Effects of mechanical tension on the formation of hypertrophic scars in rabbit ears and transforming growth factor-β1/Smad signaling pathway
-
摘要:
目的 探讨机械张力对兔耳增生性瘢痕的形成及转化生长因子β1（TGF-β1）/Smad信号通路的影响。 方法 采用实验研究方法。取6只3~5个月龄雌雄不拘新西兰大白兔，于每侧兔耳腹面制作5个全层皮肤缺损创面。观察术后0（即刻）、7、14、21、28 d所有兔耳创面外观。术后28 d，计算瘢痕形成率。将每只兔左耳的3个成熟瘢痕纳入张力组并采用螺旋扩弓器持续扩弓，将每只兔右耳的3个成熟瘢痕纳入假张力组并仅缝合螺旋扩弓器不扩弓，每组共18个瘢痕。经机械张力处理（以下简称处理）40 d，观察2组兔耳瘢痕组织颜色、质地。处理40 d，观察并计算瘢痕增生指数（SEI），分别行苏木精-伊红染色观察组织形态、Masson染色观察胶原形态，采用实时荧光定量反转录PCR法检测瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α平滑肌肌动蛋白（α-SMA）的mRNA表达，采用蛋白质印迹法检测瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平。以上实验各组样本数均为3。对数据行独立样本t检验。 结果 术后0 d，所有兔耳均形成5个新鲜创面；术后7 d，可见创面结痂；术后14 d，大部分创面已上皮化；术后21 d，可见全部创面上皮化；术后28 d，形成明显的增生性瘢痕。术后28 d，瘢痕形成率为75%（45/60）。处理40 d，张力组的兔耳瘢痕组织凸起较假张力组明显，瘢痕组织较硬，颜色较红润；张力组兔耳瘢痕的SEI为2.02±0.08，明显高于假张力组的1.70±0.08（t=5.07，P<0.01）。处理40 d，与假张力组相比，张力组兔耳瘢痕组织角质层变厚，真皮层可见大量新生的毛细血管、炎症细胞和成纤维细胞；胶原排列更加紊乱，呈结节状或旋涡状分布。处理40 d，张力组兔耳瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α-SMA的mRNA表达量分别为1.81±0.25、5.71±0.82、7.86±0.56、4.35±0.28、5.89±0.47，分别明显高于假张力组的1.00±0.08、1.00±0.12、1.00±0.13、1.00±0.14、1.00±0.14（t值分别为5.36、9.82、20.60、18.26、17.13，P值均<0.01）；张力组兔耳瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平分别为0.865±0.050、0.895±0.042、0.972±0.027、1.012±0.057、0.968±0.087，分别明显高于假张力组的0.657±0.050、0.271±0.029、0.631±0.027、0.418±0.023、0.511±0.035（t值分别为5.08、21.27、15.55、16.70、8.40，P值均<0.01）。 结论 机械张力会刺激瘢痕增生，抑制真皮层胶原纤维的正常排列，加剧胶原纤维的沉积，从而对兔耳增生性瘢痕的消退起抑制作用，其机制可能与机械张力激活TGF-β1/Smad信号通路有关。 Abstract:Objective To explore the effects of mechanical tension on the formation of hypertrophic scars in rabbit ears and transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. Methods The experimental research method was adopted. Six New Zealand white rabbits, male or female, aged 3-5 months were used and 5 full-thickness skin defect wounds were made on the ventral surface of each rabbit ear. The appearance of all rabbit ear wounds was observed on post surgery day (PSD) 0 (immediately), 7, 14, 21, and 28. On PSD 28, the scar formation rate was calculated. Three mature scars in the left ear of each rabbit were included in tension group and the arch was continuously expanded with a spiral expander. Three mature scars in the right ear of each rabbit were included in sham tension group and only the spiral expander was sutured without expansion. There were 18 scars in each group. After mechanical tension treatment (hereinafter referred to as treatment) for 40 days, the color and texture of scar tissue in the two groups were observed. On treatment day 40, the scar elevation index (SEI) was observed and calculated; the histology was observed after hematoxylin eosin staining, and the collagen morphology was observed after Masson staining; mRNA expressions of TGF-β1, Smad3, collagen Ⅰ, collagen Ⅲ, and α-smooth muscle actin (α-SMA) in scar tissue were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction; and the protein expressions of TGF-β1, collagen