SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤<sup>*</sup>

doi:10.13523/j.cb.2106024

中国生物工程杂志

2021, Vol. 41

Issue (11): 1-13 DOI: 10.13523/j.cb.2106024

研究报告

SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤^*

张晨阳¹,黑常春²,袁仕林¹,周玉佳¹,曹美玲¹,秦亦欣¹,杨笑^3,^**(

)

1 宁夏医科大学临床医学院银川 750004
2 宁夏医科大学基础医学院人体解剖与组织胚胎学系银川 750004
3 宁夏医科大学总医院神经内科银川 750004

SIRT3 Inhibits Mitophagy and Alleviates Neuronal Oxygen Deprivation/Reoxygenation Injury Aggravated by High Glucose

Chen-yang ZHANG¹,Chang-chun HEI²,Shi-lin YUAN¹,Yu-jia ZHOU¹,Mei-ling CAO¹,Yi-xin QIN¹,Xiao YANG^3,^**(

)

1 School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China
2 Department of Human Anatomy, Histology and Embryology, Ningxia Medical University, Yinchuan 750004, China
3 Neuroscience Center, General Hospital of Ningxia Medical University, Yinchuan 750004, China

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摘要：

目的:探讨SIRT3调控的线粒体自噬对高糖加重神经元缺氧再灌注损伤的影响及机制。方法:高糖(50 mmol/L)干预HT22细胞后,构建细胞缺氧/复氧模型,利用SIRT3抑制剂3-TYP抑制SIRT3表达。倒置显微镜观察细胞形态改变,CCK8法检测细胞存活率,流式细胞术检测细胞凋亡率,TMRE荧光试剂盒检测细胞线粒体膜电位,RT-qPCR、Western blot检测相关分子的基因和蛋白质表达。结果:高糖使神经元缺氧再灌注后的细胞碎片进一步增加,细胞存活率降低,细胞凋亡率升高(P<0.05)。此外,高糖降低了神经元缺氧再灌注后的线粒体膜电位(P<0.05)。进一步研究发现,高糖上调神经元缺氧再灌注后线粒体分裂相关蛋白DRP1的表达水平,降低了线粒体融合相关蛋白OPA1和线粒体外膜蛋白TOM20的表达;并且增加了自噬相关蛋白LC3Ⅱ、Beclin-1和线粒体自噬相关蛋白PINK1、Parkin的表达;同时,高糖升高了SIRT3的基因和蛋白质表达(P<0.05)。而SIRT3抑制剂3-TYP使神经元高糖缺氧再灌注损伤加重,同时进一步上调DRP1、LC3Ⅱ和PINK1的蛋白质表达(P<0.05)。结论:高糖可显著加重神经元缺氧再灌注损伤,破坏细胞线粒体功能,激活细胞线粒体自噬;SIRT3可抑制PINK1-Parkin通路介导的线粒体自噬并减轻神经元高糖缺氧再灌注损伤。

关键词： 缺氧再灌注; 高糖; 线粒体自噬; 线粒体融合分裂; SIRT3

Abstract:

Objective: To investigate whether high glucose can aggravate oxygen deprivation/reoxygenation (OD/R) injury in neurons through regulating mitophagy and the specific molecular mechanism of regulating mitophagy. Methods: HT22 cells are used to establish a model of cellular oxygen deprivation/reoxygenation to simulate cerebral ischemia/reperfusion injury in vivo. And cells are treated with high glucose medium (HG 50 mmol/L). 3-TYP is given to inhibit the expression of SIRT3. CCK8 is used to examine cell viability; flow cytometry is used to detect cell apoptosis, and TMRE fluorescence detection kit is used to detect cell MMP; Western blot is used to detect the expression of mitochondrial division/fusion related protein (DRP1, OPA1, TOM20), autophagy related protein (LC3Ⅱ, Beclin-1), mitophagy related protein (PINK1, Parkin) and SIRT3. Results: Compared with OD groups, the cell density is further reduced, the cell contour is more blurred, the synapses are shortened, the floating cells increase in HG+OD groups, and cells in HG+OD groups have lower survival rate and higher apoptosis rate (P<0.05). Compared with OD groups, cells in HG+OD groups have lower MMP (P<0.05). Compared with OD groups, the expression of P-DRP1 in HG+OD groups is increased, but OPA1 and TOM20 are decreased (P<0.05); the expression of Beclin-1 and LC3Ⅱ as well as PINK1 and Parkin in HG+OD groups are also increased (P<0.05). Furthermore, SIRT3 protein and gene are further increased in HG+OD groups (P<0.05). 3-TYP treatment can further aggravate the OD/R injury of neurons in HG groups. And 3-TYP also increases the expression of DRP1, LC3Ⅱ and PINK1. Conclusions: High glucose can aggravate oxygen deprivation/reoxygenation injury in neurons by exacerbation of cell apoptosis, reduction of MMP and activation of mitophagy. In addition, SIRT3 can inhibit PINK1-Parkin pathway-mediated mitophagy and alleviate high glucose aggravated-neuronal OD/R injury.

Key words: Oxygen deprivation/reoxygenation High glucose Mitophagy Mitochondrial fusion and fission SIRT3

收稿日期: 2021-06-15 出版日期: 2021-12-01

ZTFLH:

基金资助: * 国家自然科学基金(82060237);国家自然科学基金(81560226);国家自然科学基金(81860250);宁夏自然科学基金(2018AAC03134);宁夏自然科学基金(2021AAC03371)

通讯作者: 杨笑 E-mail: cckk606@sina.com

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引用本文:

张晨阳,黑常春,袁仕林,周玉佳,曹美玲,秦亦欣,杨笑. SIRT3抑制线粒体自噬并减轻高糖加重的神经元缺氧再灌注损伤^*[J]. 中国生物工程杂志, 2021, 41(11): 1-13.

Chen-yang ZHANG,Chang-chun HEI,Shi-lin YUAN,Yu-jia ZHOU,Mei-ling CAO,Yi-xin QIN,Xiao YANG. SIRT3 Inhibits Mitophagy and Alleviates Neuronal Oxygen Deprivation/Reoxygenation Injury Aggravated by High Glucose. China Biotechnology, 2021, 41(11): 1-13.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2106024 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I11/1

表1 高糖缺氧细胞模型所需溶液的各成分浓度

图1 模型制备的实验流程

表2 引物序列

图2 高糖对神经元缺氧再灌注损伤的影响

图3 高糖对神经元缺氧再灌注后线粒体膜电位的影响

图4 高糖对神经元缺氧再灌注后线粒体动态平衡的影响

图5 高糖对神经元缺氧再灌注后线粒体自噬的影响

图6 高糖对神经元缺氧再灌注后SIRT3的影响

图7 3-TYP对神经元高糖缺氧再灌注后SIRT3的影响

图8 抑制SIRT3对神经元高糖缺氧再灌注损伤的影响

图9 抑制SIRT3对神经元高糖神经元缺氧再灌注后线粒体自噬的影响

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