AMPK激动剂预处理对肝缺血再灌注损伤大鼠模型的影响及相关机制

潘宁波; 张旭阳; 张玉; 杨龙灿; 余曦; 李继维; 唐天豪; 黄平; 张莹

doi:10.3969/j.issn.1001-5256.2021.05.034

AMPK激动剂预处理对肝缺血再灌注损伤大鼠模型的影响及相关机制

DOI: 10.3969/j.issn.1001-5256.2021.05.034

潘宁波^1a,
张旭阳²,
张玉^1b,
杨龙灿³,
余曦^1a,
李继维⁴,
唐天豪^1a,
黄平^1a,
张莹^1a, ,

1a.
贵州省人民医院肝胆外科，贵阳550000
1b.
贵州省人民医院信息科，贵阳550000
2.
遵义医科大学附属第二医院，贵州遵义 563000
3.
铜仁市人民医院肝胆外科，贵州铜仁 554300
4.
毕节市第一人民医院肝胆外科，贵州毕节 551700

基金项目:

贵州省科技计划项目 (黔科合基础〔2020〕1Y425);

北京医卫健康基金 (〔2020〕B20251CS);

贵州省卫生计生委科学技术基金 (gzwjkj2018-1-046)

利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：潘宁波负责课题设计，资料分析，撰写论文；张旭阳、张玉、杨龙灿、余曦、李继维、唐天豪参与收集数据，修改论文；黄平、张莹负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

作者简介:
潘宁波(1994—)，男，医师，主要从事肝缺血再灌注损伤研究

通信作者:
张莹，zhangyingdoc@163.com

中图分类号: R575
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出版历程
- 收稿日期: 2020-11-03
- 录用日期: 2020-12-30
- 出版日期: 2021-05-20

Effect of pretreatment with adenosine monophosphate-activated protein kinase agonist on a rat model of hepatic ischemia-reperfusion injury and related mechanism

1a.
Department of Hepatobiliary Surgery, Guizhou Provincial People's Hospital, Guiyang 550000, China
1b.
Department of Information, Guizhou Provincial People's Hospital, Guiyang 550000, China
2.
The Second Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou 563000, China
3.
Department of Hepatobiliary Surgery, Tongren People's Hospital, Tongren, Guizhou 554300, China
4.
Department of Hepatobiliary Surgery, Bijie First People's Hospital, Bijie, Guizhou 551700, China

