Email Alerts
RSS
Volume 39 Issue 2
Feb.  2023
Turn off MathJax
Article Contents
Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2023  >  39(2): 122-131
Zhao SH,Jin LB,Zhang JH,et al.Effects of tumor necrosis factor-alpha/extracellular signal-regulated kinase pathway on migration ability of HaCaT cells and full-thickness skin defects in mice[J].Chin J Burns Wounds,2023,39(2):122-131.DOI: 10.3760/cma.j.cn501225-20221019-00460.
Citation: Zhao SH,Jin LB,Zhang JH,et al.Effects of tumor necrosis factor-alpha/extracellular signal-regulated kinase pathway on migration ability of HaCaT cells and full-thickness skin defects in mice[J].Chin J Burns Wounds,2023,39(2):122-131.DOI: 10.3760/cma.j.cn501225-20221019-00460.
shu

Effects of tumor necrosis factor-alpha/extracellular signal-regulated kinase pathway on migration ability of HaCaT cells and full-thickness skin defects in mice

doi: 10.3760/cma.j.cn501225-20221019-00460

  • Department of Plastic and Cosmetic Surgery, the Second Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400037, China

Funds:

General Program of National Natural Science Foundation of China 81971844

More Information
  • Corresponding author: Fan Dongli, Email: fdltmmu@sina.com
  • Received Date: 2022-10-19
    Abstract
  •   Objective  To investigate the effects of tumor necrosis factor-alpha (TNF-α)/extracellular signal-regulated kinase (ERK) pathway on the migration ability of HaCaT cells and full-thickness skin defects in mice.  Methods  The experimental research method was adopted. According to the random number table (the same below), HaCaT cells were divided into the normal oxygen group and the hypoxia group cultured under hypoxia (with oxygen volume fraction of 1%, the same below) condition. After 24 hours of culture, the significantly differentially expressed genes between the 2 groups were screened using the microarray confidence analysis software SAM4.01. The significance of the number of each gene in the signaling pathway was analyzed through the Kyoto encyclopedia of genes and genomes to screen the significantly differentially signaling pathways (n=3). HaCaT cells were cultured for 0 (immediately), 3, 6, 12, and 24 h under hypoxia condition. The secretion level of TNF-α was detected by enzyme-linked immunosorbent assay (ELISA), and the number of samples was 5. HaCaT cells were divided into normal oxygen group, hypoxia alone group, and hypoxia+inhibitor group cultured with FR180204 (an ERK inhibitor) and under hypoxia condition. The cells were cultured for 3, 6, 12, and 24 h. The migration ability of the cells was detected by scratch test (n=12). The expressions of phosphorylated nuclear factor kappa B (p-NF-κB), phosphorylated p38 (p-p38), phosphorylated ERK1/2 (p-ERK1/2), N-cadherin, and E-cadherin in HaCaT cells were detected by Western blotting under hypoxic condition for 0, 3, 6, 12, and 24 h (n=3). Sixty-four BALB/c male mice aged 6 to 8 weeks were used to make a full-thickness skin defect wound model on the dorsum of the mice. The mice were divided into the blank control group and the inhibitor group treated with FR180204, with 32 mice in each group being treated accordingly. On post injury day (PID) 0, 3, 6, 9, 12, and 15, the wound conditions of mice were observed and the healing rate was calculated (n=8). On PID 1, 3, 6, and 15, hematoxylin-eosin staining was used to observe neovascularization, inflammatory cell infiltration, and epidermal regeneration on wound, Masson staining was used to observe collagen deposition on wound, the expressions of p-NF-κB, p-p38, p-ERK12, N-cadherin, and E-cadherin in wound tissue were detected by Western blotting (n=6), the number of Ki67 positive cells and the absorbance value of vascular endothelial growth factor (VEGF) were detected by immunohistochemistry (n=5), the protein expressions of interleukin 6 (IL-6), IL-10, IL-1β, and CCL20 in wound tissue were detected by ELISA (n=6). Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, factorial design analysis of variance, Tukey test, least significant difference test, and independent sample t test.  Results  After 24 hours of culture, compared with normal oxygen group, 7 667 genes were up-regulated and 7 174 genes were down-regulated in cells in hypoxic group. Among the above differentially expressed genes, the TNF-α signaling pathway had significant change (P<0.05) with large number of genes. Under hypoxia condition, the expression of TNF-α at 24 h of cell culture was (11.1±2.1) pg/mL, which was significantly higher than (1.9±0.3) pg/mL at 0 h (P<0.05). Compared with normal oxygen group, the migration ability of cells in hypoxia alone group was significantly enhanced at 6, 12, and 24 h of cell culture (with t values of 2.27, 4.65, and 4.67, respectively, P<0.05). Compared with hypoxia alone group, the migration ability of cells in hypoxia+inhibitor group was significantly decreased at 3, 6, 12, and 24 h of cell culture (with t values of 2.43, 3.06, 4.62, and 8.14, respectively, P<0.05). Under hypoxia condition, the expressions of p-NF-κB, p-ERK1/2, and N-cadherin were increased significantly at 12 and 24 h of cell culture compared with 0 h of culture (P<0.05), the expression of p-p38 was significantly increased at 3, 6, 12, and 24 h of cell culture (P<0.05), the expression of E-cadherin was significantly decreased at 6, 12, and 24 h of cell culture (P<0.05), the expression of p-ERK1/2, p-NF-κB, and E-cadherin was time-dependent. Compared with blank control group, on PID 3, 6, 9, 12, and 15, the wound healing rate of mice in inhibitor group was significantly decreased (P<0.05); there were more inflammatory cell infiltration around the wound edge of mice in inhibitor group on PID 3, 6, and 15, especially on PID 15, a large number of tissue necrosis and discontinuous new epidermal layer were observed on the wound surface, and collagen synthesis and new blood vessels were reduced; the expression of p-NF-κB in the wound of mice in inhibitor group was significantly decreased on PID 3 and 6 (with t values of 3.26 and 4.26, respectively, P<0.05) but significantly increased on PID 15 (t=3.25, P<0.05), the expressions of p-p38 and N-cadherin were significantly decreased on PID 1, 3, and 6 (with t values of 4.89, 2.98, 3.98, 9.51, 11.69, and 4.10, respectively, P<0.05), the expression of p-ERK1/2 was significantly decreased on PID 1, 3, 6, and 15 (with t values of 26.69, 3.63, 5.12, and 5.14, respectively, P<0.05), the expression of E-cadherin was significantly decreased on PID 1 (t=20.67, P<0.05) but significantly increased on PID 6 (t=2.90, P<0.05); the number of Ki67 positive cells and absorbance value of VEGF of wound in inhibitor group were significantly decreased on PID 3, 6, and 15 (with t values of 4.20, 7.35, 3.34, 4.14, 3.20, and 3.73, respectively, P<0.05); the expression of IL-10 in the wound tissue of the inhibitor group was significantly decreased on PID 6 (t=2.92, P<0.05), the expression of IL-6 was significantly increased on PID 6 (t=2.73, P<0.05), the expression of IL-1β was significantly increased on PID 15 (t=3.46, P<0.05), and CCL20 expression levels were significantly decreased on PID 1 and 6 (with t values of 3.96 and 2.63, respectively, P<0.05) but significantly increased on PID 15 (t=3.68, P<0.05).  Conclusions  The TNF-α/ERK pathway can promote the migration of HaCaT cells, and regulate the healing of full-thickness skin defect wounds in mice by affecting the expression of inflammatory cytokines and chemokines.

     

