PI3K/AKT/mTOR与STAT3通路在IL-10拮抗瘢痕纤维化新功能中的作用及机制研究

Hypertrophic scar (HS), as a serious skin fibrotic disease, has always been a major problem for clinical treatment. The current research about the biological functions of interleukin 10 (IL-10) mainly focus on immunosuppressive and anti-inflammatory, and have elucidated the important regulation mechanisms in inflammatory and immune response. Reports and our research showed that IL-10 plays a pivotal role in wound healing, and has been identified as a prospective scar-improving therapeutic cytokine. These data demonstrate that IL-10 has a novel biological role in anti-fibrosis in wound healing. However, relatively little is known about the molecular mechanism on IL-10-mediated anti-fibrosis and scar-improvement effects. In accordance with extensive publication, our result revealed that the phosphorylation levels of AKT and STAT3 in HS tissue and HS fibroblasts (HSFs) have significant differences with that in corresponding normal skin (NS) tissue and NS fibroblasts (NSFs). And we founded that IL-10 could dose-dependently regulate the phosphorylation levels of AKT and STAT3 in HSFs. It is widely believed that PI3K/AKT/mTOR pathway is involved in the regulation of cell proliferation, differentiation, apoptosis and other physiological activities, and IL-10 plays biological function through STAT3 pathway via specific receptor (IL-10R) on cell surface. For these reasons, HSFs with IL-10 over-expression (HSFs-IL10+) and IL-10 silence (HSFs-IL10-) will be used in this study to confirm the anti-fibrosis role of IL-10; to explore the regulatory effect of IL-10 on PI3K/AKT/mTOR and STAT3 pathways; and further to validate the anti-fibrosis role of IL-10 in both cellular and rabbit ear HS model by molecular inhibitors/siRNAs. This study will elucidate the novel molecular mechanism of IL-10 in protection against scar fibrosis, and further lay foundation for the prevention, treatment and drug screening of HS.

增生性瘢痕（HS）作为临床治疗的难题，是研究的重点和难点。目前对IL-10功能的研究主要集中在抗炎反应与免疫抑制两个方面，但最新文献及我们的研究表明：IL-10能改善瘢痕外观，拮抗瘢痕的形成，赋予了IL-10抗瘢痕纤维化的新功能。我们深入的研究表明：HS及HSFs的pAKT和pSTAT3较NS及NSFs存在明显差异；IL-10呈量─效关系调控HSFs中pAKT及pSTAT3；且AKT与STAT3交互调控。因此，有必要深入阐明IL-10拮抗瘢痕纤维化的作用机制。鉴于此，本研究拟用IL-10过表达及沉默的HSFs，证实IL-10拮抗瘢痕纤维化之功能；分析HSFs的pAKT及pSTAT3，探讨IL-10对AKT/mTOR与STAT3通路关键分子的调控作用；并用通路抑制剂/siRNA对细胞及动物验证。该课题的实施有望阐明IL-10拮抗瘢痕纤维化的作用机制，为HS的预防、治疗及药物筛选奠定基础。

增生性瘢痕（HS）是一种严重的纤维化皮肤病，一直是临床治疗的难题，也是研究的重点和难点。IL-10是一种多功能细胞因子，其功能的研究目前主要集中在抗炎反应与免疫抑制两个方面，但最新文献及我们的研究表明IL-10能改善瘢痕外观，拮抗瘢痕的形成，赋予了IL-10抗瘢痕纤维化的新功能。我们深入的研究表明：HS及HSFs的pAKT和pSTAT3较NS及NSFs存在明显差异；IL-10呈量─效关系调控HSFs中pAKT及pSTAT3；且AKT与STAT3交互调控。因此，有必要深入阐明IL-10拮抗瘢痕纤维化的作用机制。鉴于此，本研究用过表达及沉默的IL-10载体感染HSFs，证实IL-10拮抗瘢痕纤维化之功能；通过免疫组化、免疫荧光和qRT-PCR等技术，分析评估HS和HSF中IL-10受体的表达；用IL-10受体的功能阻断抗体（IL-10RB）及PI3K抑制剂（LY294002）等分析通路关键分子及纤维化相关因子的变化；并用3D培养细胞及动物瘢痕模型验证。研究结果表明LY294002可以阻断AKT和STAT磷酸化，上调IL-10-处理细胞中Col I Col III和α-SMA的表达水平；IL-10RB则能够降低STAT3及AKT磷酸化并阻断IL-10介导的HSFs细胞的纤维化。单独或联合使用抑制剂IL10RB、LY294002、rapamycin和cryptotanshinone，不但能够阻断pAKT、pmTOR和pSTAT3等关键分子的磷酸化，而且还能够影响IL-10抗纤维化功能。这些结果表明IL-10抑制纤维化不仅经由IL-10R-STAT3及AKT-mTOR途径，且两条通路存在交叉对话，提出并证实了AKT-STAT3-mTOR环路在IL-10拮抗瘢痕纤维化中发挥重要作用。体外3D成纤维细胞培养结果表明IL-10明显改善FPCL的收缩及其胶原网格结构。动物模型研究结果证实IL-10可改善小鼠切口愈合及兔耳瘢痕模型瘢痕的外观，改善真皮组织胶原的结构及排列，减少瘢痕的形成。该课题初步阐明了IL-10拮抗瘢痕纤维化的分子机制，为HS的预防、治疗及药物筛选奠定了基础，提供了理论指导。