降钙素基因相关肽对血清饥饿作用下MC3T3-E1成骨细胞凋亡和自噬的影响

Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation

目的研究血清饥饿条件下降钙素基因相关肽(CGRP)对小鼠MC3T3-E1成骨细胞凋亡、自噬的影响以及二者之间的关系,以进一步明确CGRP对成骨细胞的保护机制。方法体外培养小鼠MC3T3-E1成骨细胞。采用流式细胞术和蛋白质印迹检测正常血清、无血清(血清饥饿)、3-MA预处理+血清饥饿培养的成骨细胞的凋亡和微管相关蛋白1轻链3(LC3)蛋白的表达。采用蛋白质印迹检测正常血清、血清饥饿、血清饥饿+不同浓度(10^(-10)、10^(-9)、10^(-8)、10^(-7) mol·L^(-1))CGRP培养的成骨细胞的LC3和P62蛋白表达;采用蛋白质印迹检测正常血清、血清饥饿、血清饥饿+10^(-8) mol·L^(-1) CGRP培养不同时间(2、6、12、24、48、72 h)的成骨细胞的LC3蛋白表达,流式细胞数检测细胞凋亡,MDC染色检测细胞自噬泡。采用流式细胞术检测正常血清、血清饥饿、血清饥饿+10^(-8) mol·L^(-1) CGRP、3-MA预处理+血清饥饿、3-MA预处理+血清饥饿+10^(-8) mol·L^(-1) CGRP培养24 h的成骨细胞的凋亡。结果血清饥饿培养时成骨细胞的LC3Ⅱ蛋白表达及细胞凋亡较正常血清时增加,3-MA预处理+血清饥饿培养时成骨细胞的凋亡较血清饥饿时增加(P<0.01)。与正常血清相比,血清饥饿、血清饥饿+CGRP培养时LC3Ⅱ蛋白表达增加,P62蛋白表达降低,以血清饥饿+10^(-8) mol·L^(-1) CGRP培养24 h时LC3Ⅱ/Ⅰ比值最高;血清饥饿+10^(-8) mol·L^(-1) CGRP培养能抑制成骨细胞的凋亡,促进自噬泡的合成。3-MA预处理后MC3T3-E1成骨细胞凋亡增加,CGRP部分逆转3-MA预处理所增加的成骨细胞凋亡。结论 CGRP能够增强血清饥饿下MC3T3-E1成骨细胞的自噬活性,并可能通过促进自噬抑制成骨细胞的凋亡。

Objective To study the effect of calcitonin gene-related peptide （CGRP） on apoptosis and autophagy of mouse MC3T3-E1 osteoblast and their interaction and to further clarify protective mechanism of CGRP on osteoblasts. Methods MC3T3-E1 osteoblasts of mouse were cultured in vitro. Western blot and flow cytometry were used to detect expressions of microtubule-associated protein 1 light chain 3 （LC3） and P62 protein of MC3T3-E1 osteoblasts cultured with serum culture and serum-free （serum starvation） culture. Western blot was also used to detect expressions of LC3 and P62 protein of MC3T3-E1 osteoblast cultured at different concentrations （10^-10, 10^-9, 10^-8, and 10^-7 mol·L^-1） or without added CGRP. MC3T3-E1 osteoblasts were treated with 10^-8 mol·L^-1 CGRP at different times （2, 6, 12, 24, 48, and 72 h）, protein expression levels of LC3 were assessed by Western blot and flow cytometry, and changes in autophagosome in cells were detected by monodansylcadaverin staining. Autophagy inhibitor 3-methyladenine （3-MA） was used to pretreat MC3T3-E1 osteoblasts. Cells were then treated with or without CGRP for 24 h. Flow cytometry was used to detect apoptosis level. Results Under serum starvation conditions, LC3Ⅱ expression and apoptosis of osteoblasts increased compared with that of serum culture. Under 3-MA pretreatment and serum starvation conditions, LC3Ⅱ expression of osteoblasts increased compared with that of serum culture （P〈0.01）. Compared with serum culture, serum starvation culture with or without CGRP significantly increased expression level of LC3 and reduced expression level of P62. LC3Ⅱ/Ⅰ of osteoblasts was the highest under serum starvation and 10^-8 mol·L^-1 CGRP conditions. Serum starvation and 10^-8 mol·L^-1 CGRP culture inhibited apoptosis of osteoblasts and promoted synthesis of autophagosome. Apoptosis of osteoblasts increased after 3-MA pretreatment, and CGRP reversed inhibitory effects of 3-MA CGRP on apoptosis. Conclusion CGRP can increase autophagy of MC3T3-E1 osteoblasts under serum starvation conditions. CGRP may also inhibit apoptosis of MC3T3-E1 osteoblasts by promoting autophagy.