    • FullText(HTML)
  • loading
    • References(33)
  • [1]
    WilkinsonHN,HardmanMJ.Wound healing: cellular mechanisms and pathological outcomes[J].Open Biol,2020,10(9):200223.DOI: 10.1098/rsob.200223.
    [2]
    ZhangJ,YanY,LiY,et al.Topical effect of benzalkonium bromide on wound healing and potential cellular and molecular mechanisms[J].Int Wound J,2021,18(5):566-576.DOI: 10.1111/iwj.13555.
    [3]
    LiaoF,ChenL,LuoP,et al.PC4 serves as a negative regulator of skin wound healing in mice[J/OL].Burns Trauma,2020,8:tkaa010[2022-10-19].https://pubmed.ncbi.nlm.nih.gov/32373645/.DOI: 10.1093/burnst/tkaa010.
    [4]
    LiL,HeY,ZhaoM,et al.Collective cell migration: Implications for wound healing and cancer invasion[J/OL].Burns Trauma,2013,1(1):21-26[2022-10-19].https://pubmed.ncbi.nlm.nih.gov/27574618/.DOI: 10.4103/2321-3868.113331.
    [5]
    KiwanukaE,HacklF,CatersonEJ,et al.CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway[J].J Surg Res,2013,185(2):e109-119.DOI: 10.1016/j.jss.2013.05.065.
    [6]
    RuthenborgRJ,BanJJ,WazirA,et al.Regulation of wound healing and fibrosis by hypoxia and hypoxia-inducible factor-1[J].Mol Cells,2014,37(9):637-643.DOI: 10.14348/molcells.2014.0150.
    [7]
    SternD,CuiH.Crafting Polymeric and peptidic hydrogels for improved wound healing[J].Adv Healthc Mater,2019,8(9):e1900104.DOI: 10.1002/adhm.201900104.
    [8]
    ZhangYM,ZhangZQ,LiuYY,et al.Requirement of Gαi1/3-Gab1 signaling complex for keratinocyte growth factor-induced PI3K-AKT-mTORC1 activation[J].J Invest Dermatol,2015,135(1):181-191.DOI: 10.1038/jid.2014.326.
    [9]
    ChenX,ZhouX,MaoTC,et al.Effect of microtubule-associated protein-4 on epidermal cell migration under different oxygen concentrations[J].J Dermatol,2016,43(6):674-681.DOI: 10.1111/1346-8138.13192.
    [10]
    ChenX,ZhouX,ShiX,et al.MAP4 regulates Tctex-1 and promotes the migration of epidermal cells in hypoxia[J].Exp Dermatol,2018,27(11):1210-1215.DOI: 10.1111/exd.13763.
    [11]
    SchremlS,SzeimiesRM,PrantlL,et al.Oxygen in acute and chronic wound healing[J].Br J Dermatol,2010,163(2):257-268.DOI: 10.1111/j.1365-2133.2010.09804.x.
    [12]
    TaiG,TaiM,ZhaoM.Electrically stimulated cell migration and its contribution to wound healing[J/OL].Burns Trauma,2018,6:20[2022-10-19].https://pubmed.ncbi.nlm.nih.gov/30003115/.DOI: 10.1186/s41038-018-0123-2.
    [13]
    FuX.Wound healing center establishment and new technology application in improving the wound healing quality in China[J/OL].Burns Trauma,2020,8:tkaa038[2022-10-19].https://pubmed.ncbi.nlm.nih.gov/33134399/.DOI: 10.1093/burnst/tkaa038.
    [14]
    黄跃生.调控生物电场与氧微环境促进创面再生修复[J].中华烧伤杂志,2021,37(1):5-8.DOI: 10.3760/cma.j.cn501120-20201123-00492.
    [15]
    RamaswamyP,GoswamiK,Dalavaikodihalli NanjaiahN,et al.TNF-α mediated MEK-ERK signaling in invasion with putative network involving NF-κB and STAT-6: a new perspective in glioma[J].Cell Biol Int,2019,43(11):1257-1266.DOI: 10.1002/cbin.11125.
    [16]
    BalkwillF.Tumour necrosis factor and cancer[J].Nat Rev Cancer,2009,9(5):361-371.DOI: 10.1038/nrc2628.
    [17]
    LuoJL,MaedaS,HsuLC,et al.Inhibition of NF-kappaB in cancer cells converts inflammation- induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression[J].Cancer Cell,2004,6(3):297-305.DOI: 10.1016/j.ccr.2004.08.012.
    [18]
    AshcroftGS,JeongMJ,AshworthJJ,et al.Tumor necrosis factor-alpha (TNF-α) is a therapeutic target for impaired cutaneous wound healing[J].Wound Repair Regen,2012,20(1):38-49.DOI: 10.1111/j.1524-475X.2011.00748.x.
    [19]
