Nrf2对C2C12细胞肌浆网钙调节的影响及机制
Effects of Nrf2 on Sarcoplasmic Reticulum Calcium Regulation in C2C12 Cells and Its Mechanism

摘要：目的：Nrf2是调节机体内氧化应激的重要的核转录因子，本研究探讨Nrf2在小鼠成肌细胞系C2C12细胞中[Ca2+]i调节中的作用及其机制。方法：体外培养C2C12细胞，分别采用Nrf2的激动剂t-BHQ和抑制剂Brusatol干预，将细胞分为空白对照组（Control，C组）、激动剂组（Stimulants，S组）和抑制剂组（Inhibitors，I组）。荧光比色法检测C2C12细胞ROS水平，采用Fluo-4 AM钙离子染料负载，激光扫描共聚焦显微镜检测C2C12细胞[Ca2+]i变化，Western blot方法检测C2C12细胞肌浆网钙调节蛋白RyR、FKBP12、CaM、PKA、CaMKⅡ、SERCA1和SERCA2的蛋白表达。结果：1）与C组相比，I组C2C12细胞ROS水平显著增加（P<0.01）；2）在H2O2氧化应激刺激下，与C组相比，I组C2C12细胞[Ca2+]i在185.4秒后非常显著增加（P<0.01），而S组[Ca2+]i在61.8秒后显著降低（P<0.01）；3）与C组相比，I组Nrf2蛋白表达显著降低（P<0.05），S组无显著性差异；4）与C组相比，肌浆网钙调节蛋白RyR、FKBP12、PKA显著增加（P<0.05），SERCA1和SERCA2蛋白显著降低（P<0.05），而Nrf2激动剂组蛋白表达无显著变化。结论：1）Nrf2在H2O2介导的C2C12细胞[Ca2+]i异常增加过程中起保护作用；2）Nrf2被抑制后可引起C2C12细胞RyR、PKA蛋白表达增加，从而增强肌浆网钙释放功能；同时引起C2C12细胞SERCA蛋白表达减少，使肌浆网钙回收功能减弱。
Abstract: Objective: As an important nuclear transcription factor in vivo, Nrf2 is a key regulator of oxidative stress. The purpose of this study was to investigate the role and mechanism of Nrf2 in regulating line intracellular free Ca2+ ([Ca2+]i) in C2C12 cell of mouse’ skeletal muscle. Methods: C2C12 cells were cultivated in vitro, and intervened by Nrf2 agonist t-BHQ and inhibitor Brusatol, and divided into blank control group (C), agonist group (S) and inhibitor group (I). The level of ROS in C2C12 cells was detected by fluorescence colorimetric assay. The change of [Ca2+]i in C2C12 cells was measured by laser scanning confocal microscopy after Fluo-4 AM loaded on C2C12 cells. The expression of Nrf2, RyR1, SERCA1, SERCA2, FKBP12, CaM, CaMKⅡ and PKA protein in C2C12 cells were detected by Western Blot. Results: 1) Compared with C group, the ROS level of C2C12 cells in I group was significantly increased. 2) Compared with C group, the [Ca2+]i of the C2C12 cells in I group was significantly increased after 185.4 seconds under the stimulation of H2O2 oxidative stress, while the [Ca2+]i of I group was significantly decreased after 61.8 seconds. 3) Compared with C group, the protein expression of Nrf2 was significantly lower in I group (P <0.05), but no change in S group. 4) Compared with C group, the expressions of RyR, FKBP12 and PKA of I group were significantly increased (P <0.05), SERCA1 and SERCA2 protein were significantly decreased (P <0.05), while the expressions of S group did not change significantly. Conclusions: 1) Nrf2 plays a protective role in the process of oxidative stress-induced skeletal muscle [Ca2+]i increase. 2) Nrf2 inhibition can increase the expression of RyR and PKA protein in C2C12 cells, enhance the release function of sarcoplasmic reticulum calcium, decrease the expression of SERCA protein,