Ⅰ, collagen Ⅲ, and α-SMA, and phosphorylation level of Smad3 in scar tissue were detected by Western blotting. The number of samples of each group in the experiments was 3. Data were statistically analyzed with independent sample t test. Results On PSD 0, 5 fresh wounds were formed on all the rabbit ears; on PSD 7, the wounds were scabbed; on PSD 14, most of the wounds were epithelialized; on PSD 21, all the wounds were epithelialized; on PSD 28, obvious hypertrophic scars were formed. The scar formation rate was 75% (45/60) on PSD 28. On treatment day 40, the scar tissue of rabbit ears in tension group was more prominent than that in sham tension group, the scar tissue was harder and the color was more ruddy; the SEI of the scar tissue of rabbit ears in tension group (2.02±0.08) was significantly higher than 1.70±0.08 in sham tension group (t=5.07, P<0.01). On treatment day 40, compared with those in sham tension group, the stratum corneum of scar tissue became thicker, and a large number of new capillaries, inflammatory cells, and fibroblasts were observed in the dermis, and collagen was more disordered, with nodular or swirling distribution in the scar tissue of rabbit ears in tension group. On treatment day 40, the mRNA expressions of TGF-β1, Smad3, collagen Ⅰ, collagen Ⅲ, and α-SMA in the scar tissue of rabbit ears in tension group were respectively 1.81±0.25, 5.71±0.82, 7.86±0.56, 4.35±0.28, and 5.89±0.47, which were significantly higher than 1.00±0.08, 1.00±0.12, 1.00±0.13, 1.00±0.14, and 1.00±0.14 in sham tension group (with t values of 5.36, 9.82, 20.60, 18.26, and 17.13, respectively, all P<0.01); the protein expressions of TGF-β1, collagen Ⅰ, collagen Ⅲ, and α-SMA, and phosphorylation level of Smad3 in the scar tissue of rabbit ears in tension group were respectively 0.865±0.050, 0.895±0.042, 0.972±0.027, 1.012±0.057, and 0.968±0.087, which were significantly higher than 0.657±0.050, 0.271±0.029, 0.631±0.027, 0.418±0.023, and 0.511±0.035 in sham tension group (with t values of 5.08, 21.27, 15.55, 16.70, and 8.40, respectively, all P<0.01). Conclusions Mechanical tension can inhibit the regression of hypertrophic scars in rabbit ears through stimulating the hyperplasia of scars, inhibiting the normal arrangement of dermal collagen fibers, and intensifying the deposition of collagen fibers, and the mechanism may be related to the activation of TGF-β1/Smad signaling pathway by mechanical tension. -
Key words:
- Cicatrix /
- Transforming growth factor beta1 /
- Smad proteins /
- Mechanical tension
-
参考文献
