miR-582-5p靶向调控FOXO1对新生大鼠缺血缺氧性脑病神经元损伤的影响

doi:10.11958/20230591

天津医药 ›› 2024, Vol. 52 ›› Issue (4): 356-361.doi: 10.11958/20230591

miR-582-5p靶向调控FOXO1对新生大鼠缺血缺氧性脑病神经元损伤的影响

颜海峰¹(), 吴小红¹, 林欲庆¹, 霍开明¹, 王莹莹²

1 海南医学院第二附属医院儿科二区（邮编570311）
2 海南医学院第二附属医院检验科（邮编570311）

收稿日期:2023-04-19 修回日期:2023-06-16 出版日期:2024-04-15 发布日期:2024-04-19
作者简介:颜海峰（1980），男，副主任医师，主要从事新生儿（早产儿）医学、重症医学、营养与儿童健康方面研究。E-mail：vj3byi2@163.com
基金资助:
2020年度海南省卫生健康行业科研项目(琼卫科教（2020）9号389)

Effect of miR-582-5p targeting regulation of FOXO1 on neuronal damage in neonatal rats with hypoxic ischemic encephalopathy

YAN Haifeng¹(), WU Xiaohong¹, LIN Yuqing¹, HUO Kaiming¹, WANG Yingying²

1 Department of Pediatric Area II, the Second Affiliated Hospital of Hainan Medical College, Haikou 570311, China
2 Department of Laboratory, the Second Affiliated Hospital of Hainan Medical College, Haikou 570311, China

Received:2023-04-19 Revised:2023-06-16 Published:2024-04-15 Online:2024-04-19

颜海峰, 吴小红, 林欲庆, 霍开明, 王莹莹. miR-582-5p靶向调控FOXO1对新生大鼠缺血缺氧性脑病神经元损伤的影响[J]. 天津医药, 2024, 52(4): 356-361.

YAN Haifeng, WU Xiaohong, LIN Yuqing, HUO Kaiming, WANG Yingying. Effect of miR-582-5p targeting regulation of FOXO1 on neuronal damage in neonatal rats with hypoxic ischemic encephalopathy[J]. Tianjin Medical Journal, 2024, 52(4): 356-361.

摘要/Abstract

摘要：

目的探讨微小RNA-582-5p（miR-582-5p）靶向调控叉头框转录因子1（FOXO1）对新生大鼠缺血缺氧性脑病（HIE）神经元损伤的影响。方法 90只新生大鼠按照随机数字表法均分为对照（NC）组、模型（HIE）组、miRNA对照（LV-miRNA-NC）组、miR-582-5p过表达（LV-miR-582-5p）组、miR-582-5p过表达+mRNA对照（LV-miR-582-5p+LV-NC）组、miR-582-5p过表达+FOXO1过表达（LV-miR-582-5p+LV-FOXO1）组。除NC组外的各组大鼠建立HIE模型，对大鼠进行神经功能缺损评分，TTC染色测定脑梗死体积，Real-time PCR检测miR-582-5p和FOXO1表达，双萤光素酶报告基因实验检测miR-582-5p和FOXO1靶向关系，HE染色观察海马组织病理变化，TUNEL和Neu N荧光双标共定位检测海马组织神经元凋亡，免疫组化染色检测FOXO1、胱天蛋白酶3（Caspase-3）蛋白表达。结果 miR-582-5p和FOXO1具有靶向关系，与NC组比较，HIE组大鼠神经功能缺损评分、脑梗死体积、FOXO1表达、神经元凋亡率、FOXO1、Caspase-3蛋白表达增加，miR-582-5p表达降低，海马组织出现病理损伤（P<0.05）；与LV-miRNA-NC组比较，LV-miR-582-5p组大鼠神经功能缺损评分、脑梗死体积、FOXO1表达、神经元凋亡率、FOXO1、Caspase-3蛋白表达降低，miR-582-5p表达增加，海马组织病理损伤好转（P<0.05）；LV-FOXO1可以逆转LV-miR-582-5p对于HIE大鼠神经元损伤的保护作用。结论 miR-582-5p可以直接靶向负调控FOXO1表达，减少HIE新生大鼠神经元凋亡，对神经损伤具有保护作用。