摘要

摘要: 目的初步探讨腺苷酸活化蛋白激酶(AMPK)激动剂预处理对大鼠肝缺血再灌注损伤大鼠模型的影响及可能机制。方法 54只健康SPF级SD雄性大鼠随机平均分为3组。5-氨基咪唑-4-甲酰胺核苷酸(AICAR)组(实验组)、缺血再灌注组(对照组)和假手术组, 每组18只。每组分别于肝缺血再灌注术后12、24、72 h取材，检测ALT、AST、TBil、TNFα和IL-6浓度，肝三磷酸腺苷(ATP)水平，HE染色观察肝组织学变化，实时荧光定量PCR检测AMPK、哺乳动物雷帕霉素靶蛋白(mTOR)、葡萄糖转运蛋白4型(GLUT4)、多药耐药蛋白2(MRP2)的mRNA相对表达水平，Western Blot检测磷酸化AMPK、磷酸化mTOR、磷酸化GLUT4、MRP2的蛋白表达水平。多组间比较采用重复测量资料的方差分析，进一步两两比较采用LSD-t检验。结果 HE染色结果显示，实验组肝损伤程度在各时点都较对照组轻。术后12、24、72 h，对照组血清ALT、AST、TBil、IL-6、TNFα均高于实验组和假手术组，而实验组均高于假手术组(P值均 < 0.05)。术后12、24、72 h，实验组肝组织ATP水平均高于对照组和假手术组，而对照组均低于假手术组(P值均 < 0.05)。术后12、24、72 h，实验组AMPK、GLUT4、MRP2 mRNA相对表达水平及磷酸化AMPK、磷酸化GLUT4、MRP2蛋白表达水平均高于对照组和假手术组(P值均 < 0.05)；对照组AMPK mRNA相对表达水平及磷酸化AMPK蛋白表达水平均高于假手术组，而GLUT4、MRP2 mRNA相对表达水平及磷酸化GLUT4、MRP2蛋白表达水平均低于假手术组(P值均 < 0.05)。实验组mTOR mRNA相对表达水平及其磷酸化mTOR蛋白表达水平均低于对照组和假手术组(P值均 < 0.05)；对照组mTOR mRNA相对表达水平及其磷酸化mTOR蛋白表达水平均高于假手术组(P值均 < 0.05)。结论 AICAR预处理能激活AMPK信号通路，改善能量代谢途径，减轻肝脏炎症反应，从而降低肝缺血再灌注损伤程度。
- 肝疾病 /
- 缺血再灌注损伤 /
- AMP活化蛋白激酶类 /
- 大鼠, Sprague-Dawley
Abstract: Objective To investigate the effect of pretreatment with adenosine monophosphate-activated protein kinase (AMPK) agonist on rats with hepatic ischemia-reperfusion injury (HIRI) and the possible mechanism. Methods A total of 54 healthy specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into 5-aminimidazole-4-formamide nucleotide (AICAR) treatment group (experimental group), ischemia-reperfusion group (control group), and sham-operation group. Samples were collected at 12, 24, and 72 hours after hepatic ischemia-reperfusion surgery to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil), tumor necrosis factor-α (TNFα), and interleukin-6 (IL-6) and the level of adenosine triphosphate (ATP) in the liver. HE staining was used to observe liver histological changes; quantitative real-time PCR was used to measure the relative mRNA expression levels of AMPK, mammalian target of rapamycin (mTOR), glucose transporter type 4 (GLUT4), and multidrug resistance-associated protein 2 (MRP2); Western blot was used to measure the protein expression levels of phosphorylated AMPK, phosphorylated mTOR, phosphorylated GLUT4, and MRP2. The repeated-measures analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results HE staining showed that the experimental group had milder liver injury than the control group at each time point. At 12, 24, and 72 hours after surgery, the control group had significantly higher serum levels of ALT, AST, TBil, IL-6, and TNFα than the experimental group and the sham-operation group, while the experimental group had significantly higher levels than the sham-operation group (all P < 0.05). At 12, 24, and 72 hours after surgery, the experimental group had a significantly higher level of ATP in liver tissue than the control group and the sham-operation group, and the control group had a significantly lower level than the sham-operation group (all P < 0.05). At 12, 24, and 72 hours after surgery, compared with the control group and the sham-operation group, the experimental group had significantly higher relative mRNA expression levels of AMPK, GLUT4, and MRP2 and protein expression levels of phosphorylated AMPK, phosphorylated GLUT4, and MRP2 (all P < 0.05); compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of AMPK and protein expression level of phosphorylated AMPK, as well as significantly lower relative mRNA expression levels of GLUT4 and MRP2 and protein expression levels of phosphorylated GLUT4 and MRP2 (all P < 0.05). The experimental group had significantly lower relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR than the control group and the sham-o peration group (all P < 0.05), and compared with the sham-operation group, the control group had significantly higher relative mRNA expression level of mTOR and protein expression level of phosphorylated mTOR (both P < 0.05). Conclusion AICAR pretreatment can activate the AMPK signaling pathway, improve energy metabolism pathway, alleviate liver inflammation, and thus reduce the severity of HIRI.
- Liver Diseases /
- Ischemia Reperfusion Injury /
- AMP-Activated Protein Kinases /
- Rats, Sprague-Dawley

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图 1 各组大鼠肝组织HE染色结果(×200)

下载: 全尺寸图片幻灯片

图 2 三组大鼠术后12、24、72 h各蛋白相对表达结果

下载: 全尺寸图片幻灯片

表 1 引物序列

蛋白	正向引物	反向引物
AMPK	5′-AATCCAAACACCAAGGCGTAC-3′	5′-GCTCTACACACTTCTGCCATG-3′
mTOR	5′-GACCGGCTGAGTGGAAAGAT-3′	5′-TCGAGACCGGTAACCTCCAT-3′
GLUT4	5′-CTGCCCGAAAGAGTCTAAAGC-3′	5′-GACGCTCTCTTTCCAACTTCC-3′
MRP2	5′-TTGGAGAGACGGTGAACTTGA-3′	5′-CAACACCTGCTAAGATGGACG-3′
GAPDH	5′-GGTTGTCTCCTGCGACTTCA-3′	5′-TGGTCCAGGGTTTCTTACTCC-3′