    YenYH,PuCM,LiuCW,et al.Curcumin accelerates cutaneous wound healing via multiple biological actions: the involvement of TNF-α, MMP-9, α-SMA, and collagen[J].Int Wound J,2018,15(4):605-617.DOI: 10.1111/iwj.12904.
    [20]
    YanC,GrimmWA,GarnerWL,et al.Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-alpha through bone morphogenic protein-2[J].Am J Pathol,2010,176(5):2247-2258.DOI: 10.2353/ajpath.2010.090048.
    [21]
    HaY,LeeWH,JeongJ,et al.Pyropia yezoensis extract suppresses IFN-gamma- and TNF-alpha-induced proinflammatory chemokine production in HaCaT cells via the down-regulation of NF-κB[J].Nutrients,2020,12(5)DOI: 10.3390/nu12051238.
    [22]
    WenLJ,HuXL,LiCY,et al.Myosin 1b promotes migration, invasion and glycolysis in cervical cancer via ERK/HIF-1α pathway[J].Am J Transl Res,2021,13(11):12536-12548.
    [23]
    张灿,张琼,张均辉,等.组蛋白脱乙酰酶6抑制剂Tubastatin A对人皮肤成纤维细胞增殖和运动的影响及其分子机制[J].中华烧伤杂志,2021,37(9):853-859.DOI: 10.3760/cma.j.cn501120-20200519-00274.
    [24]
    LiB,TangH,BianX,et al.Calcium silicate accelerates cutaneous wound healing with enhanced re-epithelialization through EGF/EGFR/ERK-mediated promotion of epidermal stem cell functions[J/OL].Burns Trauma,2021,9:tkab029[2022-10-19].https://pubmed.ncbi.nlm.nih.gov/34604395/.DOI: 10.1093/burnst/tkab029.
    [25]
    BrunerHC,DerksenP.Loss of E-cadherin-dependent cell-cell adhesion and the development and progression of Cancer[J].Cold Spring Harb Perspect Biol,2018,10(3):a029330.DOI: 10.1101/cshperspect.a029330.
    [26]
    NiemanMT,PrudoffRS,JohnsonKR,et al.N-cadherin promotes motility in human breast cancer cells regardless of their E-cadherin expression[J].J Cell Biol,1999,147(3):631-644.DOI: 10.1083/jcb.147.3.631.
    [27]
    WalkerA,FreiR,LawsonKR.The cytoplasmic domain of N-cadherin modulates MMP-9 induction in oral squamous carcinoma cells[J].Int J Oncol,2014,45(4):1699-1706.DOI: 10.3892/ijo.2014.2549.
    [28]
    ZhangH,SunP,WangYL,et al.MiR-214 promotes proliferation and inhibits apoptosis of oral cancer cells through MAPK/ERK signaling pathway[J].Eur Rev Med Pharmacol Sci,2020,24(7):3710-3716.DOI: 10.26355/eurrev_202004_20834.
    [29]
    KhanNM,HaseebA,AnsariMY,et al.Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes[J].Free Radic Biol Med,2017,106:288-301.DOI: 10.1016/j.freeradbiomed.2017.02.041.
    [30]
    DongQ,JieY,MaJ,et al.Renal tubular cell death and inflammation response are regulated by the MAPK-ERK-CREB signaling pathway under hypoxia-reoxygenation injury[J].J Recept Signal Transduct Res,2019,39(5-6):383-391.DOI: 10.1080/10799893.2019.1698050.
    [31]
    FurueK,ItoT,TsujiG,et al.The CCL20 and CCR6 axis in psoriasis[J].Scand J Immunol,2020,91(3):e12846.DOI: 10.1111/sji.12846.
    [32]
    DuttaP,TaA,ThakurBK,et al.Biphasic Ccl20 regulation by Toll-like receptor 9 through the activation of ERK-AP-1 and non-canonical NF-κB signaling pathways[J].Biochim Biophys Acta Gen Subj,2017,1861(1 Pt A):3365-3377.DOI: 10.1016/j.bbagen.2016.08.019.
    [33]
    刘中阳,程旭,张景霞,等.Vγ4T细胞在应用雷帕霉素的小鼠全层皮肤缺损创面愈合障碍中的作用及其机制[J].中华烧伤与创面修复杂志,2022,38(5):462-470.DOI: 10.3760/cma.j.cn501120-20201209-00523.
    • Relative Articles
    • Supplements(0)
    • Cited By
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(6)  / Tables(1)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (2730) PDF downloads(40) Cited by()
    Proportional views
    Related

    Copyright © Chinese Journal of Burns京ICP备07035254号-14

    E-mail：shaoshangzazhi@163.com

    Website：https://zhsszz.xml-journal.net/

    Supported by: Beijing Renhe Information Technology Co. Ltd    

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return