关键词: 缺氧缺血, 脑, 创伤, 神经系统, 叉头框蛋白O1, 微小RNA-582-5p

Abstract:

Objective To investigate the effect of microRNA-582-5p (miR-582-5p) on neuronal damage in neonatal rats with hypoxic ischemic encephalopathy (HIE) through targeted regulation of forkhead box transcription factor O1 (FOXO1). Methods Newborn rats (n=90) were randomly grouped into the control (NC) group, the model (HIE) group, the miRNA non-specific control (LV-miRNA-NC) group, the miR-582-5p overexpression (LV-miR-582-5p) group, the miR-582-5p overexpression+mRNA control (LV-miR-582-5p+LV-NC) group and the miR-582-5p overexpression+FOXO1 overexpression (LV-miR-582-5p+LV-FOXO1) group. HIE model was established in all groups except the NC group, and neurological deficits were scored on rats. TTC staining was applied to measure the volume of cerebral infarction. Real-time PCR was applied to detect miR-582-5p and FOXO1 expression. Dual fluorescence reporter gene experiment was applied to detect the targeting relationship between miR-582-5p and FOXO1. HE staining method was applied to observe pathological changes in hippocampal tissue. TUNEL and NeuN fluorescence dual labeling co localization were applied to detect neuronal apoptosis in hippocampal tissue. Immunohistochemistry was applied to detect expressions of FOXO1 and Caspase-3 proteins. Results There was a targeting relationship between MiR-582-5p and FOXO1. Compared with the NC group, the neurological deficit score, cerebral infarction volume, FOXO1 expression, neuronal apoptosis rate, FOXO1 and Caspase-3 protein expression were increased in the HIE group, and the miR-582-5p expression obviously decreased, the hippocampal tissue showed obvious pathological damage (P<0.05). Compared with the LV-miRNA-NC group, the neurological deficit score, cerebral infarction volume, FOXO1 expression, neuronal apoptosis rate, FOXO1 and Caspase-3 protein expression obviously decreased in the LV-miR-582-5p group, and the miR-582-5p expression obviously increased, the pathological damage of hippocampus tissue was obviously improved (P<0.05). LV-FOXO1 was able to reverse the protective effect of LV-miR-582-5p on neuronal damage in HIE rats. Conclusion MiR-582-5p can directly target FOXO1, negatively regulate FOXO1 expression, reduce neuronal apoptosis in HIE neonatal rats, and have a protective effect on neural injury.

Key words: hypoxia-ischemia, brain, trauma, nervous system, forkhead box protein O1, microRNA-582-5p

中图分类号:

R342，R722.1

图/表 8

图1 各组大鼠脑梗死体积变化（TTC染色）

Fig.1 Cerebral infarction volume of rats in each group (TTC staining)

表1 各组大鼠神经功能缺损评分、脑梗死体积比较（$\bar{x}±s$）

Tab.1 Comparison of neurological deficit score and cerebral infarction volume between six groups

组别	神经功能缺损评分/分（n=15）	脑梗死体积/ %（n=5）
NC组	0.00±0.00	0.00±0.00
HIE组	2.54±0.42^a	31.14±4.41^a
LV-miRNA-NC组	2.32±0.34	32.02±3.95
LV-miR-582-5p组	1.51±0.32^b	15.32±2.33^b
LV-miR-582-5p+LV-NC组	1.32±0.36	16.10±2.46
LV-miR-582-5p+LV-FOXO1组	2.15±0.40^c	23.01±4.40^c
F	114.531^＊＊	64.997^＊＊

表2 各组大鼠海马组织miR-582-5p和FOXO1表达比较（n=5，$\bar{x}±s$）

Tab.2 Comparison of miR-582-5p and FOXO1 expression in hippocampal tissue of rats between six groups