下载: 导出CSV

表 2 三组大鼠术后12、24、72 h血清指标水平比较

指标	时间	假手术组(n=18)	对照组(n=18)	实验组(n=18)	F值	P值
ALT(U/L)	12 h	55.05±5.36	291.78±35.04¹⁾	182.40±19.48¹⁾²⁾	205.91	< 0.05
	24 h	54.62±5.57	569.52±55.96¹⁾	308.26±26.79¹⁾²⁾	410.08	< 0.05
	72 h	52.33±5.24	425.56±39.67¹⁾	290.67±15.62¹⁾²⁾	464.63	< 0.05
AST(U/L)	12 h	58.64±6.49	285.77±33.61¹⁾	176.30±23.98¹⁾²⁾	177.26	< 0.05
	24 h	59.66±7.79	520.33±36.66¹⁾	372.92±32.09¹⁾²⁾	545.54	< 0.05
	72 h	57.50±8.16	451.43±36.02¹⁾	314.65±20.20¹⁾²⁾	541.81	< 0.05
TBil (μmol /L)	12 h	2.28±0.44	7.52±0.87¹⁾	4.34±0.44¹⁾²⁾	146.90	< 0.05
	24 h	2.12±0.41	10.88±0.83¹⁾	7.35±0.80¹⁾²⁾	312.36	< 0.05
	72 h	2.20±0.38	8.99±0.79¹⁾	6.39±0.61¹⁾²⁾	245.30	< 0.05
IL-6(pg/ml)	12 h	26.88±3.84	69.71±5.98¹⁾	57.16±6.02¹⁾²⁾	100.54	< 0.05
	24 h	26.44±4.69	131.32±16.84¹⁾	100.43±10.99¹⁾²⁾	122.57	< 0.05
	72 h	26.36±3.72	98.82±9.07¹⁾	82.72±9.91¹⁾²⁾	134.08	< 0.05
TNFα(pg/ml)	12 h	28.30±6.71	65.06±9.05¹⁾	49.91±5.85¹⁾²⁾	39.86	< 0.05
	24 h	26.99±6.43	102.85±14.11¹⁾	83.71±9.37¹⁾²⁾	80.90	< 0.05
	72 h	28.53±6.38	89.18±8.30¹⁾	71.46±9.02¹⁾²⁾	93.45	< 0.05
注：1)与假手术组相比，P < 0.05；2)与对照组相比，P < 0.05。

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表 3 三组大鼠术后12、24、72 h肝ATP水平比较

指标	时间	假手术组(n=18)	对照组(n=18)	实验组(n=18)	F值	P值
ATP(nmol/L)	12 h	1024.86±157.94	701.28±90.72¹⁾	1245.56±124.99¹⁾²⁾	27.64	< 0.05
	24 h	1033.20±135.56	764.28±78.29¹⁾	1404.69±178.95¹⁾²⁾	32.93	< 0.05
	72 h	1005.38±148.16	806.89±65.32¹⁾	1394.00±141.88¹⁾²⁾	22.54	< 0.05
注：1)与假手术组相比，P < 0.05；2)与对照组相比，P < 0.05。

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表 4 三组大鼠术后12、24、72 h各蛋白mRNA相对表达水平比较

