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已知神经生长因子(nerve growth factor,NGF)对糖尿病外周神经病变(diabetic peripheral neuropathy,DPN)患者具有良好的治疗效果,内质网应激(endoplasmic reticulum stress,ERS)在调控DPN的发生发展方面扮演着重要的角色。然而,二者间的关系未知。本研究以30 mmol/L的高糖处理RSC 96大鼠雪旺细胞(RSC96 Schwann cells,SCs),模拟DPN患者外周神经的内环境。研究结果证实,在高糖条件下,NGF通过抑制SCs内ERS的过度激活进而保护SCs的存活且这种抑制作用依靠P13K/AKT/GSK-3β和ERK1/2两条信号通路的调节实现。MTT检测细胞的存活率,结果显示高糖环境培养的SCs在24 h发生最佳程度的抑制,且此时间点加入的NGF浓度为50 ng/m L时,其存活率最高。Western印迹检测ERS和相关蛋白质的表达揭示,高糖能够过度激活SCs内ERS蛋白(GRP-78、ATF-6、ATF-4、XBP-1、CHOP),给予50 ng/m L的NGF处理后,ERS蛋白的表达水平大幅下调并接近正常,且此时p-AKT、p-ERK1/2、p-GSK3β蛋白的检测水平明显升高。流式细胞术检测细胞凋亡显示,NGF能显著抑制SCs的早期凋亡。上述结果证明,高糖诱导SCs的凋亡增加是由于自身的ERS被过度激活,NGF可通过调节P13K/AKT/GSK-3β和ERK1/2两条信号通路来抑制ERS的过度激活,达到保护SCs存活的目的。此机制为临床治疗DPN提供新的理论基础。
It is known that nerve growth factor (NGF) has a good therapeutic effect on patients with diabetic peripheral neuropathy (DPN). Endoplasmic reticulum stress (ERS) plays an important role in regulating the occurrence and development of DPN play an important role. However, the relationship between the two is unknown. In this study, RSC 96 Schwann cells (SCs) were treated with 30 mmol / L high glucose to simulate the internal environment of peripheral nerve in DPN patients. The results confirm that under high glucose conditions, NGF protects the survival of SCs by inhibiting ERS over-activation in SCs, and this inhibition relies on the regulation of two signaling pathways, P13K / AKT / GSK-3β and ERK1 / 2. MTT assay of cell viability, the results showed that cultured in high glucose environment SCs occurred optimal degree of inhibition at 24 h, and the time of adding NGF concentration of 50 ng / m L, the highest survival rate. Detection of ERS and related proteins by Western blotting revealed that high glucose could overexpress ERS proteins (GRP-78, ATF-6, ATF-4, XBP-1, CHOP) in SCs after treatment with 50 ng / , The expression of ERS protein was significantly down-regulated and close to normal, and the levels of p-AKT, p-ERK1 / 2 and p-GSK3β were significantly increased at this time. Flow cytometry showed that NGF could significantly inhibit the early apoptosis of SCs. The above results demonstrate that the increased apoptosis of SCs induced by high glucose is due to the over-activation of ERS itself. NGF can inhibit the over-activation of ERS by regulating the two signaling pathways of P13K / AKT / GSK-3β and ERK1 / 2 to protect SCs The purpose of survival. This mechanism provides a new theoretical basis for the clinical treatment of DPN.