组别	FOXO1	miR-582-5p
NC组	1.00±0.02	1.00±0.02
HIE组	1.54±0.12^a	0.38±0.04^a
LV-miRNA-NC组	1.52±0.13	0.35±0.05
LV-miR-582-5p组	0.70±0.08^b	0.89±0.08^b
LV-miR-582-5p+LV-NC组	0.68±0.07	0.76±0.06
LV-miR-582-5p+LV-FOXO1组	1.12±0.09^c	0.73±0.04
F	84.133^＊＊	131.981^＊＊

图2 miR-582-5p与FOXO1基因的靶向关系验证

Fig.2 Verification of the targeting relationship between miR-582-5p and FOXO1 gene

图3 大鼠海马组织病理学变化（HE染色，×200）

Fig.3 Histopathological changes of rat hippocampus (HE staining, ×200)

图4 海马组织神经元凋亡（×200） Neu N和TUNEL荧光双染，Neu N为红色，TUNEL为绿色。

Fig.4 Hippocampal neuron apoptosis (×200)

表3 各组大鼠海马组织FOXO1、Caspase-3蛋白阳性表达比较（n=5，%，$\bar{x}±s$）

Tab.3 Comparison of positive expressions of FOXO1 and Caspase-3 in hippocampal tissue of rats between six groups

组别	FOXO1	Caspase-3
NC组	8.52±1.01	6.25±1.14
HIE组	26.21±3.42^a	21.32±3.02^a
LV-miRNA-NC组	25.34±3.05	21.03±3.42
LV-miR-582-5p组	11.02±1.78^b	8.77±1.25^b
LV-miR-582-5p+LV-NC组	11.55±1.56	9.02±1.44
LV-miR-582-5p+LV-FOXO1组	19.01±2.44^c	17.32±1.74^c
F	52.857^＊＊	47.163^＊＊

图5 海马组织FOXO1、Caspase-3蛋白表达（免疫组化，×200） A、B、C、D、E、F依次为NC组、HIE组、LV-miRNA-NC组、LV-miR-582-5p组、LV-miR-582-5p+LV-NC组、LV-miR-582-5p+LV-FOXO1组。

Fig.5 Expressions of FOXO1 and Caspase-3 in hippocampus (immunohistochemistry, ×200)

