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浙江大学学报(医学版)  2021, Vol. 50 Issue (3): 352-360    DOI: 10.3724/zdxbyxb-2021-0164
原著     
盐诱导激酶2对脑缺血再灌注大鼠脑组织能量代谢的影响
张冉12(),刘云12,张翠1,马梦尧1,李曙12,*(),洪云3,*()
1.皖南医学院病理生理学教研室,安徽 芜湖 241002
2.皖南医学院临床医学院,安徽 芜湖 241002
Salt-inducible kinase 2 regulates energy metabolism in rats with cerebral ischemia-reperfusion
ZHANG Ran12(),LIU Yun12,ZHANG Cui1,MA Mengyao1,LI Shu12,*(),HONG Yun3,*()
1. Department of Pathophysiology, Wannan Medical College, Wuhu 241002, Anhui Province, China;
2. Clinical Colloge of Wannan Medical College, Wuhu 241002, Anhui Province, China; 3. Department of Ultrasonic Medicine, Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China
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摘要:

目的:探究盐诱导激酶2(SIK2)对脑缺血再灌注大鼠能量代谢相关物质表达的影响。方法:选用成年SD雄性大鼠(240~260?g),参照改良Zea-Longa线栓法建立大鼠短暂性中动脉栓塞模型(MCAO),在构建MCAO模型前8?d予以右侧脑室注射7?μL腺病毒使大鼠脑组织SIK2过表达,随后建立缺血2?h再灌注24?h模型。实验分为手术对照组、缺血对照组、缺血再灌注组、腺病毒空载组及SIK2过表达组。苏木精-伊红(HE)染色观察大鼠神经细胞损伤的病理学改变;氯化三苯基四氮唑(TTC)染色观察大鼠脑组织梗死情况;酶联免疫吸附试验(ELISA)检测各组大鼠脑组织中腺苷三磷酸(ATP)、腺苷二磷酸(ADP)的含量;荧光定量聚合酶链反应和蛋白质印迹法检测各组大鼠脑组织中SIK2及缺氧诱导因子1α(HIF-1α)的表达量。结果:与手术对照组比较,缺血对照组及缺血再灌注组SIK2表达减少,且缺血再灌注组较缺血对照组减少更明显(P<0.05);与手术对照组和缺血对照组比较,缺血再灌注组病理损伤较重,梗死体积较大;与缺血再灌注组和腺病毒空载组比较,SIK2过表达组病理损伤较轻,梗死体积减少。与手术对照组比较,缺血对照组和缺血再灌注组HIF-1α表达均增加,其中缺血对照组增加更明显(均P<0.05);SIK2过表达组HIF-1α表达比缺血再灌注组和腺病毒空载组均增多(均P<0.05)。与手术对照组比较,缺血对照组和缺血再灌注组ATP含量均减少,且缺血再灌注组较缺血对照组减少更为显著(P<0.05);ADP含量在缺血对照组和缺血再灌注组均增多,且缺血再灌注组较缺血对照组明显增多(P<0.05);与缺血再灌注组和腺病毒空载组比较,SIK2过表达组ATP含量增加(均P<0.05),ADP含量减少(均P<0.05)。结论:SIK2可通过上调HIF-1α的表达,增加大鼠脑组织中ATP的含量,减少ADP含量,减轻大鼠脑缺血再灌注损伤。

关键词: 缺血再灌注能量代谢盐诱导激酶2缺氧诱导因子1α腺苷三磷酸腺苷二磷酸SD大鼠    
Abstract:

Objective:To investigate the effects of salt-inducible kinase 2 (SIK2) on energy metabolism in rats with cerebral ischemia-reperfusion. Methods: Adult SD male rats (240-260?g) were divided into 5 groups: sham group, ischemia group, reperfusion group, adenovirus no-load group, and SIK2 overexpression group with 5 animals in each group. The middle cerebral artery occlusion (MCAO) was induced with the modified Zea-Longa line thrombus method to establish the cerebral ischemia reperfusion model. Eight days before the MCAO, SIK2 overexpression was induced by injecting 7 μL adenovirus in the right ventricle, then MCAO was performed for 2?h, followed by reperfusion 24?h. HE staining was used to observe the pathological changes of cerebral tissue in rats; TTC staining was used to observe the volume of cerebral infarct. The levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) in rat brain tissue were detected by ELISA; the levels of SIK2 and hypoxia-inducible factor 1α (HIF-1α) in the rat brain tissues were detected by RT-qPCR and Western blotting. Results:Compared with the sham group, SIK2 level was decreased in the ischemia group, and it was further declined in the reperfusion group (P<0.05). Compared with the sham group and ischemic group, the pathological injury in reperfusion group were more severe, and the infarct size was larger; compared with the reperfusion group and adenovirus no-load group, the pathological injury of the SIK2 overexpression group was milder, and the infarct size is less. Compared with the sharn group, HIF-1α was increased in both ischemia group and reperfusion group, especially in ischemia group (allP<0.05); HIF-1α level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (allP<0.05). ATP level in ischemia group and reperfusion group was lower than that in the sham group, and the reperfusion group decreased more significantly than the ischemia group (P<0.05); ADP content was increased in the ischemia and reperfusion group, and the ADP content in reperfusion group was significantly higher than that in the ischemia group (P<0.05). ATP level in the SIK2 overexpression group was higher than that in the reperfusion group and adenovirus no-load group (allP<0.05), and ADP was decreased in the SIK2 overexpression group (allP<0.05).Conclusion:SIK2 can up-regulate the ATP level and down-regulate the ADP level in rat brain tissue and alleviate cerebral ischemia-reperfusion injury by increase the level of HIF-1α.

