Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (35): 5642-5648.doi: 10.12307/2023.818

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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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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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