(43) [1] YuanB, UptonZ, LeavesleyD, et al. Vascular and collagen target: a rational approach to hypertrophic scar management[J]. Adv Wound Care (New Rochelle), 2022,12(1):38-55. DOI: 10.1089/wound.2020.1348. [2] TuLX, LinZW, HuangQ, et al. USP15 enhances the proliferation, migration, and collagen deposition of hypertrophic scar-derived fibroblasts by deubiquitinating TGF-βR1 in vitro[J]. Plast Reconstr Surg, 2021,148(5):1040-1051. DOI: 10.1097/PRS.0000000000008488. [3] SharpPA, PanB, YakuboffKP, et al. Development of a best evidence statement for the use of pressure therapy for management of hypertrophic scarring[J]. J Burn Care Res, 2016,37(4): 255-264. DOI: 10.1097/BCR.0000000000000253. [4] GuSC, HuangX, XuXW, et al. Inhibition of CUB and sushi multiple domains 1 (CSMD1) expression by miRNA-190a-3p enhances hypertrophic scar-derived fibroblast migration in vitro[J]. BMC Genomics, 2021,22(1): 613. DOI: 10.1186/s12864-021-07920-8. [5] TejiramS, ZhangJ, TravisTE, et al. Compression therapy affects collagen type balance in hypertrophic scar[J]. J Surg Res, 2016,201(2):299-305. DOI: 10.1016/j.jss.2015.10.040. [6] ZhangZW, HuangX, YangJH, et al. Identification and functional analysis of a three-miRNA ceRNA network in hypertrophic scars[J]. J Transl Med, 2021,19(1): 451. DOI: 10.1186/s12967-021-03091-y. [7] MengXX,YuZX,XuWY, et al. Control of fibrosis and hypertrophic scar formation via glycolysis regulation with IR780[J/OL].Burns Trauma,2022,10:tkac015[2021-12-13]. https://pubmed.ncbi.nlm.nih.gov/35769829/.DOI: 10.1093/burnst/tkac015. [8] KravezE, VilligerM, BoumaB, et al. Prediction of scar size in rats six months after burns based on early post-injury polarization-sensitive optical frequency domain imaging[J]. Front Physiol, 2017,8:967. DOI: 10.3389/fphys.2017.00967. [9] RuZ,HuY,HuangSH,et al.Bioflavonoid Galangin suppresses hypertrophic scar formation by the TGF-β/Smad signaling pathway[J].Evid Based Complement Alternat Med,2021,2021:2444839.DOI: 10.1155/2021/2444839. [10] SohrabiC,GoutosI.The use of botulinum toxin in keloid scar management: a literature review[J].Scars Burn Heal,2020,6:2059513120926628.DOI: 10.1177/2059513120926628. [11] TanJL,WuJ. Current progress in understanding the molecular pathogenesis of burn scar contracture[J/OL].Burns Trauma,2017,5:14[2021-12-13]. https://pubmed.ncbi.nlm.nih.gov/28546987/.DOI: 10.1186/s41038-017-0080-1. [12] WongVW, RustadKC, AkaishiS, et al. Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling[J]. Nat Med, 2011,18(1):148-152. DOI: 10.1038/nm.2574. [13] ShaoT,TangW,LiY,et al.Research on function and mechanisms of a novel small molecule WG449E for hypertrophic scar[J].J Eur Acad Dermatol Venereol,2020,34(3):608-618.DOI: 10.1111/jdv.16028. [14] JiXJ, TangZ, ShuaiWW, et al. Endogenous peptide LYENRL prevents the activation of hypertrophic scar-derived fibroblasts by inhibiting the TGF-β1/Smad pathway[J]. Life Sci, 2019,231:116674. DOI: 10.1016/j.lfs.2019.116674. [15] 李荟元, 刘建波, 兰海. 建立增生性瘢痕动物实验模型[J]. 第四军医大学学报, 1998,19(6):655-657. [16] 李荟元,刘建波,夏炜,等.增生性瘢痕动物实验模型的建立与应用[J].中华整形外科杂志,2001,17(5):276-278,插图5-1.DOI: 10.3760/j.issn:1009-4598.2001.05.006. [17] 李希军,柳大烈,王吉慧.兔耳增生性瘢痕模型建立方法的探讨[J].中国美容医学,2006,15(5):499-500,中插1.DOI: 10.3969/j.issn.1008-6455.2006.05.006. [18] PaternoJ, VialIN, WongVW, et al. Akt-mediated mechanotransduction in murine fibroblasts during hypertrophic scar formation[J]. Wound Repair Regen, 2011,19(1):49-58. DOI: 10.1111/j.1524-475X.2010.00643.x. [19] AarabiS, BhattKA, ShiY, et al. Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis[J]. FASEB J, 2007,21(12): 3250-3261. DOI: 10.1096/fj.07-8218com. [20] 李虎,李小静,宁金龙,等.兔耳增生性瘢痕模型的建立及微血管构筑在病理性瘢痕形成和发展过程中的意义[J].中国临床康复,2006,10(4):116-118,封三.DOI: 10.3321/j.issn:1673-8225.2006.04.049. [21] 闫伦, 李辉超, 王大雷, 等. 咪喹莫特抑制兔耳瘢痕增生的机制研究[J]. 现代生物医学进展, 2013,13(7):1214-1218. DOI: 10.13241/j.cnki.pmb.2013.07.009. [22] 何香,李洁,刘佳琦,等.自噬相关基因在博来霉素诱导小鼠皮肤纤维化中的表达及作用[J].中华烧伤杂志,2020,36(5):346-356.DOI: 10.3760/cma.j.cn501120-20200210-00047. [23] ElmelegyNG,HegazyAM,SadakaMS,et al.Electrophotobiomodulation in the treatment of facial post-burn hypertrophic scars in pediatric patients[J].Ann Burns Fire Disasters,2018,31(2):127-132. [24] AtiyehB, IbrahimA. Nonsurgical management of hypertrophic scars: evidence-based therapies, standard practices, and emerging methods: an update[J]. Aesthetic Plast Surg, 2020,44(4):1345-1347. DOI: 10.1007/s00266-020-01766-3. [25] ZhangH,WangHY,WangDL,et al.Effect of pressure therapy for treatment of hypertrophic scar[J].Medicine (Baltimore),2019,98(26):e16263.DOI: 10.1097/MD.0000000000016263. [26] Amini-NikS, YousufY, JeschkeMG. Scar management in burn injuries using drug delivery and molecular signaling: Current treatments and future directions[J]. Adv Drug Deliv Rev, 2018,123: 135-154. DOI: 10.1016/j.addr.2017.07.017. [27] LiJ,LiY,WangYC,et al.Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts[J/OL].Burns Trauma,2021,9:tkab038[2021-12-13]. https://pubmed.ncbi.nlm.nih.gov/34859108/. DOI: 10.1093/burnst/tkab038. [28] DengXW, ChenQ, QiangLJ, et al. Development of a porcine full-thickness burn hypertrophic scar model and investigation of the effects of shikonin on hypertrophic scar remediation[J]. Front Pharmacol, 2018,9: 590. DOI: 10.3389/fphar.2018.00590. [29] 赵文鲁 积雪草甙对兔耳瘢痕模型TGF-β1基因表达的影响 青岛 青岛大学 2009 赵文鲁. 积雪草甙对兔耳瘢痕模型TGF-β1基因表达的影响[D]. 青岛:青岛大学, 2009.
[30] BermanB,MaderalA,RaphaelB.Keloids and hypertrophic scars: pathophysiology, classification, and treatment[J].Dermatol Surg,2017,43 Suppl 1:S3-18.DOI: 10.1097/DSS.0000000000000819. [31] HarnHI, OgawaR, HsuCK, et al. The tension biology of wound healing[J]. Exp Dermatol, 2019,28(4):464-471. DOI: 10.1111/exd.13460. [32] JimiS, SaparovA, KoizumiS, et al. A novel mouse wound model for scar tissue formation in abdominal muscle wall[J]. J Vet Med Sci, 2021,83(12): 1933-1942. DOI: 10.1292/jvms.21-0464. [33] 赵倩楠, 周粤闽, 孙朝阳. 机械张力对创伤后增生性瘢痕形成的影响研究进展[J]. 中华烧伤杂志, 2021,37(6): 586-590. DOI: 10.3760/cma.j.cn501120-20200315-00167. [34] ZhouQD, GongJX, BiJN, et al. KGF-2 regulates STAP-2-mediated signal transducer and activator of transcription 3 signaling and reduces skin scar formation[J]. J Invest Dermatol, 2022,142(7): 2003-2013.e5. DOI: 10.1016/j.jid.2021.12.018. [35] SeoCH, CuiHS, KimJB. Calpastatin-mediated inhibition of calpain ameliorates skin scar formation after burn injury[J]. Int J Mol