人脂肪间充质干细胞来源的外泌体通过 miR-192-5p/IL-17RA/Smad 轴减轻增生性瘢痕纤维化。

BACKGROUND: Hypertrophic scar (HS) is a fibro-proliferative disorder of dermis after burn or trauma and usually leads to esthetic disfiguration and functionary impairment for patients. Emerging evidences demonstrated ADSC-Exo could alleviate the visceral fibrosis, but little attention had been paid to its role in skin fibrosis. In the study, we would explore the effect of ADSC-Exo on HS and investigated the exact mechanism underlying the properties. METHODS: ADSC-Exo were isolated, identified, and internalized by HS-derived fibroblasts (HSFs). The effect of ADSC-Exo on the proliferation and migration of HSFs were detected by flow cytometry and Ki67 immunofluorescence staining, or scratch and trans-wells assays, respectively. RT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry staining were used to evaluate the expression of IL-17RA, Col1, Col3, α-SMA, SIP1, and p-Smad2/p-Smad3 in HSFs stimulated with ADSC-Exo, miR-192-5p mimics, or inhibitors, IL-17RA siRNA and their negative controls. Digital morphology, H&E, Masson's trichrome staining, and immunohistochemistry staining were performed to measure the effect of ADSC-Exo and Lv-IL-17RA shRNA on excisional wound of BALB/c mice. RESULTS: The verified ADSC-Exo effectively inhibited the proliferation and migration of HSFs, decreased the expression of Col1, Col3, α-SMA, IL-17RA, and p-Smad2/p-Smad3 and increased the levels of SIP1 in HSFs. Besides, the mice in ADSC-Exo-treated group demonstrated faster wound healing and less collagen deposition. Furthermore, miR-192-5p was highly expressed in ADSC-Exo and ADSC-Exosomal miR-192-5p ameliorated hypertrophic scar fibrosis. Meanwhile, miR-192-5p targeted the expression of IL-17RA to decrease the pro-fibrotic proteins levels. Moreover, IL-17RA was overexpressed in HS and HSFs, and knockdown IL-17RA alleviated the expression of Col1, Col3, α-SMA, and p-Smad2/p-Smad3 and increased the expression of SIP1 in HSFs. Most importantly, IL-17RA silence also facilitated wound healing, attenuated collagen production, and modulated Smad pathway in HSFs. CONCLUSIONS: This study illustrated ADSC-Exo attenuated the deposition of collagen, the trans-differentiation of fibroblasts-to-myofibroblasts, and the formation of hypertrophic scar by in vitro and in vivo experiments. ADSC-Exosomal miR-192-5p targeted IL-17RA to regulate Smad pathway in hypertrophic scar fibrosis. ADSC-Exo could be a promising therapeutic strategy for clinical treatment of hypertrophic scar and the anti-fibrotic properties could be achieved by miR-192-5p/IL-17RA/Smad axis.

背景：增生性瘢痕（HS）是烧伤或创伤后真皮的纤维增生性疾病，通常会导致患者出现美容畸形和功能障碍。越来越多的证据表明 ADSC-Exo 可以减轻内脏纤维化，但很少有人关注其在皮肤纤维化中的作用。在这项研究中，我们将探讨 ADSC-Exo 对 HS 的影响，并研究其特性的具体机制。

方法：ADSC-Exo 被 HS 衍生的成纤维细胞（HSFs）分离、鉴定并内化。通过流式细胞术和 Ki67 免疫荧光染色或划痕和 Transwell 测定分别检测 ADSC-Exo 对 HSFs 增殖和迁移的影响。通过 RT-PCR、免疫印迹、免疫荧光和免疫组织化学染色，评估 ADSC-Exo 刺激的 HSFs 中 IL-17RA、Col1、Col3、α-SMA、SIP1 和 p-Smad2/p-Smad3 的表达。通过数字形态学、H&E、Masson 三色染色和免疫组织化学染色，测量 ADSC-Exo 和 Lv-IL-17RA shRNA 对 BALB/c 小鼠切创的影响。

结果：经过验证的 ADSC-Exo 可有效抑制 HSFs 的增殖和迁移，降低 Col1、Col3、α-SMA、IL-17RA 和 p-Smad2/p-Smad3 的表达，并增加 HSFs 中 SIP1 的水平。此外，ADSC-Exo 治疗组的小鼠伤口愈合更快，胶原沉积更少。此外，miR-192-5p 在 ADSC-Exo 中高表达，ADSC-Exosomal miR-192-5p 可改善增生性瘢痕纤维化。同时，miR-192-5p 靶向 IL-17RA 表达，降低促纤维化蛋白水平。此外，IL-17RA 在 HS 和 HSFs 中过度表达，沉默 IL-17RA 可降低 HSFs 中 Col1、Col3、α-SMA 和 p-Smad2/p-Smad3 的表达，增加 SIP1 的表达。最重要的是，IL-17RA 沉默也促进了伤口愈合，减轻了胶原产生，并调节了 HSFs 中的 Smad 通路。

结论：本研究通过体外和体内实验表明，ADSC-Exo 通过减少胶原沉积、成纤维细胞向肌成纤维细胞的转分化以及增生性瘢痕的形成，减轻了增生性瘢痕的形成。ADSC-Exosomal miR-192-5p 通过靶向 IL-17RA 调节增生性瘢痕纤维化中的 Smad 通路。ADSC-Exo 可能成为治疗增生性瘢痕的一种有前途的临床治疗策略，其抗纤维化特性可通过 miR-192-5p/IL-17RA/Smad 轴实现。