中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (35): 5642-5648.doi: 10.12307/2023.818

• 皮肤粘膜组织构建 skin and mucosal tissue construction • 上一篇    下一篇

紫草素调控MicroRNA-382-5p抑制人增生性瘢痕成纤维细胞纤维化

唐玉婷1，2，3，贺  茜1，2，3，万  瑀1，2，3，王建军1，2，3，杨安宁2，4，吴  凯4，焦  运5，白志刚6，姜怡邓2，4，沈江涌3   

  1. 宁夏医科大学，1临床医学院，2国家卫生健康委代谢性心血管疾病研究重点实验室，4基础医学院，宁夏回族自治区银川市  750004；宁夏医科大学总医院，3烧伤整形外科，5感染科，宁夏回族自治区银川市  750004；6宁夏回族自治区人民医院骨科，宁夏回族自治区银川市  750004
  • 收稿日期:2022-09-20 接受日期:2022-11-08 出版日期:2023-12-18 发布日期:2023-06-02
  • 通讯作者: 沈江涌，博士，副教授，宁夏医科大学总医院烧伤整形外科，宁夏回族自治区银川市 750004
  • 作者简介:唐玉婷，女，1995年生，四川省成都市人，汉族，宁夏医科大学在读硕士，主要从事增生性瘢痕形成的机制研究。
  • 基金资助:
    国家自然科学基金(U21A20343)，项目负责人：姜怡邓；国家自然科学基金(81860555)，项目负责人：沈江涌；中国医学科学院中央级公益性科研院所基本科研业务费项目(2019PT330002)，项目负责人：姜怡邓；宁夏回族自治区重点研发计划重点项目(2020BFH02003)，项目负责人：杨安宁；宁夏回族自治区重点研发计划重点项目(2020BFH02001)，项目负责人：白志刚；宁夏回族自治区重点研发计划重点项目(2021BEG02028)，项目负责人：吴凯；宁夏高等学校一流学科建设(宁夏医科大学国内一流建设学科临床医学)资助项目(NXYLXK2017A05)，项目负责人：沈江涌

Shikonin inhibits fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p

Tang Yuting1, 2, 3, He Xi1, 2, 3, Wan Yu1, 2, 3, Wang Jianjun1, 2, 3, Yang Anning2, 4, Wu Kai4, Jiao Yun5, Bai Zhigang6, Jiang Yideng2, 4, Shen Jiangyong3   

  1. 1Clinical College of Medicine, 2National Key Laboratory of Metabolic Cardiovascular Disease Research, 4Basic Medical College, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Department of Burn and Plastic Surgery, 5Department of Infection, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 6Department of Orthopedics, Ningxia Hui Autonomous Region People’s Hospital, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Received:2022-09-20 Accepted:2022-11-08 Online:2023-12-18 Published:2023-06-02
  • Contact: Shen Jiangyong, MD, Associate professor, Department of Burn and Plastic Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Tang Yuting, Master candidate, Clinical College of Medicine, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; National Key Laboratory of Metabolic Cardiovascular Disease Research, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Department of Burn and Plastic Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    National Natural Science Foundation of China, Nos. U21A20343 (to JYD) and 81860555 (to SJY); Fundamental Research Funds for Central Level Public Welfare Research Institutes of the Chinese Academy of Medical Sciences, No. 2019PT330002 (to JYD); Key Projects of Ningxia Hui Autonomous Region Key Research and Development Plan, Nos. 2020BFH02003 (to YAN), 2020BFH02001 (to BZG), and 2021BEG02028 (to WK); Ningxia University First-class Discipline Construction Project (Ningxia Medical University Domestic First-class Discipline Clinical Medicine), No. NXYLXK2017A05 (to SJY) 

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摘要：


文题释义：

紫草素：是紫草根的主要化学成分，具有抗增殖、抗肿瘤、抗炎等多种功能活性。
增生性瘢痕：组织过度增生形成的一种病理性瘢痕，表现为创面局部异常增生高出于皮面的红色瘢痕组织。成纤维细胞的过度增殖及细胞外基质的过度沉积是瘢痕的病理特征主要表现。
微小RNA(MicroRNA)：小的非编码RNA，已成为基因表达调控中必不可少的转录后调节因子。
细胞迁移：是活细胞普遍存在的一种运动形式，细胞迁移对于免疫防御、伤口修复和组织稳态等多种生理过程至关重要。

