褪黑素对心肌缺血再灌注损伤的保护作用及机制

doi:10.11958/20231005

摘要/Abstract

摘要：

目的探讨褪黑素调控蛋白激酶B（Akt）/叉头框转录因子O4（FOXO4）/B细胞淋巴瘤2样11蛋白（BIM）信号通路对心肌缺血再灌注损伤（IRI）的保护作用及其作用机制。方法建立心肌细胞IRI模型。细胞分为正常对照组（CTRL组）、IRI组、褪黑素组（IRI+MEL组）和Akt抑制剂组（IRI+MEL+MK2206组）。应用MTT法检测各组细胞存活率，流式细胞术检测各组细胞凋亡变化，酶标仪检测细胞乳酸脱氢酶（LDH）释放量；荧光定量PCR检测炎性因子白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）和单核细胞趋化蛋白-1（MCP-1）基因的表达，Western blot法检测各组磷酸化Akt（p-Akt）、磷酸化FOXO4（p-FOXO4）和BIM的蛋白表达变化。结果与CTRL组比较，IRI组细胞存活率下降，LDH释放量升高，细胞早期和总凋亡率升高，IL-6、TNF-α和MCP-1基因表达水平升高，p-Akt蛋白表达水平降低，p-FOXO4和BIM蛋白表达水平升高（P<0.05）。与IRI组比较，IRI+MEL组细胞存活率升高，LDH释放量降低，细胞早期和总凋亡率减少，IL-6、TNF-α和MCP-1基因表达水平降低，p-Akt蛋白表达水平升高，p-FOXO4和BIM蛋白表达水平降低（P<0.05）。Akt抑制剂MK2206可部分逆转褪黑素对IRI心肌细胞的保护作用。结论褪黑素对IRI的心肌细胞具有保护作用，其机制可能与调控Akt/FOXO4/BIM信号通路、降低细胞凋亡和炎症反应有关。

关键词: 褪黑激素, 再灌注损伤, 信号传导, 炎症, 心肌梗死

Abstract:

Objective To investigate the protective effect of melatonin on myocardial ischemia reperfusion injury(IRI) by regulating Akt/FOXO4/BIM signaling pathway and its mechanism. Methods Myocardial IRI model was established, and cells were divided into the control group (CTRL group), the ischemia reperfusion group (IRI group), the melatonin treatment group (IRI+MEL group) and the Akt inhibitor group (IRI+MEL+MK2206 group). MTT method was used to detect the cell survival rate. The level of released LDH was measured with microplate reader. Flow cytometry was used to detect cell apoptosis. PCR method was used to detect the inflammatory gene levels of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and monocyte chemotactic protein-1 (MCP-1). Western blot assay was used to detect the protein expression changes of p-Akt, p-FOXO4 and BIM. Results Compared with the CTRL group, the cell survival rate decreased and LDH content increased, the early stage and total apoptosis rates were increased, expression levels of IL-6, TNF-α and MCP-1 genes were increased, p-Akt protein expression decreased, and p-FOXO4 and BIM protein expression levels increased in the IRI group (P<0.05). Compared with the IRI group, melatonin therapy increased the cell survival rate and p-Akt protein expression, decreased LDH content, cell apoptosis, IL-6, TNF-α, MCP-1 gene level and p-FOXO4 and BIM protein expression (P<0.05). Compared with the IRI+MEL group, MK2206 partially reversed the protective effect of melatonin on IRI cardiomyocytes (P<0.05). Conclusion Melatonin has protective effect on ischemia reperfusion injured myocardial cells, and its mechanism may be related to the regulation of Akt/FOXO4/BIM signaling pathway to reduce apoptosis and cell inflammation.

Key words: melatonin, reperfusion injury, signal transduction, inflammation, myocardial infarction

中图分类号:

R542.2+2

图/表 7

参考文献 16

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基因名称	引物序列（5′→3′）	产物大小/bp
IL-6	上游：ACAACCACGGCCTTCCCTACTT	129
	下游：CACGATTTCCCAGAGAACATGTG	129
TNF-α	上游：GTAGCCCACGTCGTAGCAAA	214
	下游：TAGCAAATCGGCTGACGGTG	214
MCP-1	上游：ACCTGCTGCTACTCATTCACC	238
	下游：AAGACCTTAGGGCAGATGCAG	238
GAPDH	上游：TTCACCACCATGGAGAAGGC	237
	下游：GGCATGGACTGTGGTCATGA	237

基因名称	引物序列（5′→3′）	产物大小/bp
IL-6	上游：ACAACCACGGCCTTCCCTACTT	129
	下游：CACGATTTCCCAGAGAACATGTG	129
TNF-α	上游：GTAGCCCACGTCGTAGCAAA	214
	下游：TAGCAAATCGGCTGACGGTG	214
MCP-1	上游：ACCTGCTGCTACTCATTCACC	238
	下游：AAGACCTTAGGGCAGATGCAG	238
GAPDH	上游：TTCACCACCATGGAGAAGGC	237
	下游：GGCATGGACTGTGGTCATGA	237

组别	细胞存活率/%	LDH释放量/（U/mL）
CTRL组	100.00±9.77	0.24±0.05
IRI组	50.49±6.89^a	0.58±0.08^a
IRI+MEL组	77.39±4.60^b	0.35±0.06^b
IRI+MEL+MK2206组	56.25±7.88^c	0.59±0.07^c
F	53.762^＊	44.228^＊

组别	细胞存活率/%	LDH释放量/（U/mL）
CTRL组	100.00±9.77	0.24±0.05
IRI组	50.49±6.89^a	0.58±0.08^a
IRI+MEL组	77.39±4.60^b	0.35±0.06^b
IRI+MEL+MK2206组	56.25±7.88^c	0.59±0.07^c
F	53.762^＊	44.228^＊

组别	早期凋亡率	晚期凋亡率	总凋亡率
CTRL组	5.18±2.25	2.37±0.30	7.55±2.48
IRI组	31.50±2.38^a	4.04±2.87	35.54±4.99^a
IRI+MEL组	13.35±5.96^b	3.04±0.58	16.38±6.53^b
IRI+MEL+MK2206组	31.13±1.91^c	2.41±0.40	33.55±1.64^c
F	40.525^＊	2.363	34.462^＊