Neuritin通过NMDA受体促进骨髓间充质干细胞迁移治疗糖尿病周围神经病变的作用机制

The migration ability of bone marrow mesenchymal stem cells in diabetes reduced by the damage of diabetic bone marrow neuropathy is the key factors of diabetic peripheral neuropathy deterioration. Our previous studies showed that bone marrow-specific neurotrophic factor neuritin in type 2 diabetic (db/db) mice can improve peripheral neuropathy and bone marrow glutamatergic nerve, but its mechanism is unclear. Neuritin can repair damaged nerve and enhance the migration ability of bone marrow mesenchymal stem cells. Our recent experiments show that glutamate promotes the migration of bone marrow mesenchymal stem cells via N-methyl-D-aspartate receptor (NMDA receptor, a glutamate receptor and ion channel protein) to enhance SDF-1α/CXCR4 axis and PI3K/Akt signaling pathway. We speculate that neuritin repairs bone marrow glutamatergic nerve to modulate glutamate release, via NMDA receptor and SDF-1α/CXCR4 axis to promote bone marrow mesenchymal stem cell migration is the improvement mechanism of neuritin on diabetic peripheral neuropathy. This study was designed to observe the effects of bone marrow-specific neuritin overexpression and neuritin knockout, and GFP transgenic mice parabiosis on diabetic peripheral neuropathy, bone marrow glutamatergic nerve and migration of bone marrow mesenchymal stem cells in vivo. In vitro, bone marrow mesenchymal stem cells were co-cultured with neurons, the regulative effects of glutamate, NMDA receptor, SDF-1α/CXCR4 axis, and PI3K/Akt signaling pathway on the migration of bone marrow mesenchymal stem cells and the intervention of neuritin were measured. This study will clarify the effect of neuritin on the migration of bone marrow mesenchymal stem cells via repairing bone marrow glutamatergic nerve, glutamate release, NMDA receptor, the SDF-1α/CXCR4 axis, and PI3K/Akt signaling pathway to improve diabetic peripheral neuropathy, which will provide a theoretical basis for self-repairing strategy for diabetic peripheral neuropathy.

糖尿病骨髓神经病变导致骨髓间充质干细胞(BMSC)迁移能力下降是糖尿病周围神经病变(DPN)恶化的关键因素。我们前期研究证明2型糖尿病(db/db)小鼠骨髓特异性高表达神经营养因子neuritin可改善DPN和骨髓谷氨酸能神经，但其机制不清。鉴于neuritin可修复神经且增强BMSC迁移，预实验发现谷氨酸通过其离子型NMDA受体调控SDF-1α/CXCR4轴和PI3K/Akt信号通路促进BMSC迁移。推测neuritin修复骨髓谷氨酸能神经以调控谷氨酸释放，通过NMDA受体、SDF-1α/CXCR4轴促进BMSC迁移是改善DPN的作用机制。为此该研究观察骨髓特异性高表达、敲除neuritin和连体实验对小鼠DPN、骨髓谷氨酸能神经和BMSC迁移的影响；体外用BMSC与神经细胞共培养，探讨NMDA受体调控BMSC迁移的机制。该研究可进一步阐明BMSC迁移的机制，可望为治疗DPN提供新方法。

糖尿病骨髓神经病变导致骨髓间充质干细胞迁移能力下降是糖尿病周围神经病变恶化的关键因素。本项目采用CMV-loxP-STOP-loxP系统高表达neuritin转基因小鼠（由赛业广州生物科技有限公司提供）与Lyz-cre小鼠（骨髓特异性表达Cre酶）杂交，获得骨髓特异性高表达neuritin转基因小鼠。再与从美国Jackson实验室引进的可育的db/m（杂合子）小鼠杂交，获得骨髓特异性高表达neuritin的2型糖尿病(db/db)小鼠。观察骨髓特异性高表达neuritin对2型糖尿病周围神经病变小鼠的骨髓病理结构改变、骨髓神经末梢、骨髓脂肪积聚和骨髓间充质干细胞迁移能力等的影响。在原代培养的骨髓间充质干细胞中，使用拮抗剂抑制SDF-1α/CXCR4-PI3K/Akt信号通路。Real-time PCR、免疫荧光和western blot技术检测证实，本项目成功构建了骨髓特异性高表达neuritin小鼠。与基础饲料喂养的db/m小鼠比较，骨髓高表达neuritin可降低2型糖尿病血糖，改善糖尿病周围神经病变、坐骨神经超微结构和表皮神经纤维密度，促进雪旺细胞增殖和坐骨神经再髓鞘化；改善骨髓脂肪积聚、脂肪细胞数量、血清和骨髓中谷氨酸含量、骨髓血管和神经密度；降低血清和骨髓中的谷氨酸含量；恢复骨髓、血液和坐骨神经之间的梯度SDF-1α含量，促进受损的糖尿病骨髓间充质干细胞迁移。细胞实验证实，neuritin通过SDF-1α/CXCR4-PI3K/Akt信号通路促进骨髓间充质干细胞迁移。可见，骨髓高表达neuritin可以改善糖尿病骨髓中的神经病变，从而提高骨髓间充质干细胞的迁移能力，进而修复糖尿病周围神经病变，这至少部分是通过SDF-1α/CXCR4轴激活PI3K/Akt通路实现的。该研究可进一步阐明骨髓间充质干细胞迁移的机制，可望为治疗糖尿病周围神经病变提供新方法。