角质形成细胞源性Sema3A合成及分泌异常介导糖尿病小纤维神经病变的作用及机制研究

Small fibers are the earliest and most prone to damage in diabetic patients, but the relationship between secretion of keratinocytes and diabetic small-fiber neuropathy is still unclear.Our recent study indicated that Sema3A expression in epidermal keratinocytes is higher in patients with diabetes mellitus, while decreased nerve fiber densities are found in the epidermis. The effect of keratinocyte-conditioned medium on the growth cone of dorsal root ganglion neurons was reversed by anti-Sema3A antibody. Based on this knowledge, we speculate that increased Sema3A expression in diabetic keratinocytes might be involved in the pathogenesis of diabetic small-fiber neuropathy through actting on epidermal nerve fiber. In this study, we aim to demonstrate high glucose upregulated Sema3A synthesis via mTORC1 pathway through the analysis of 5’ TOP sequences of Sema3A. On the basis of providing evidence for the interaction between Sema3A and Rictor (an important component of the mTORC2 complex), this project describes the mechanism of increased secretion of Sema3A promoted by its synthesis through inhibition of the mTORC2 activity. From the perspective of cellular interactions and with mTORC1/mTORC2 as the core, the project shed new light on the mechanism that increased Sema3A secreted and produced by keratinocytes contributes to diabetic small-fiber neuropathy.

小纤维神经病变是糖尿病最早最易出现的周围神经病变。角质形成细胞可分泌多种活性物质调控神经纤维生长，但其分泌功能异常与糖尿病小纤维神经病变的关系尚不清楚。我们发现糖尿病患者角质形成细胞源性 Sema3A异常增加，而表皮神经纤维密度却明显下降；抗 Sema3A抗体能逆转高糖微环境下角质形成细胞对神经纤维的损伤。据此提出假说：角质形成细胞源性Sema3A通过作用于周围的神经纤维，介导糖尿病小纤维神经病变的发生。本项目拟在分析Sema3A含 5’ TOP特殊结构的基础上，阐述高糖通过mTORC1促Sema3A合成的机制；在确认Sema3A与Rictor相互作用的基础上，阐述异常合成的Sema3A抑制mTORC2活性进而促Sema3A自身分泌的调控机制。我们从细胞相互作用的角度以mTORC1/ mTORC2为核心，力求揭示角质形成细胞源性Sema3A合成及分泌异常介导糖尿病小纤维神经病变的新机制。

小纤维神经病变是糖尿病最早最易出现的周围神经病变。角质形成细胞分泌功能异常与糖尿病小纤维神经病变的关系尚未见报道。我们推测：异常增加的角质形成细胞源性Sema3A通过作用于表皮神经纤维，介导糖尿病小纤维神经病变发生。我们实验发现：糖尿病小纤维神经病变患者皮肤组织中Sema3A表达上调，mTOR信号通路被过度激活；高糖、重组人Sema3A 干预HaCaT细胞可以使mTOR信号通路上调，及Sema3A表达的增加, 而雷帕霉素可以阻止其增加；mTOR信号通路可以调控角质形成细胞Sema3A合成及分泌；STZ诱导糖尿病大鼠成模8周出现机械痛及热痛阈值下降，同时伴有皮肤活检评估表皮神经纤维密度减少，提示存在糖尿病小纤维神经病变；在糖尿病小纤维神经病变大鼠足底皮肤组织中Sema3A表达上调，mTOR/p70 S6K及mTOR/4E-BP1信号通路被过度激活；雷帕霉素可以降低上调的Sema3A，增加皮肤活检评估表皮神经纤维密度，改善糖尿病大鼠痛觉过敏，逆转糖尿病小纤维神经病变。