盐诱导激酶<strong>2</strong>对脑缺血再灌注大鼠脑组织能量代谢的影响

doi:10.3724/zdxbyxb-2021-0164

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 Shu^12,*(

),HONG Yun^3,*(

)

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 或 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

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

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

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

表 4 各组脑组织中腺苷三磷酸和腺苷二磷酸含量比较

1	盛桂芝, 王　波. 超早期溶栓治疗脑梗塞的疗效及影响因素探析[J]. 系统医学, 2019, 4(3): 62-64 SHENG Guizhi, WANG Bo. Analysis of the efficacy and influencing factors of ultra-early thrombolytic therapy for cerebral infarction[J]. Systems Medicine, 2019, 4(3): 62-64. (in Chinese)
2	刘　磊, 刘丽华, 马玉奎. 脑缺血再灌注损伤机制研究进展[J]. 药学研究, 2016, 35(9): 542-544 LIU Lei, LIU Lihua, MA Yukui. Research progress on mechanisms of cerebral ischemia-reperfusion injury[J]. Journal of Pharmaceutical Research, 2016, 35(9): 542-544. (in Chinese)
3	郭敏敏, 蔡　乐, 王　勇. 脑缺血再灌注损伤发病机制的研究进展[J]. 世界最新医学信息文摘, 2019, 19(30): 80-81 GUO Minmin, CAI Le, WANG Yong. Research progress on pathogenesis of cerebral ischemia-reperfusion injury[J]. World Latest Medicine Information, 2019, 19(30): 80-81. (in Chinese)
4	UEBIT, ITOHY, HATANOO, et al.Involvement of SIK3 in glucose and lipid homeostasis in mice[J/OL]PLoS ONE, 2012, 7( 5): e37803. doi: 10.1371/journal.pone.0037803
5	KER, XUQ, LIC, et al.Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism[J]Cell Biol Int, 2018, 42( 4): 384-392. doi: 10.1002/cbin.10915
6	O’NEILLH M, MAARBJERGS J, CRANEJ D, et al.AMP-activated protein kinase (AMPK) beta1beta2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise[J]Proc Natl Acad Sci U S A, 2011, 108( 38): 16092-16097. doi: 10.1073/pnas.1105062108
7	LIZCANOJ M, G?RANSSONO, TOTHR, et al.LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1[J]EMBO J, 2004, 23( 4): 833-843. doi: 10.1038/sj.emboj.7600110
8	JALEELM, MCBRIDEA, LIZCANOJ M, et al.Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate[J]FEBS Lett, 2005, 579( 6): 1417-1423. doi: 10.1016/j.febslet.2005.01.042
9	YANGF C, TANB C M, CHENW H, et al.Reversible acetylation regulates salt-inducible kinase (SIK2) and its function in autophagy[J]J Biol Chem, 2013, 288( 9): 6227-6237. doi: 10.1074/jbc.M112.431239
10	赵　雷. 缺氧诱导因子-1α对脑缺血缺氧性损伤治疗作用的研究[D]. 石家庄: 河北医科大学, 2013 ZHAO Lei. Study on the therapeutic effect of hypoxia-inducible factor-1α on cerebral ischemia and hypoxic injury[D]. Shijiazhuang: Hebei Medical University, 2013. (in Chinese)
11	宋慧琨. 缺氧诱导因子-1α在脑缺血中的作用及其机制研究[D]. 唐山: 华北理工大学, 2015 SONG Huikun. The role and mechanism of hypoxia-inducible factor-1α in cerebral ischemia[D]. Tangshan: North China University of Science and Technology, 2015. (in Chinese)
