical and clinical evidence for stem cell therapies as treatment for diabetic wounds [J]. Drug Discov Today, 2015, 20(6): 703-717.
32
Lobmann R, Pap T, Ambrosch A, et al. Differential effects of PDGF-BB on matrix metalloproteases and cytokine release in fibroblasts of Type 2 diabetic patients and normal controls in vitro [J]. J Diabetes Complications, 2006, 20(2): 105-112.
33
Armstrong DG, Jude EB. The role of matrix metalloproteinases in wound healing [J]. J Am Podiatr Med Assoc, 2002, 92(1): 12-18.
34
Martins VL, Caley M, O'Toole EA. Matrix metalloproteinases and epidermal wound repair [J]. Cell Tissue Res, 2013, 351(2): 255-268.
35
Liu D, Wu MQ, Lu Y, et al. Protective effects of 6-Gingerol on vascular endothelial cell injury induced by high glucose via activation of PI3K-AKT-eNOS pathway in human umbilical vein endothelial cells [J]. Biomed Pharmacother, 2017, 93: 788-795.
[1] 刘晨鹭, 刘洁, 张帆, 严彩英, 陈倩, 陈双庆. 增强MRI 影像组学特征生境分析在预测乳腺癌HER-2 表达状态中的应用[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 339-345.
[2] 刘琴, 刘瀚旻, 谢亮. 基质金属蛋白酶在儿童哮喘发生机制中作用的研究现状[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 564-568.
[3] 韩肖燕, 杨桦. 中孕期孕妇血清胎盘生长因子水平低与胎儿不良预后的关系[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(04): 398-402.
[4] 邵小丽, 林燕, 张玲玲, 韩亚琴. 超声引导下子宫肌瘤注射聚桂醇硬化术联合术后米非司酮治疗临床疗效分析[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(03): 353-360.
[5] 刘昌玲, 张金丽, 张志, 李孝建, 汤文彬, 胡逸萍, 陈宾, 谢晓娜. 负载人脂肪干细胞外泌体的甲基丙烯酰化明胶水凝胶对人皮肤成纤维细胞增殖和迁移的影响[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 517-525.
[6] 李星月, 董伟, 徐永波, 张文法, 胡晓璇, 钟玉绪, 褚海波. 浅表血栓性静脉炎管壁基质金属蛋白酶及其抑制剂表达研究[J/OL]. 中华普通外科学文献(电子版), 2024, 18(05): 338-343.
[7] 李雪, 韩萌萌, 冯雪园, 马宁. 人表皮生长因子受体2低表达乳腺癌的研究进展及挑战[J/OL]. 中华普通外科学文献(电子版), 2024, 18(04): 308-312.
[8] 黄福, 王黔, 金相任, 唐云川. VEGFR2、miR-27a-5p在胃癌组织中的表达与临床病理参数及预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(05): 558-561.
[9] 郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[10] 朱佳琳, 方向, 贵诗雨, 黄丹, 周小雨, 郭文恺. 大鼠切口疝腹膜前间隙补片修补术后血清中VEGF 和Ang-1 的表达情况[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 703-707.
[11] 张敏龙, 杨翠平, 王博, 崔云杰, 金发光. MiR-200b-3p 通过抑制HIF-1α 表达减轻海水吸入诱导的肺水肿作用及机制[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 696-700.
[12] 井发红, 李丽娜, 高婷, 高艳梅, 杨楠, 李卓, 慕玉东. 肺癌立体定向放疗血清SAP 和MMPs 表达及临床意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 707-713.
[13] 赖淼, 景鑫, 李桂珍, 李怡. 非小细胞肺癌EGFR 突变亚型的临床病理和预后意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 731-737.
[14] 梁丽斯, 李洁, 贺帅, 来艳君, 刘铭, 张琳. MMP-9、MMP-2 及TLR4、HE4对非小细胞肺癌早期诊断意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 756-761.
[15] 刘先勇, 秦东梅, 张若梅, 李俊娇, 孟春芹, 邬明歆, 王玉红, 赵新鲜, 徐瑞联, 洪文文, 马玲, 仇玮, 周宇. Her2/Hes1在肠型胃癌Correa级联反应3个病理阶段中的表达及意义[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(06): 321-327.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号