背景：目前已有研究表明紫草素具有治疗增生性瘢痕的潜力，且miRNA参与增生性瘢痕的病理机制调控，猜测紫草素有可能通过影响miRNA调控增生性瘢痕的发生发展。
目的：探讨紫草素通过影响MicroRNA-382-5p对人增生性瘢痕成纤维细胞纤维化的作用机制。
方法：收集宁夏医科大学总医院烧伤整形外科提供的增生性瘢痕组织及瘢痕旁正常皮肤组织(瘢痕旁3 cm以内)，并分别分离出人增生性瘢痕成纤维细胞和正常成纤维细胞用于后续实验。苏木精-伊红染色鉴定正常皮肤和增生性瘢痕；免疫荧光鉴定成纤维细胞；采用实时荧光定量PCR从组织水平检测MicroRNA-382-5p相对表达水平。将增生性瘢痕成纤维细胞随机分为紫草素组(紫草素溶于二甲基亚砜配成浓度为13.46 μmol/L的药物干预细胞24 h)、二甲基亚砜组、MicroRNA-382-5p阴性对照组、MicroRNA-382-5p抑制组、紫草素+MicroRNA-382-5p阴性对照组及紫草素+MicroRNA-382-5p过表达组。实时荧光定量PCR检测Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白mRNA表达；Western blot检测Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的蛋白表达；CCK-8法检测细胞活性；划痕实验检测细胞迁移能力。

结果与结论：①与正常皮肤成纤维细胞相比，增生性瘢痕成纤维细胞增殖活性更强(P < 0.01)；②与二甲基亚砜组相比，紫草素组可以下调增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA：P < 0.01，蛋白：P < 0.01)，并抑制增生性瘢痕成纤维细胞迁移(P < 0.05)；③与正常皮肤相比，MicroRNA-382-5p在增生性瘢痕中呈高表达(P < 0.01)，且紫草素能下调增生性瘢痕成纤维细胞中MicroRNA-382-5p的表达(P < 0.01)；④敲低MicroRNA-382-5p能下调增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA：P < 0.01，蛋白：P < 0.01)，并抑制增生性瘢痕成纤维细胞迁移(P < 0.05)；⑤过表达MicroRNA-382-5p能上调在紫草素影响下增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA：P < 0.01，蛋白：P < 0.01)，并促进增生性瘢痕成纤维细胞迁移(P < 0.01)；⑥提示紫草素可通过下调MicroRNA-382-5p的表达抑制增生性瘢痕成纤维细胞的纤维化和迁移。

https://orcid.org/000-0001-5880-0404(唐玉婷)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 紫草素, MicroRNA-382-5p, 增生性瘢痕, 正常皮肤, 成纤维细胞, 胶原沉积, 细胞迁移

Abstract: BACKGROUND: Previous studies have shown that shikonin has the potential to treat hypertrophic scar and that microRNAs are involved in the regulation of the pathological mechanism of hypertrophic scar. It is speculated that shikonin may regulate the occurrence and development of hypertrophic scar through microRNAs regulation. 
OBJECTIVE: To investigate the mechanism of shikonin on the fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p. 
METHODS: Hypertrophic scar tissue and normal skin tissue adjacent to the scar (within 3 cm around the scar) were provided by the Department of Burn and Plastic Surgery, General Hospital of Ningxia Medical University to extract human hypertrophic scar fibroblasts and human normal skin fibroblasts, respectively. Hematoxylin-eosin staining was used to identify normal skin and hypertrophic scar and immunofluorescence was used to identify fibroblasts. The relative expression level of MicroRNA-382-5p was detected by quantitative real-time PCR at the tissue level. Hypertrophic scar fibroblasts were randomly divided into shikonin group (shikonin was dissolved in dimethyl sulfone to make drug solution at a concentration of 13.46 μmol/L, which was used for cell culture for 24 hours), dimethyl sulfone group, MicroRNA-382-5p negative control group, MicroRNA-382-5p inhibitor group, shikonin+MicroRNA-382-5p negative control group and shikonin+MicroRNA-382-5p overexpression group. Real-time fluorescence quantitative PCR and western blot were used to detect the expression of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin at mRNA and protein levels, respectively. Cell counting kit-8 was used to detect cell viability. Cell scratch test was used to detect the migration ability of cells.
RESULTS AND CONCLUSION: Compared with normal skin fibroblasts, hypertrophic scar fibroblasts had stronger proliferative activity (P < 0.01). Compared with the dimethyl sulfone group, shikonin down-regulated the expression levels of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in human hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and inhibited the migration of hypertrophic scar fibroblasts (P < 0.05). Compared with normal skin, MicroRNA-382-5p was highly expressed in hypertrophic scar (P < 0.01), and shikonin could down-regulate the expression of MicroRNA-382-5p (P < 0.01). Inhibition of MicroRNA-382-5p down-regulated the expression level of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and inhibited the migration of hypertrophic scar fibroblasts (P < 0.05). Under the influence of shikonin, overexpression of MicroRNA-382-5p upregulated the expression levels of Collagen Type I α1, Collagen Type III α1 and α-smooth muscle actin in hypertrophic scar fibroblasts (mRNA: P < 0.01, protein: P < 0.01), and promoted the migration of hypertrophic scar fibroblasts (P < 0.01). To conclude, shikonin can inhibit the fibrosis and migration of hypertrophic scar fibroblasts by down-regulating the expression of MicroRNA-382-5p.

Key words: shikonin, MicroRNA-382-5p, hypertrophic scar, normal skin, fibroblast, collagen deposition, cell migration

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