参考文献 18

[1]	LI B, DASGUPTA C, HUANG L, et al. MiRNA-210 induces microglial activation and regulates microglia-mediated neuroinflammation in neonatal hypoxic-ischemic encephalopathy[J]. Cell Mol Immunol, 2020, 17(9):976-991. doi:10.1038/s41423-019-0257-6.
[2]	曾高, 刘雨桐, 孙澎, 等. 儿童脑动静脉畸形治疗策略[J]. 中国现代神经疾病杂志, 2023, 23(5):388-392.
	ZENG G, LIU Y T, SUN P, et al. Treatment of pediatric cerebral arteriovenous malformation[J]. Chin J Contemp Neurol Neurosurg, 2023, 23(5):388-392.
[3]	CHEN R, WANG M, FU S, et al. MicroRNA-204 may participate in the pathogenesis of hypoxic-ischemic encephalopathy through targeting KLLN[J]. Exp Ther Med, 2019, 18(5):3299-3306. doi:10.3892/etm.2019.7936.
[4]	NIU R Z, WANG L Q, YANG W, et al. MicroRNA-582-5p targeting Creb1 modulates apoptosis in cardiomyocytes hypoxia/reperfusion-induced injury[J]. Immun Inflamm Dis, 2022, 10(11):e708-e721. doi:10.1002/iid3.708.
[5]	WANG D, WANG Y, ZOU X, et al. FOXO1 inhibition prevents renal ischemia-reperfusion injury via cAMP-response element binding protein/PPAR-γ coactivator-1α-mediated mitochondrial biogenesis[J]. Br J Pharmacol, 2020, 177(2):432-448. doi:10.1111/bph.14878.
[6]	HE F, ZHANG N, LV Y, et al. Low-dose lipopolysaccharide inhibits neuronal apoptosis induced by cerebral ischemia/reperfusion injury via the PI3K/Akt/FoxO1 signaling pathway in rats[J]. Mol Med Rep, 2019, 19(3):1443-1452. doi:10.3892/mmr.2019.9827.
[7]	刘鑫, 霍世芳, 马中岭, 等. 微小RNA-126靶向调控血管内皮生长因子对新生大鼠缺氧-缺血性脑病神经元损伤的影响[J]. 解剖学报, 2021, 52(6):875-881.
	LIU X, HUO S F, MA Z L, et al. Effect of targeting vascular endothelial growth factor by microRNA-126 on neuronal damage in neonatal rats with hypoxic-ischemic encephalopathy[J]. Acta Anatomica Sinica, 2021, 52(6):875-881. doi:10.16098/j.issn.0529-1356.2021.06.006.
[8]	XIONG Q, LI X, XIA L, et al. Dihydroartemisinin attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting oxidative stress[J]. Mol Brain, 2022, 15(1):36. doi:10.1186/s13041-022-00921-y.
[9]	DIAO M, QU Y, LIU H, et al. Effect of carbamylated erythropoietin on neuronal apoptosis in fetal rats during intrauterine hypoxic-ischemic encephalopathy[J]. Biol Res, 2019, 52(1):28. doi:10.1186/s40659-019-0234-7.
[10]	AL-WARD H, LIU N, OMOROU M, et al. The relationship between miRNA-210 and SCN1B in fetal rats with hypoxic-ischemic brain injury[J]. Biosci Rep, 2023, 43(1):BSR20222016. doi:10.1042/BSR20222016.
[11]	WU Z, BAI Y, QI Y, et al. lncRNA NEAT1 downregulation ameliorates the myocardial infarction of mice by regulating the miR-582-5p/F2RL2 axis[J]. Cardiovasc Ther, 2022, 2022:4481360. doi:10.1155/2022/4481360.
[12]	ZHANG Y F, LI X X, CAO X L, et al. MicroRNA-582-5p contributes to the maintenance of neural stem cells through inhibiting secretory protein FAM19A1[J]. Front Cell Neurosci, 2022, 16:866020. doi:10.3389/fncel.2022.866020.
[13]	MEI Z G, HUANG Y G, FENG Z T, et al. Electroacupuncture ameliorates cerebral ischemia/reperfusion injury by suppressing autophagy via the SIRT1-FOXO1 signaling pathway[J]. Aging (Albany NY), 2020, 12(13):13187-13205. doi:10.18632/aging.103420.
[14]	LIU Y, JIANG J, WANG X, et al. miR-582-5p is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO1[J]. PLoS One, 2013, 8(10):e78381. doi:10.1371/journal.pone.0078381.
[15]	MA Q, ZHANG L, PEARCE W J. MicroRNAs in brain development and cerebrovascular pathophysiology[J]. Am J Physiol Cell Physiol, 2019, 317(1):C3-C19. doi:10.1152/ajpcell.00022.2019.
[16]	TOADER A M, HOTEIUC O, BIDIAN C, et al. Neuronal apoptosis can be prevented by the combined therapy with melatonin and hypothermia in a neonatal rat model of hypoxic-ischemic encephalopathy[J]. Med Pharm Rep, 2021, 94(2):197-207. doi:10.15386/mpr-1903.
[17]	RODRIGUEZ J, LI T, XU Y, et al. Role of apoptosis-inducing factor in perinatal hypoxic-ischemic brain injury[J]. Neural Regen Res, 2021, 16(2):205-213. doi:10.4103/1673-5374.290875.
[18]	WANG X X, ZHANG B, XIA R, et al. Inflammation, apoptosis and autophagy as critical players in vascular dementia[J]. Eur Rev Med Pharmacol Sci, 2020, 24(18):9601-9614. doi:10.26355/eurrev_202009_23048.