12	SHIH, CHANGY. Regulations and posttranslational modifications of hypoxia inducible factor-1[J]Acta Biophysica Sinca, 2012, 28( 5): 373-382. doi: 10.3724/SP.J.1260.2012.20032
13	张文芳, 王　东, 崔景晶. 最佳线栓头端直径建立对小鼠脑缺血再灌注模型成功率的影响[J]. 中国康复医学杂志, 2019, 34(4): 386-391 ZHANG Wenfang, WANG Dong, Cui Jingjing. The best tip diameter of the suture established the middle cerebral artery occlusion mouse model[J]. Chinese Journal of Rehabilitation Medicine, 2019, 34(4): 386-391. (in Chinese)
14	李振宗, 赵育梅, 袁　辉. 大鼠线栓法局灶性脑缺血模型的改良[J]. 中国微侵袭神经外科杂志, 2018, 23(9): 419-422 LI Zhenzhong, ZHAO Yumei, YUAN Hui. Improvement of the cerebral ischemia model by intraluminal filament in rats[J]. Chinese Journal of Minimally Invasive Neurosurgery, 2018, 23(9): 419-422. (in Chinese)
15	LONGAE Z, WEINSTEINP R, CARLSONS, et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J]Stroke, 1989, 20( 1): 84-91. doi: 10.1161/01.STR.20.1.84
16	郑建峰. 缺血性脑卒中相关大脑中动脉梗阻模型制备的研究[D]. 福州: 福建医科大学, 2016 ZHEN Jianfeng. Study on the preparation of a model of middle cerebral artery obstruction associated with ischemic stroke[D]. Fuzhou: Fujian Medical University, 2016. (in Chinese)
17	方永奇, 莫镇涛. 脑能量代谢调节机制研究新进展[J]. 广州中医药大学学报, 2011, 28(3): 328-331 FANG Yongqi, MO Zhentao. Research progress on the regulation mechanism of brain energy metabolism[J]. Journal of Guangzhou University of Traditional Chinese Medicine, 2011, 28(3): 328-331. (in Chinese)
18	HARDIED G, ROSSF A, HAWLEYS A. AMPK: a nutrient and energy sensor that maintains energy homeostasis[J]Nat Rev Mol Cell Biol, 2012, 13( 4): 251-262. doi: 10.1038/nrm3311
19	杜　静, 杨　超, 晏耀明, 等. SIK2抑制3T3-L1脂肪细胞脂肪合成的效应及机制[J]. 中国实验诊断学, 2012, 16(8): 1354-1357 DU Jing, YANG Chao, YAN Yaoming, et al. The inhibition effect and mechanism of SIK2 on lipogenesis in 3T3-L1 adipocyte[J]. Chinese Journal of Laboratory Diagnosis, 2012, 16(8): 1354-1357. (in Chinese)
20	王建校, 李珊珊, 赵珊珊, 等. 盐诱导激酶2的研究进展[J]. 生物物理学报, 2014, 30(2): 93-100 WANG Jianxiao, LI Shanshan, ZHAO Shanshan, et al. Research progress of salt-induced kinase 2[J]. Acta Biophysica Sinica, 2014, 30(2): 93-100. (in Chinese)
21	ZHANGZ, YAOL, YANGJ, et al.PI3K/Akt and HIF?1 signaling pathway in hypoxia?ischemia (Review)[J]Mol Med Rep, 2018, 18( 4): 3547-3554. doi: 10.3892/mmr.2018.9375
22	张爱民, 蒋宗滨. 高压氧预处理通过HIF-1α/VEGF通路减轻大脑缺血再灌注损伤[J]. 中国病理生理杂志, 2018, 34(11): 2048-2053 ZHANG Aiming, JIANG Zongbing. Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reper fusion injury by hyperbaric oxygen pretreatment[J]. Chinese Journal of Pathophysiology, 2018, 34(11): 2048-2053. (in Chinese)
23	张永春, 谷　江, 董安涛, 等. 三白脂素-8抑制缺氧诱导因子-1α对肾癌细胞能量代谢的影响[J]. 贵州医药, 2018, 42(8): 926-929 ZHANG Yongchun, GU Jiang, DONG Antao, et al. Effect of Man A on energy metabolism of renal carcinoma cells when HIF-1α inhibited[J]. Guizhou Medical Journal, 2018, 42(8): 926-929. (in Chinese)

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