Naa20通过抑制AIM2表达减轻脓毒症心肌损伤

doi:10.11958/20230338

天津医药 ›› 2023, Vol. 51 ›› Issue (10): 1065-1070.doi: 10.11958/20230338

Naa20通过抑制AIM2表达减轻脓毒症心肌损伤

刘畅(), 范彩红, 刘佳, 刘亚珊, 张诗琪, 刘士铭, 申艳娜^△()

天津医科大学医学检验学院（邮编300203）

收稿日期:2023-03-15 修回日期:2023-06-30 出版日期:2023-10-15 发布日期:2023-10-18
通讯作者: ^∆E-mail：shenyanna@tmu.edu.cn
作者简介:刘畅（1992），女，讲师，主要从事感染免疫方面研究。E-mail：liuchang419@tmu.edu.cn
基金资助:
天津市教委科研计划项目(2020KJ206)

Naa20 alleviated myocardial injury in sepsis by inhibiting AIM2 expression

LIU Chang(), FAN Caihong, LIU Jia, LIU Yashan, ZHANG Shiqi, LIU Shiming, SHEN Yanna^△()

School of Medical Laboratory, Tianjin Medical University, Tianjin 300203, China

Received:2023-03-15 Revised:2023-06-30 Published:2023-10-15 Online:2023-10-18
Contact: ^∆E-mail：shenyanna@tmu.edu.cn

刘畅, 范彩红, 刘佳, 刘亚珊, 张诗琪, 刘士铭, 申艳娜. Naa20通过抑制AIM2表达减轻脓毒症心肌损伤[J]. 天津医药, 2023, 51(10): 1065-1070.

LIU Chang, FAN Caihong, LIU Jia, LIU Yashan, ZHANG Shiqi, LIU Shiming, SHEN Yanna. Naa20 alleviated myocardial injury in sepsis by inhibiting AIM2 expression[J]. Tianjin Medical Journal, 2023, 51(10): 1065-1070.

摘要/Abstract

摘要：

目的探究Naa20在脂多糖（LPS）脓毒症心肌损伤中的调控作用及机制。方法 12只雄性C57BL/6J小鼠随机分为对照组和模型组，每组6只。模型组采用LPS一次性尾静脉注射方法造模，对照组注射同等剂量的磷酸盐缓冲液（PBS）。LPS注射12 h后采用心脏多普勒超声检测各组小鼠心脏功能；酶联免疫吸附试验（ELISA）检测各组小鼠血清中肌酸激酶同工酶（CK-MB）表达水平；苏木精-伊红（HE）染色观察各组小鼠心肌损伤程度。在体外分离、培养SD大鼠乳鼠心肌细胞，随机分为对照组和LPS处理组。荧光探针检测心肌细胞中线粒体膜电位和活性氧（ROS）释放情况；Western blot检测心肌细胞中黑色素瘤缺失因子2（AIM2）、消皮素D（GSDMD）、活化的GSDMD（GSDMD-N）、白细胞介素（IL）-1β、活化的IL-1β（p17）和Naa20的蛋白表达水平；ELISA检测心肌细胞上清液中IL-1β含量；实时荧光定量PCR（qPCR）检测心肌细胞中Naa20的表达。H9c2心肌细胞分为对照组、Naa20对照组、LPS组和Naa20+LPS组，四甲基偶氮唑盐（MTT）和乳酸脱氢酶（LDH）实验检测心肌细胞活力；免疫共沉淀检测Naa20与AIM2的结合情况。结果与对照组相比，模型组小鼠左心室射血分数（LVEF）、短轴缩短率（FS）降低，血清中CK-MB含量增加（P<0.01），小鼠心肌出现病理改变。体外实验结果表明，与对照组相比，LPS处理组心肌细胞线粒体膜电位降低，ROS释放增多，AIM2、GSDMD、GSDMD-N、IL-1β、p17和Naa20的蛋白表达水平增加，上清液中IL-1β含量增多（P<0.05）。免疫共沉淀证实Naa20与AIM2结合。与LPS组相比，Naa20+LPS组心肌细胞活力增强，AIM2乙酰化修饰增多，AIM2、GSDMD和GSDMD-N的蛋白表达水平降低，上清液中IL-1β含量降低（P<0.05）。结论 Naa20可通过调节AIM2蛋白表达调控心肌细胞焦亡，减轻心肌损伤。