Key words: Ischemia reperfusion    Energy metabolism    Salt-inducible kinase 2    Hypoxia-inducible factor-1α    Adenosine triphosphate    Adenosine diphosphate    SD rat
收稿日期: 2021-01-11 出版日期: 2021-08-16
CLC:  R363  
基金资助: 安徽高校自然科学研究重大项目(KJ2020ZD55);安徽省高校优秀青年人才支持计划(gxyq2018044);皖南医学院校级中青年基金(WK201914)
通讯作者: 李曙,洪云     E-mail: 18255366693@163.com;yxx2003@126.com
作者简介: 张 冉,硕士研究生,主要从事心脑血管病理生理学研究;E-mail:18255366693@163.com;https://orcid.org/0000-0002-2417-2574
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引用本文:

张冉,刘云,张翠,马梦尧,李曙,洪云. 盐诱导激酶2对脑缺血再灌注大鼠脑组织能量代谢的影响[J]. 浙江大学学报(医学版), 2021, 50(3): 352-360.

ZHANG Ran,LIU Yun,ZHANG Cui,MA Mengyao,LI Shu,HONG Yun. Salt-inducible kinase 2 regulates energy metabolism in rats with cerebral ischemia-reperfusion. J Zhejiang Univ (Med Sci), 2021, 50(3): 352-360.

链接本文:

http://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0164        http://www.zjujournals.com/med/CN/Y2021/V50/I3/352

图 1  手术对照组、缺血对照组和缺血再灌注组脑组织中盐诱导激酶(SIK)2蛋白表达电泳图

组 别

n

mRNA

蛋白

手术对照组

4

0.73±0.09

0.98±0.05

缺血对照组

4

0.58±0.05

0.96±0.04

缺血再灌注组

4

0.46±0.10*

0.85±0.02*#

表 1  手术对照组、缺血对照组和缺血再灌注组脑组织盐诱导激酶2表达量比较
图 2  各组脑组织病理学表现(HE染色)手术对照组神经细胞形态规则,细胞质丰富,细胞核呈圆形清晰可见,未见空泡状结构及淡染区;缺血对照组缺血中心区淡染,可见少部分神经细胞液化性坏死,出现空泡样结构呈筛网状;缺血再灌注组出现大面积神经细胞液化性坏死,高倍镜下整个视野布满空泡样结构;腺病毒空载组亦出现大面积神经细胞液化性坏死,高倍镜下整个视野布满空泡样结构;SIK2过表达组部分皮质区淡染,细胞核增多,高倍镜下可见空泡样结构.红色区域为细胞质,蓝色为细胞核.
图 3  各组脑组织梗死情况比较手术对照组脑组织染色呈正常的红色,无梗死灶;缺血对照组脑组织存在白色梗死灶,面积较小;缺血再灌注组脑组织有明显的白色梗死灶且面积较大;腺病毒空载组脑组织有明显的白色梗死灶且面积较大;SIK2过表达组出现白色梗死灶,面积较小. 红色为正常脑组织,白色为组织梗死区域.
图 4  各组脑组织中缺氧诱导因子1α蛋白表达电泳图

组 别

n

mRNA

蛋 白

手术对照组

5

0.92±0.11

0.77±0.10

缺血对照组

5

1.29±0.62*

1.00±0.00*

缺血再灌注组

5

1.03±0.10#

0.86±0.08#

表 2  缺血及再灌注对大鼠脑组织中缺氧诱导因子1α表达量的影响

组 别

n

mRNA

蛋白

缺血再灌注组

5

0.92±0.14

0.73±0.09

腺病毒空载组

5

1.00±0.07

0.75±0.08

SIK2过表达组

5

1.27±0.07*#

1.00±0.09*#

表 3  盐诱导激酶2过表达对缺血再灌注大鼠脑组织中缺氧诱导因子1α表达量的影响

组 别

n

腺苷三磷酸

腺苷二磷酸

手术对照组

5

6187±306

0.98±0.14

缺血对照组

5

5877±244

2.08±1.18

缺血再灌注组

5

5122±233*#

7.38±3.25*#

腺病毒空载组

5

5491±295

7.08±2.45

SIK2过表达组

5

7296±214△▲

0.58±0.28△▲

表 4  各组脑组织中腺苷三磷酸和腺苷二磷酸含量比较
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