Sci, 2021,22(11): 5771. DOI: 10.3390/ijms22115771. [36] TianS, ZhengYJ, XiaoSC, et al. Ivermectin inhibits cell proliferation and the expression levels of type I collagen, α-SMA and CCN2 in hypertrophic scar fibroblasts[J]. Mol Med Rep, 2021,24(1): 488. DOI: 10.3892/mmr.2021.12127. [37] HsiehSC, WuCC, HsuSL, et al. Gallic acid attenuates TGF-β1-stimulated collagen gel contraction via suppression of RhoA/Rho-kinase pathway in hypertrophic scar fibroblasts[J]. Life Sci, 2016,161:19-26. DOI: 10.1016/j.lfs.2016.07.011. [38] HwangboC, TaeN, LeeS, et al. Syntenin regulates TGF-β1-induced Smad activation and the epithelial-to-mesenchymal transition by inhibiting caveolin-mediated TGF-β type I receptor internalization[J]. Oncogene, 2016,35(3): 389-401. DOI: 10.1038/onc.2015.100. [39] RippaAL, KalabushevaEP, VorotelyakEA. Regeneration of dermis: scarring and cells involved[J]. Cells, 2019,8(6):607. DOI: 10.3390/cells8060607. [40] ZhengZ, ZhangXL, DangC, et al. Fibromodulin is essential for fetal-type scarless cutaneous wound healing[J]. Am J Pathol, 2016,186(11): 2824-2832. DOI: 10.1016/j.ajpath.2016.07.023. [41] HuangD,LiuYP,HuangYJ,et al.Mechanical compression upregulates MMP9 through SMAD3 but not SMAD2 modulation in hypertrophic scar fibroblasts[J].Connect Tissue Res,2014,55(5/6):391-396.DOI: 10.3109/03008207.2014.959118. [42] WangXX, GuC, ShangF, et al. Inhibitory effect of the LY2109761 on the development of human keloid fibroblasts[J]. Anal Cell Pathol (Amst), 2021,2021:8883427. DOI: 10.1155/2021/8883427. [43] 周孝亮, 刘德伍, 毛远桂, 等. 粉防己碱对兔耳瘢痕增生组织Ⅰ、Ⅲ型胶原与TGF-β1基因表达的影响[J]. 中华整形外科杂志, 2013,29(6):406-412. DOI: 10.3760/cma.j.issn.1009-4598.2013.06.002. -
2 兔耳增生性瘢痕模型的构建及经机械张力处理40 d的2组兔耳瘢痕的大体外观。2A.术后0 d（即刻），可见5个直径为1 cm的圆形新鲜创面；2B.术后7 d，可见各创面已结痂；2C.术后14 d，可见部分创面上皮化；2D.术后21 d，可见全部创面上皮化；2E.术后28 d，形成明显的增生性瘢痕，外观明显凸起、颜色红润，其增生范围不超过原创缘；2F、2G.分别为处理前假张力组、张力组瘢痕，均明显凸起；2H、2I.分别为处理40 d假张力组、张力组瘢痕，图2I瘢痕凸起更加明显且颜色红润
3 经机械张力处理40 d的2组兔耳增生性瘢痕组织形态观察。3A.假张力组可见少量新生毛细血管 HE×20；3B.图3A中方框处放大图，可见少量炎症细胞和Fb HE×100；3C.张力组可见角质层较图3A变厚，真皮层可见大量新生的毛细血管 HE×20；3D.图3C中方框处放大图，可见大量炎症细胞和Fb HE×100；3E.假张力组可见少量胶原，排列较紊乱 Masson×20；3F.图3E中方框处放大图，可见少量排列较紊乱的胶原 Masson×100；3G.张力组可见胶原排列图较图3E更加紊乱，呈结节状或旋涡状分布 Masson×20；3H.图3G中方框处放大图，可见大量纤维化和胶原，胶原排列更加紊乱 Masson×100
注：HE为苏木精-伊红，Fb为成纤维细胞，胶原Masson阳性染色为蓝色
4 蛋白质印迹法检测经机械张力处理40 d的2组兔耳增生性瘢痕组织中TGF-β1、Ⅰ型胶原、Ⅲ型胶原、α-SMA的蛋白表达和Smad3磷酸化水平
注：TGF-β1为转化生长因子β1，α-SMA为α平滑肌肌动蛋白，条带上方1、2分别指假张力组、张力组
表1 采用实时荧光定量反转录PCR法检测兔耳增生性瘢痕组织中TGF-β1、Smad3、Ⅰ型胶原、Ⅲ型胶原、α-SMA的mRNA表达的引物序列及产物大小
基因名称 引物序列（5'→3'） 产物大小（bp） GAPDH 上游：GCTTCTTCTCGTGCAGTGCTA下游：ATGACCAGCTTCCCGTTCTC 239 TGF-β1 上游：CCAAGTGGACATCAACGGGA下游：CTCTGTGGAGCTGAAGCAGT 175 Smad3 上游：AGACCTGACATCCGAGACGA下游：CATCAGGACAGCCGAACAGT 105 Ⅰ型胶原 上游：GCAAGAACGGAGATGACGGA下游：TTGGCACCATCCAAACCACT 151 Ⅲ型胶原 上游：CCGAACCGTGCCAAATATGC下游：AACAGTGCGGGGAGTAGTTG 158 α-SMA 上游：AACAATGTGCTCTCAGGGGG下游：TCTGGAGGGGCAATTATCTTGA 116 注：GAPDH为3-磷酸甘油醛脱氢酶，TGF-β1为转化生长因子β1，α-SMA为α平滑肌肌动蛋白 
下载: 导出CSV
-
图(5) / 表(1)
计量
- 文章访问数: 576
- HTML全文浏览量: 166
- PDF下载量: 48
- 被引次数: 0