关键词: 细胞焦亡, Naa20, AIM2, 心肌损伤

Abstract:

Objective To explore the regulatory effect and mechanism of Naa20 on lipopolysaccharide (LPS) induced myocardial injury in sepsis. Methods Twelve male C57BL/6J mice were divided into the control group and the myocardial injury model group (n=6 in each group). Mice of the myocardial injury model group were treated with a single intravenous injection of LPS, while mice of the control group were injected with the same dose of PBS. Cardiac function of mice in each group was evaluated by cardiac Doppler ultrasonography 12 h after LPS injection. The serum content of CK-MB was detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin staining (HE) was used to evaluate myocardial injury in mice. Cardiomyocytes from SD rats were isolated and cultured in vitro and divided into the control group and the LPS group. Mitochondrial membrane potential and reactive oxygen species (ROS) release in cardiomyocytes were detected by fluorescent probe. The protein expression levels of AIM2, GSDMD, activated GSDMD (GSDMD-N), IL-1β, activated IL-1β (p17) and Naa20 were detected by Western blot assay. The content of IL-1β in supernatant was determined by ELISA. The expression level of Naa20 in cardiomyocytes was detected by quantitative real-time PCR (qPCR). H9c2 cells were randomly divided into the control group, the Naa20 group, the LPS group and the Naa20+LPS group. Methyl thiazolyl tetrazolium (MTT) assay and lactate dehydrogenase (LDH) assay were used to detect the viability of H9c2 cells. The interaction between Naa20 and AIM2 was detected by immunoprecipitation. Results Compared with the control group, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS) were decreased in the myocardial injury model group (P<0.01), the serum content of CK-MB was increased (P<0.01), and HE staining showed pathological changes in heart of mice. In vitro, compared with the control group, mitochondrial membrane potential was decreased, while ROS production, expressions of AIM2, GSDMD, GSDMD-N, IL-1β, p17 and Naa20 and the content of IL-1β in supernatant were significantly increased in the LPS group (P<0.05). Results of immunocoprecipitation confirmed that Naa20 interacted with AIM2. Compared with the LPS group, the viability of H9c2 cells and the acetylation of AIM2 were increased, while the protein expression levels of AIM2, GSDMD, GSDMD-N and the content of IL-1β in supernatant were decreased in the Naa20+LPS group (P<0.05). Conclusion Naa20 can regulate AIM2-mediacted pyroptosis by inhibiting AIM2 expression, thereby alleviating sepsis-induced myocardial injury.

Key words: pyroptosis, Naa20, AIM2, myocardial injury

中图分类号:

R631.3

图/表 14

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

基因	引物序列（5′→3′）	产物大小/bp
Naa20	上游：GACGACCTGTTCCGCTTCAA 下游：TCTGCTTTGCCCATAACCAA	148
GAPDH	上游：CATGAGAAGTATGACAACAGCT 下游：AGTCCTTCCACGATACCAAAGT	197

图1 2组小鼠心脏组织病理学变化（HE染色，×100）

Fig.1 Heart histopathological changes in two groups of mice (HE staining, ×100)

表2 2组小鼠LVEF、FS、CK-MB水平比较 (n=6,$\bar{x}±s$)

Tab.2 Comparison of left ventricular ejection fraction, fraction shortening and CK-MB between two groups of mice

组别	LVEF	FS/%	CK-MB/（ng/L）
对照组	0.70±0.04	37.93±3.48	345.84±250.09
模型组	0.40±0.05	18.88±3.05	7 573.23±1 194.54
t	10.765^＊＊	10.084^＊＊	14.506^＊＊

图2 2组乳鼠心肌细胞线粒体膜电位（JC-1染色，×100）

Fig.2 The mitochondrial membrane potential of neonatal rat cardiomyocytes in two groups (JC-1 staining, ×100)

图3 2组乳鼠心肌细胞ROS水平（DCFH-DA染色，×100）

Fig.3 ROS levels of neonatal rat cardiomyocytes in two groups (DCFH-DA staining, ×100)

