BNDF调控SIRT1/PGC—1α通路拮抗Aβ25～35诱导的神经元细胞氧化损伤(1)
[摘要] 目的 探讨脑源性神经营养因子(BDNF)对Aβ25～35诱导的神经元细胞氧化损伤的影响及作用机制。 方法 分离新生SD大鼠皮层神经元细胞，并分为空白对照组、Aβ25～35组、1 μg/L BDNF+Aβ25～35组、10 μg/L BDNF+Aβ25～35组、100 μg/L BDNF+Aβ25～35组、BDNF+siRNA-scramble+Aβ25～35组、BDNF+siRNA-TrkB+Aβ25～35组、BDNF+siRNA-SIRT1+Aβ25～35组。不同浓度BDNF诱导培养细胞48 h，siRNA-scramble、siRNA-TrkB或siRNA-SIRT1采用Lipofectamine 2000转染细胞，诱导培养24 h。MTT法和流式细胞术检测神经元细胞的细胞存活率和凋亡率，利用硫代巴比妥酸法和黄嘌呤氧化酶法检测各组细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性，qRT-PCR和Western blot检测TrkB、SIRT1、PGC-1α、Bcl-2、Bax的表达。 结果 与空白对照组比较，Aβ25～35组细胞存活率明显降低，细胞凋亡率增加，MDA含量升高，SOD活性降低(P < 0.05)。与Aβ25～35组比较，不同浓度BDNF均能提高细胞存活率，降低凋亡率，升高细胞内SOD活性，降低MDA含量，上调TrkB、SIRT1、PGC-1α、Bcl-2表达，减少Bax表达(P < 0.05)，并且呈现剂量依赖性。沉默SIRT1表达后抑制BDNF对Aβ25～35诱导细胞凋亡的保护作用；沉默TrkB表达后抑制BDNF对SIRT1/PGC-1α信号通路的激活作用。 结论 BDNF能够保护Aβ25～35诱导神经元细胞的氧化损伤，并且通过TrkB受体调控SIRT1/PGC-1α信号通路。
[关键词] 脑源性神经营养因子；神经元细胞；氧化损伤；TrkB；SIRT1/PGC-1α
[中图分类号] R318 [文献标识码] A [文章编号] 1673-7210(2016)07(b)-0016-06
[Abstract] Objective To explore the effect and potential mechanism of brain-derived neurotrophic factor (BDNF) pretreatment on neuronal oxidative damage induced by Aβ25-35. Methods The rat cortical neurons obtained from new born SD rats were cultured in vitro for 7 days. After the identification， neurons were randomly divided into control group， Aβ25-35 group， 1 μg/L BDNF+Aβ25-35 group， 10 μg/L BDNF+Aβ25-35 group， 100 μg/L BDNF+Aβ25-35 group， BDNF+siRNA-scramble+Aβ25-35 group， BDNF+siRNA-TrkB+Aβ25-35 group， BDNF+siRNA-SIRT1+Aβ25-35 group. Cells was cultured with different concentrations of BDNF for 48 hours， while siRNA-scramble， siRNA-TrkB and siRNA-SIRT1 were transfected into cells with Lipofectamine 2000 and cultured for 24 hours. The cell survival rate and apoptotic rate of different groups were measured by MTT method and flow cytometry， respectively. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected respectively by the thiobarbituric method and xanthinoxidase method. The expression of TrkB， SIRT1， PGC-1α， Bcl-2 and Bax was detected by quantitative Real-time PCR and Western blot， respectively. Results Compared with the control group， the cell survival rate was decreased， the apoptotic rate was increased， MDA contents were increased and SOD activity was decreased in Aβ25-35 group (P < 0.05). Compared with the Aβ25-35 group， the cell survival rates were increased， the apoptotic rates were decreased， MDA contents were decreased and SOD activity was increased after pre-treatment with different concentrations of BDNF. Furthermore， the expression of TrkB， SIRT1， PGC-1α and Bcl-2 protein was also increased， while the expression of Bax protein was decreased in a dose-dependent manner (P < 0.05). Silencing SIRT1 gene antagonized the protective effect of BDNF on the apoptotic induced by Aβ25-35， silencing TrkB gene antagonized the activation of BDNF on SIRT1/PGC-1α pathways. Conclusion BDNF has a protective effect on neurons against oxidative damage induced by Aβ25-35， and modulates the SIRT1/PGC-1α pathways via TrkB. (姚婕 张红梅 阎丽萍)
[关键词] 脑源性神经营养因子；神经元细胞；氧化损伤；TrkB；SIRT1/PGC-1α
[中图分类号] R318 [文献标识码] A [文章编号] 1673-7210(2016)07(b)-0016-06
[Abstract] Objective To explore the effect and potential mechanism of brain-derived neurotrophic factor (BDNF) pretreatment on neuronal oxidative damage induced by Aβ25-35. Methods The rat cortical neurons obtained from new born SD rats were cultured in vitro for 7 days. After the identification， neurons were randomly divided into control group， Aβ25-35 group， 1 μg/L BDNF+Aβ25-35 group， 10 μg/L BDNF+Aβ25-35 group， 100 μg/L BDNF+Aβ25-35 group， BDNF+siRNA-scramble+Aβ25-35 group， BDNF+siRNA-TrkB+Aβ25-35 group， BDNF+siRNA-SIRT1+Aβ25-35 group. Cells was cultured with different concentrations of BDNF for 48 hours， while siRNA-scramble， siRNA-TrkB and siRNA-SIRT1 were transfected into cells with Lipofectamine 2000 and cultured for 24 hours. The cell survival rate and apoptotic rate of different groups were measured by MTT method and flow cytometry， respectively. The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected respectively by the thiobarbituric method and xanthinoxidase method. The expression of TrkB， SIRT1， PGC-1α， Bcl-2 and Bax was detected by quantitative Real-time PCR and Western blot， respectively. Results Compared with the control group， the cell survival rate was decreased， the apoptotic rate was increased， MDA contents were increased and SOD activity was decreased in Aβ25-35 group (P < 0.05). Compared with the Aβ25-35 group， the cell survival rates were increased， the apoptotic rates were decreased， MDA contents were decreased and SOD activity was increased after pre-treatment with different concentrations of BDNF. Furthermore， the expression of TrkB， SIRT1， PGC-1α and Bcl-2 protein was also increased， while the expression of Bax protein was decreased in a dose-dependent manner (P < 0.05). Silencing SIRT1 gene antagonized the protective effect of BDNF on the apoptotic induced by Aβ25-35， silencing TrkB gene antagonized the activation of BDNF on SIRT1/PGC-1α pathways. Conclusion BDNF has a protective effect on neurons against oxidative damage induced by Aβ25-35， and modulates the SIRT1/PGC-1α pathways via TrkB. (姚婕 张红梅 阎丽萍)