miR-582-5p靶向调控FOXO1对新生大鼠缺血缺氧性脑病神经元损伤的影响

Effect of miR-582-5p targeting regulation of FOXO1 on neuronal damage in neonatal rats with hypoxic ischemic encephalopathy

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 8

参考文献 18

相关文章 15

编辑推荐

Metrics

本文评价

[1]	焦爱菊, 任宝龙, 张春花, 李文瑞, 赵玮婧. NIHSS评分联合血清BDNF、IL-6对脑卒中后抑郁的预测价值[J]. 天津医药, 2024, 52(9): 963-966.
[2]	范哲华, 刘建荣. 葡萄糖依赖性促胰岛素多肽与多囊卵巢综合征相关性的研究进展[J]. 天津医药, 2024, 52(9): 996-999.
[3]	周程继, 唐勇, 江朋, 胡周全, 卫伟, 王国安, 付晓飞. 棕榈酰转移酶修饰的NOD2在小鼠心肺复苏后脑损伤中的作用[J]. 天津医药, 2024, 52(8): 804-808.
[4]	王俊懿, 李宸, 吴昕岳, 丁心语, 万春晓. 早期运动干预对脑缺血大鼠脑神经髓鞘的影响及机制研究[J]. 天津医药, 2024, 52(6): 589-594.
[5]	薛晶, 元小冬, 邢爱君, 王连辉, 马倩, 符永山, 张萍淑. 急性缺血性脑卒中患者睡眠-觉醒生物节律变化与预后的关系研究[J]. 天津医药, 2024, 52(6): 614-619.
[6]	陈惠刚, 池小锋, 封娣, 米娅莉. 黄芪甲苷抑制Fas/FasL信号通路减轻创伤性脑损伤大鼠神经功能缺损和神经元凋亡[J]. 天津医药, 2024, 52(5): 469-474.
[7]	邵红霞, 吴涓涓, 于洪志, 吴琦, 武俊平. 血行播散性肺结核合并颅内结核的临床特点[J]. 天津医药, 2024, 52(5): 495-498.
[8]	贾西瑞, 刘莉洁. 小胶质细胞在脓毒症相关性脑病中的作用及研究进展[J]. 天津医药, 2024, 52(5): 557-560.
[9]	武潇潇, 胡玉鲲, 吴江. 积雪草酸对实验性蛛网膜下腔出血大鼠脑损伤的保护作用[J]. 天津医药, 2024, 52(3): 285-289.
[10]	张文超, 杨雪辉, 尹涛, 王睿健, 张盟盟. 自发性急性脑出血患者血浆sCD163/sTWEAK比值与预后的关系[J]. 天津医药, 2024, 52(3): 297-301.
[11]	徐丹, 刘夏, 钟殿胜. 佐利替尼一线治疗EGFR突变NSCLC伴中枢神经系统转移2例报告[J]. 天津医药, 2024, 52(3): 315-318.
[12]	易娜, 肖雯, 田源, 袁李礼. BMAL1减轻H₂O₂诱导的心肌细胞损伤机制研究[J]. 天津医药, 2024, 52(2): 119-123.
[13]	周秀芬, 刘晖, 陈虹, 赵宗波. SAP患者GLI与外周血各指标变化的临床意义[J]. 天津医药, 2024, 52(12): 1286-1291.
[14]	刘鹏程, 方无杰, 王晓涛, 邓典峰. 天麻钩藤饮联合丁苯酞对急性脑梗死患者症状改善及血管弹性的影响[J]. 天津医药, 2024, 52(12): 1326-1330.
[15]	丁宁, 李亚杰, 刘敏琦, 李奇, 邢政, 褚晓蕾, 徐卫国. 慢性疼痛患者脑电时间-频率信号变化研究进展[J]. 天津医药, 2024, 52(12): 1340-1344.