图4 Western blot检测2组AIM2、GSDMD、IL-1β及p17蛋白表达水平

Fig.4 Expression levels of AIM2, GSDMD, IL-1β and p17 in two groups detected by Western blot assay

表3 2组乳鼠心肌细胞AIM2、GSDMD、IL-1β、p17蛋白表达和上清液中IL-1β水平比较 (n=3,$\bar{x}±s$)

Tab.3 Comparison of AIM2, GSDMD, IL-1β, p17 protein expression levels and IL-1β content in supernatant between two groups

组别		AIM2		GSDMD		GSDMD-N
对照组		1.00±0.00		1.00±0.00		1.00±0.00
LPS处理组		3.65±1.49		2.47±0.16		1.57±0.21
t		3.103^＊		15.777^＊＊		4.637^＊
组别	IL-1β		p17		IL-1β/（ng/L）
对照组	1.00±0.00		1.00±0.00		1.07±0.35
LPS处理组	2.76±0.38		2.52±0.27		30.24±2.53
t	7.958^＊＊		9.782^＊＊		19.759^＊＊

表4 各组乳鼠心肌细胞中Naa20水平比较 (n=3,$\bar{x}±s$)

Tab.4 Comparison of Naa20 levels between four groups of neonatal rat cardiomyocytes

组别	Naa20 mRNA	Naa20蛋白
对照组	1.01±0.21	1.00±0.00
LPS 1 mg/L组	1.71±0.16^a	2.19±0.54^a
LPS 5 mg/L组	1.70±0.02^a	2.28±0.39^a
LPS 10 mg/L组	2.13±0.20^a	2.34±0.47^a
F	23.662^＊＊	7.296^＊

图5 各组乳鼠心肌细胞中 Naa20的蛋白表达水平

Fig.5 The expression levels of Naa20 protein in neonatal rat cardiomyocytes in four groups

表5 各组H9c2细胞活力和LDH比较 (n=3,$\bar{x}±s$)

Tab.5 Comparison of H9c2 cell viability and LDH levels between four groups

组别	细胞活力/%	LDH/（U/mg）
对照组	97.16±2.81	141.43±3.04
Naa20对照组	95.52±1.83	147.63±19.12
LPS组	46.99±3.96^a	254.22±14.92^a
Naa20+LPS组	74.44±3.15^b	182.20±5.94^b
F	178.801^＊＊	50.825^＊＊

图6 不同种属来源的AIM2氨基酸序列

Fig.6 Amino acid sequence analysis of different species AIM2

图7 Naa20与AIM2结合

Fig.7 Naa20 interacted with AIM2

图8 各组H9c2心肌细胞中AIM2乙酰化及AIM2、GSDMD、GSDMD-N的蛋白表达水平 A：对照组；B：Naa20对照组；C：LPS组；D：Naa20+LPS组。

Fig.8 The expression levels of ace-AIM2 and AIM2, GSDMD protein in four groups of H9c2 cells

表6 各组H9c2心肌细胞中AIM2乙酰化，AIM2、GSDMD、GSDMD-N蛋白及上清液IL-1β表达水平比较 (n=3,$\bar{x}±s$)

Tab.6 Comparison of ace-AIM2 and AIM2, GSDMD, GSDMD-N protein, and IL-1β content in supernatant between four groups of H9c2 cells

组别	Ac	AIM2	GSDMD	GSDMD-N	IL-1β/（ng/L）
对照组	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.07±0.30
Naa20对照组	0.97±0.27	0.92±0.23	0.96±0.24	1.12±0.20	1.07±0.18
LPS组	8.01±1.55^a	2.43±0.39^a	3.35±0.97^a	3.94±0.88^a	28.06±2.18^a
Naa20+LPS组	14.98±4.49^b	1.21±0.38^b	1.43±0.52^b	1.44±0.36^b	16.48±4.00^b
F	23.841^＊＊	16.919^＊＊	12.134^＊	24.692^＊＊	98.943^＊＊

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Naa20通过抑制AIM2表达减轻脓毒症心肌损伤

Naa20 alleviated myocardial injury in sepsis by inhibiting AIM2 expression

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