硫化氢对瘢痕疙瘩纤维化的影响及机制研究

Keloid is a pathological healing process of skin wound featuring extensive fibrosis. Transforming growth factor β1 (TGF-β1)/Smad3 signaling pathway plays a significant role. Hydrogen sulfide (H2S) as an important endogenous gasotransmitter maintains several physiological functions. Our preliminary experiments showed that H2S generation was deficiency in keloid tissue, while TGF-β1 expression was enhanced. Proliferation, TGF-β1 expression and phosphorylation of Smad3 were significantly suppressed, while CREB phosphorylation was enhanced by exogenous administration of H2S in fibroblasts of keloid. We will perform experiments on skin fibroblasts from human keloid and CSE-/- mouse with technology of gene silencing and plasmid transfection, combined with keloid model on nude mice and skin wound model on CSE-/- mouse. The experiments are performed to validate the scientific hypothesis that H2S inhibits the TGF-β1/Smad3 expression in keloid, or reduces the affinity of CBP (co-activator of Smad3) to Smad3 by enhancing phosphorylation of CREB and its competitively combination with CBP, regulates the target gene expression related with TGF-β1/Smad3 pathway and finally delays the progress of keloid fibrosis. The significance of our research is to discover effects and mechanisms of H2S on fibrosis of keloid and to provide novel ideas for prevention and treatment of keloid.

瘢痕疙瘩是皮肤创伤的病理愈合过程，以过度纤维化为特征，转化生长因子-β1（TGF-β1）/Smad3通路在其中发挥关键作用。硫化氢（H2S）是具有多种生理功能的内源性气体信号分子。预实验发现瘢痕疙瘩组织中H2S生成障碍，TGF-β1表达增加；外源性H2S抑制瘢痕疙瘩成纤维细胞增殖，减少TGF-β1表达及Smad3磷酸化，促进CREB磷酸化。本研究拟利用人瘢痕疙瘩成纤维细胞和CSE-/-小鼠皮肤成纤维细胞，联合基因沉默和质粒转染等手段，结合裸鼠瘢痕疙瘩模型和CSE-/-小鼠皮肤创伤模型，验证科学假说：H2S抑制瘢痕疙瘩中TGF-β1/Smad3表达，或通过磷酸化CREB进而增加与Smad3辅激活因子CBP的结合来降低CBP与Smad3的亲和力，调节TGF-β1/Smad3相关靶基因的表达，改善瘢痕疙瘩纤维化性状。研究有利于明确H2S对瘢痕疙瘩纤维化的调节作用及机制，为瘢痕疙瘩防治提供新思路。

瘢痕疙瘩是皮肤创伤的病理愈合过程，以过度纤维化为特征，转化生长因子-β1（TGF-β1）/Smad3通路在其中发挥关键作用。硫化氢（H2S）是具有多种生理功能的内源性气体信号分子。本研究在临床水平发现：瘢痕疙瘩患者的血浆H2S含量和瘢痕疙瘩组织H2S生成率、胱硫醚-γ-裂解酶（CSE）表达水平降低，证实瘢痕疙瘩患者全身及皮肤局部H2S生成障碍；H2S供体NaHS可抑制人瘢痕疙瘩皮肤成纤维细胞增殖和裸鼠荷人瘢痕疙瘩组织模型中瘢痕疙瘩移植物的生长，下调TGF-β1/Smad3表达，促进CREB磷酸化，增加与Smad3的辅激活因子CBP结合，降低CBP与Smad3的亲和力，进而调节TGF-β1/Smad3相关靶基因的表达，表明H2S通过调节TGF-β1-Smad3-CREB信号通路改善瘢痕疙瘩的纤维化。此外本项目还发现：CSE基因敲除（KO）小鼠皮肤创伤愈合延缓，创伤血流量减少；NaHS预处理后加速小鼠创面愈合，增加创伤血流量，提高皮肤创伤组织的血管内皮生长因子（VEGF）基因和蛋白表达，抑制皮肤创伤组织超氧阴离子产生，降低丙二醛水平，增加总抗氧化能力。NaHS降低创伤组织中发动相关蛋白1（DRP1）表达，增加视神经萎缩蛋白1（OPA1）蛋白表达。细胞实验发现：CSE KO小鼠皮肤成纤维细胞在划痕和TGF-β1刺激下迁移和增殖变慢，提示内源性缺失H2S可阻碍皮肤创伤愈合；NaHS可促进划痕后皮肤成纤维细胞迁移和增殖，减弱划痕后皮肤成纤维细胞中活性氧生成，改善线粒体膜电位，降低DRP1表达，增加OPA1的表达。这些结果表明外源性补充H2S可促进皮肤创伤愈合，其机制可能与抑制氧化应激和增加VEGF表达有关。研究揭示了H2S对瘢痕疙瘩纤维化和皮肤创伤愈合的调节作用及机制，可为瘢痕疙瘩等皮肤纤维化疾病的防治提供新思路。