燕麦β-葡聚糖作用于Dectin-1调控表皮屏障损伤修复的机制研究

The epidermal barrier acts as a line of defense against external agents as well as helps maintaining body homeostasis. The calcium concentration gradient across the epidermal barrier is closely related to the proliferation and differentiation of keratinocytes (KC), and the regulation of these two processes is the key to the endogenous repair of epidermal barrier disruption. Our preliminary study has shown that oat β-glucan (OG) could promote the repair of epidermal barrier disruption in mice. Subsequent mechanistic study demonstrated that OG could promote KC differentiation under low calcium condition by up-regulating the expression of calcium-sensitive receptor (CaSR), which could then lead to the activation of the Catenin/PLCγ1 pathway. Further study has shown that OG could directly bind to Dectin-1, resulting in the activation of the ERK and p38 pathways, eventually upregulating CaSR expression, suggesting that oat β-glucan might upregulate CaSR expression via Dectin-1. Therefore, the proposed study plans to further examine the interaction between OG with Dectin-1 and the effect that this might have on the downstream target genes of Dectin-1. This work will reveal the molecular mechanism of OG in the repair of epidermal barrier and provide a basis for the development of OG into an epidermal barrier repair agent. In addition, it can also establish Dectin-1 as a new target for therapy that aims at the process of repairing disruption epidermal barrier. This would enable the screening of more effective polysaccharides that could potentially form a new strategy for repairing disruption epidermal barrier.

表皮屏障具有阻止外界侵害，维持机体稳态的重要功能。表皮钙离子浓度梯度与角质形成细胞（KC）增殖与分化密切相关，而调控KC的增殖与分化是表皮屏障损伤内源性修复的关键。前期的研究发现燕麦β-葡聚糖（OG）具有促进小鼠表皮屏障损伤修复的作用。机制研究表明，OG通过上调钙敏感受体（CaSR）的表达、激活Catenin/PLCγ1通路，使KC在低钙条件下正常分化。同时发现OG能够与Dectin-1直接结合，活化ERK、p38通路，上调CaSR的表达，提示燕麦β-葡聚糖可能通过结合Dectin-1发挥促进表皮屏障损伤修复的作用。因此，本项目拟围绕OG与Dectin-1的互作及其下游通路展开研究，一方面将揭示OG在表皮屏障修复中的分子作用机制，为其发展为表皮屏障修复剂奠定基础；另一方面将明确Dectin-1为调控表皮屏障内源性修复的新靶点，并基于此靶点筛选更多活性多糖，为表皮屏障内源性修复提供新的策略。

本项目主要研究目标为阐明燕麦β-葡聚糖通过作用于Dectin-1，活化ERK、p38从而上调CaSR的这一未知通路，增加角质形成细胞对Ca2+敏感性，从而调控低钙条件下角质形成细胞的分化并促进表皮屏障损伤修复的内源性修复机制。以Dectin-1为靶标、以Dectin-1介导的信号通路为依据，筛选更多有效的能够促进表皮屏障内源性修复的活性多糖，为表皮屏障损伤修复提供新的策略。迄今，整个项目按照计划实施并取得了如下的预期成果：在细胞水平上我们首先（1）利用分子互作实验验证了Dectin-1与燕麦β-葡聚糖的互作，并在明确了Dectin-1与燕麦β-葡聚糖互作的基础上，进一步（2）检测燕麦β-葡聚糖通过作用Dectin-1活化 ERK、p38通路促进 CaSR的表达以及 CaSR介导的 Catenin/PLCγ1通路的影响，进而促进角质形成细胞分化的作用而阐明了燕麦β-葡聚糖促进角质形成细胞低钙条件下的表皮屏障损伤修复的内源性修复机制；并将该研究结果应用（3）在3D表皮模型上检验了燕麦β-葡聚糖对表皮模型屏障损伤的修复作用。为了印证体外研究结果的准确性，在动物水平上我们利用基因敲除鼠（Dectin-1-/-）为实验对象施加一定剂量的燕麦β-葡聚糖,首先（4）确定了在动物体内Dectin-1在皮肤屏障损伤中的修复作用；同时（5）研究 Dectin-1 缺失是否消除燕麦β-葡聚糖屏障损伤的修复作用；进一步我们证明（6）燕麦β-葡聚糖的应用不能增加Dectin-1 缺失小鼠表皮中CaSR的表达；（7）燕麦β-葡聚糖的应用不能激活Dectin-1 缺失小鼠表皮中相关信号通路；以上结果表明，体内研究结果与体外研究结果相一致。为了发挥新靶点Dectin-1的作用，我们（8）以Dectin-1为靶标筛选活性多糖。在裙带菜岩藻多糖、银耳多糖、甘露聚糖、灵芝多糖等候选多糖中，我们筛选出裙带菜盐藻多糖同样具有促进表皮屏障损伤的修复的生物学功能。综上，本项目的研究目标全部完成，本项目的研究结果为表皮屏障损伤的修复及皮肤修复剂的研发奠定了坚实的基础。