指标	时间	假手术组(n=18)	对照组(n=18)	实验组(n=18)	F值	P值
AMPK	12 h	1.04±0.14	1.14±0.11¹⁾	2.53±0.14¹⁾²⁾	62.65	< 0.05
	24 h	1.04±0.11	1.25±0.18¹⁾	3.66±0.57¹⁾²⁾	103.79	< 0.05
	72 h	1.04±0.18	1.22±0.14¹⁾	3.21±0.45¹⁾²⁾	101.66	< 0.05
mTOR	12 h	0.98±0.06	1.51±0.27¹⁾	0.48±0.08¹⁾²⁾	59.37	< 0.05
	24 h	0.97±0.08	1.87±0.30¹⁾	0.56±0.14¹⁾²⁾	70.57	< 0.05
	72 h	0.98±0.10	1.75±0.24¹⁾	0.71±0.12¹⁾²⁾	62.11	< 0.05
GLUT4	12 h	1.00±0.13	0.78±0.10¹⁾	1.64±0.19¹⁾²⁾	60.21	< 0.05
	24 h	1.00±0.12	0.88±0.14¹⁾	2.36±0.46¹⁾²⁾	49.51	< 0.05
	72 h	0.99±0.13	0.92±0.14¹⁾	2.05±0.33¹⁾²⁾	49.85	< 0.05
MRP2	12 h	0.98±0.12	0.46±0.14¹⁾	1.57±0.24¹⁾²⁾	60.66	< 0.05
	24 h	0.99±0.12	0.65±0.12¹⁾	2.39±0.42¹⁾²⁾	75.87	< 0.05
	72 h	0.98±0.10	0.87±0.10¹⁾	1.98±0.39¹⁾²⁾	39.40	< 0.05
注：1)与假手术组相比，P < 0.05；2)与对照组相比，P < 0.05。

下载: 导出CSV

表 5 三组大鼠术后12、24、72 h各蛋白相对表达水平比较

指标	时间	假手术组(n=18)	对照组(n=18)	实验组(n=18)	F值	P值
磷酸化AMPK	12 h	0.56±0.11	1.39±0.32¹⁾	2.16±0.25¹⁾²⁾	65.65	< 0.05
	24 h	0.55±0.13	1.45±0.31¹⁾	2.56±0.43¹⁾²⁾	61.04	< 0.05
	72 h	0.57±0.18	1.35±0.18¹⁾	2.47±0.38¹⁾²⁾	78.90	< 0.05
磷酸化mTOR	12 h	2.29±0.43	3.33±0.45¹⁾	1.33±0.20¹⁾²⁾	42.62	< 0.05
	24 h	2.22±0.33	3.77±0.50¹⁾	1.46±0.24¹⁾²⁾	58.83	< 0.05
	72 h	2.15±0.38	3.71±0.48¹⁾	1.48±0.22¹⁾²⁾	55.75	< 0.05
磷酸化GLUT4	12 h	2.22±0.40	0.87±0.17¹⁾	3.12±0.40¹⁾²⁾	65.92	< 0.05
	24 h	2.19±0.56	1.13±0.27¹⁾	3.55±0.47¹⁾²⁾	43.34	< 0.05
	72 h	2.20±0.51	1.24±0.14¹⁾	3.49±0.39¹⁾²⁾	53.41	< 0.05
MRP2	12 h	1.33±0.25	0.41±0.14¹⁾	2.10±0.26¹⁾²⁾	85.21	< 0.05
	24 h	1.34±0.28	0.54±0.16¹⁾	2.50±0.33¹⁾²⁾	82.66	< 0.05
	72 h	1.32±0.30	0.62±0.14¹⁾	2.18±0.34¹⁾²⁾	51.15	< 0.05
注：1)与假手术组相比，P < 0.05；2)与对照组相比，P < 0.05。

下载: 导出CSV

参考文献(13)

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AMPK激动剂预处理对肝缺血再灌注损伤大鼠模型的影响及相关机制

DOI: 10.3969/j.issn.1001-5256.2021.05.034

作者简介:
潘宁波(1994—)，男，医师，主要从事肝缺血再灌注损伤研究

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张莹，zhangyingdoc@163.com

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Effect of pretreatment with adenosine monophosphate-activated protein kinase agonist on a rat model of hepatic ischemia-reperfusion injury and related mechanism

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AMPK激动剂预处理对肝缺血再灌注损伤大鼠模型的影响及相关机制

DOI: 10.3969/j.issn.1001-5256.2021.05.034

作者简介: 潘宁波(1994—)，男，医师，主要从事肝缺血再灌注损伤研究

通信作者: 张莹，zhangyingdoc@163.com

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Effect of pretreatment with adenosine monophosphate-activated protein kinase agonist on a rat model of hepatic ischemia-reperfusion injury and related mechanism

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指南共识

作者简介:
潘宁波(1994—)，男，医师，主要从事肝缺血再灌注损伤研究

通信作者:
张莹，zhangyingdoc@163.com