Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing 100029, China.
Department of Stomatology, China-Japan Friendship Hospital, Beijing 100029, China.
Life Sci. 2019 May 15;225:107-116. doi: 10.1016/j.lfs.2019.03.068. Epub 2019 Mar 27.
To explore the effect and mechanism of 1, 25-(OH)D on Schwann cell apoptosis induced by advanced glycation end products.
Schwann cells, isolated from rodent sciatic nerve were incubated with AGE-modified bovine serum albumin(AGE) to mimic diabetic conditions and 1,25-(OH)D was used as protector. Cell apoptosis was detected by PI/Annexin-V staining, caspase 3 activity assay and western blotting for caspase 3 and PARP. The activation of protein kinase A (PKA) and nuclear factor kappa-B (NF-κB) was evaluated by western blot. Immunofluorescent staining was used for intercellular location of NF-κB. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay.
Schwann cell apoptosis accelerated after incubating with AGE. However, if combining 1,25-(OH)D with AGE, apoptosis decreased significantly. 1,25-(OH)D enhanced PKA activity, but inhibited AGE-induced nuclear translocation of NF-κB. Furthermore, PKA activator (8-bromoadenoside cyclic adenoside monophosphate, 8-Br-cAMP) or NF-κB inhibitor (caffeic acid phenethyl ester, CAPE) could reduce the apoptosis, decreased cleaved caspase 3 and cleaved PARP, suggesting the involvement of PKA and NF-κB pathways in the protection of 1,25-(OH)D on Schwann cells. Moreover, 8-Br-cAMP and CAPE could inhibit AGE-induced secretion of interleukin(IL)-1β, prostaglandin E2(PEG2) and cyclooxygenase 2(COX2). Interestingly, 8-Br-cAMP decreased phospho-NF-κB and inhibited nucleus translocation of NF-κB. It hinted at the regulation of PKA to NF-κB. Finally, a pre-treatment of H-89 (an inhibitor of PKA) could block the protection of 1,25-(OH)D on cell apoptosis. In conclusion, 1,25-(OH)D could protect Schwann cell against AGE-induced apoptosis through PKA/NF-κB pathway.
These findings provide experimental rationales for using vitamin D for diabetic neuropathy.
探讨 1,25-(OH)D 对糖基化终产物诱导施万细胞凋亡的作用及机制。
用 AGE 修饰牛血清白蛋白（AGE）孵育分离的啮齿动物坐骨神经施万细胞，模拟糖尿病条件，并使用 1,25-(OH)D 作为保护剂。通过碘化丙啶/膜联蛋白-V 染色、半胱天冬酶 3 活性测定和半胱天冬酶 3 和多聚（ADP-核糖）聚合酶（PARP）的 Western blot 检测细胞凋亡。通过 Western blot 评估蛋白激酶 A（PKA）和核因子 kappa-B（NF-κB）的激活。免疫荧光染色用于检测 NF-κB 的细胞内定位。酶联免疫吸附试验用于评估细胞因子分泌。
AGE 孵育后施万细胞凋亡加速，但如果将 1,25-(OH)D 与 AGE 结合，凋亡明显减少。1,25-(OH)D 增强了 PKA 活性，但抑制了 AGE 诱导的 NF-κB 核转位。此外，PKA 激活剂（8-溴环腺苷单磷酸，8-Br-cAMP）或 NF-κB 抑制剂（咖啡酸苯乙酯，CAPE）可减少凋亡，减少裂解的半胱天冬酶 3 和裂解的 PARP，表明 PKA 和 NF-κB 途径参与了 1,25-(OH)D 对施万细胞的保护作用。此外，8-Br-cAMP 和 CAPE 可抑制 AGE 诱导的白细胞介素（IL）-1β、前列腺素 E2（PEG2）和环氧化酶 2（COX2）的分泌。有趣的是，8-Br-cAMP 降低了磷酸化 NF-κB 并抑制了 NF-κB 的核转位。这暗示了 PKA 对 NF-κB 的调节。最后，用 H-89（PKA 抑制剂）预处理可阻断 1,25-(OH)D 对细胞凋亡的保护作用。
1,25-(OH)D 通过 PKA/NF-κB 途径保护施万细胞免受 AGE 诱导的凋亡。
这些发现为使用维生素 D 治疗糖尿病性神经病提供了实验依据。
