线粒体自噬-NLRP3炎症小体通路在新生大鼠缺血性脑损伤中的作用研究

doi:10.11958/20230415

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1181-1186.doi: 10.11958/20230415

线粒体自噬-NLRP3炎症小体通路在新生大鼠缺血性脑损伤中的作用研究

林永文(), 陈巧媚, 敖当, 黄炳龙, 骆成珠, 李承燕^△()

广东医科大学附属医院儿童医学中心（邮编524000）

收稿日期:2023-03-24 修回日期:2023-05-26 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：chengyan_may@163.com
作者简介:林永文（1971），男，副主任医师，主要从事儿科临床及基础方面研究。E-mail:fylinywen@163.com
基金资助:
广东省基础与应用基础研究项目(2019A1515110564);湛江市科技发展专项资金竞争性分配项目(2022A01181)

Research on effect of mitophagy-NLRP3 inflammasome pathway in cerebral ischemia-reperfusion of neonatal rats

LIN Yongwen(), CHEN Qiaomei, AO Dang, HUANG Binglong, LUO Chengzhu, LI Chengyan^△()

Department of Children’s Medical Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524000, China

Received:2023-03-24 Revised:2023-05-26 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：chengyan_may@163.com

林永文, 陈巧媚, 敖当, 黄炳龙, 骆成珠, 李承燕. 线粒体自噬-NLRP3炎症小体通路在新生大鼠缺血性脑损伤中的作用研究[J]. 天津医药, 2023, 51(11): 1181-1186.

LIN Yongwen, CHEN Qiaomei, AO Dang, HUANG Binglong, LUO Chengzhu, LI Chengyan. Research on effect of mitophagy-NLRP3 inflammasome pathway in cerebral ischemia-reperfusion of neonatal rats[J]. Tianjin Medical Journal, 2023, 51(11): 1181-1186.

摘要/Abstract

摘要：

目的探讨线粒体自噬-NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症小体通路对新生大鼠缺血性脑损伤的影响。方法将42只1周龄SD大鼠分为Sham组、脑缺血-再灌注损伤（CIRI）组、CIRI+二甲基亚砜（DMSO）组、CIRI+Mdivi-1组、CIRI+MCC950组、CIRI+Ac-YVAD-cmk组，每组7只。除Sham组外，其他各组采用单侧颈总动脉阻断法建立新生大鼠缺氧缺血性脑损伤模型。采用Londa法行神经行为障碍评分，HE染色观察海马体CA1区细胞形态，透射电子显微镜观察海马神经元超微结构，Western blot检测线粒体自噬和NLRP3炎症小体通路相关蛋白。结果与CIRI+DMSO组比较，CIRI+Mdivi-1组的神经行为障碍评分增加，突触数减少及肿胀线粒体数增多（均P<0.05）；与CIRI+Mdivi-1组比较，CIRI+MCC950组及CIRI+Ac-YVAD-cmk组神经行为障碍评分降低，突触数增多及肿胀线粒体数减少（均P<0.05）。与CIRI+DMSO组比较，CIRI+Mdivi-1组PTEN诱导的激酶1（PINK1）、Parkin、LC3Ⅱ/Ⅰ表达下调，线粒体外膜转位酶20（TOMM20）、P62及NLRP3、胱天蛋白酶（caspase）-1、caspase-8蛋白表达增加（P<0.05）。与CIRI+Mdivi-1组比较，CIRI+MCC950组及CIRI+Ac-YVAD-cmk组PINK1、Parkin、LC3Ⅱ/Ⅰ蛋白表达增加（P<0.05），CIRI+MCC950组NLRP3、caspase-1、caspase-8蛋白及CIRI+Ac-YVAD-cmk组caspase-1、caspase-8蛋白表达下调。结论激活线粒体自噬或抑制NLRP3炎症小体活化对新生大鼠缺血性脑损伤具有保护作用。

关键词: 线粒体自噬, NLR家族, 热蛋白结构域包含蛋白3, 婴儿, 新生, 疾病, 再灌注损伤, 炎症小体, PTEN诱导的激酶1, Parkin

Abstract:

Objective To investigate the effect of mitophagy-NOD-like receptor heat protein domain associated protein 3 (NLRP3) inflammasome pathway on cerebral ischemic injury in neonatal rats. Methods Forty-two 7-day SD rats were divided into the sham group, the cerebral ischemia-reperfusion injury (CIRI) group, the CIRI+dimethyl sulfoxide (DMSO) group, the CIRI+Mdivi-1 group, the CIRI+MCC950 group and the CIRI+Ac-YVAD-CMK group (7 rats in each group). Except for the sham group, the neonatal rat model of hypoxic-ischemic brain injury was established by unilateral common carotid artery occlusion. Neurological symptom score was performed by Londa method. Cell morphology in CA1 region of hippocampus was observed by hematoxylin-eosin (HE) staining. Ultrastructure of hippocampal neurons was observed by transmission electron microscopy, and related proteins of mitochondrial autophagy and NLRP3 inflammasome pathway were detected by Western blot assay. Results Compared with the CIRI+DMSO group, the CIRI+Mdivi-1 group had higher scores of neurobehavioral disorders, fewer synapses and more swollen mitochondria (all P<0.05). Compared with the CIRI+Mdivi-1 group, the neurobehavioral disorder score and the number of synapses increased, and the number of swollen mitochondria decreased in the CIRI+MCC950 group and the CIRI+Ac-YVAD-cmk group (all P<0.05). Compared with the CIRI+DMSO group, the protein expressions of PINK1, Parkin and LC3 Ⅱ/Ⅰ were down-regulated in the CIRI+Mdivi-1 group, and the protein expressions of TOMM20, P62, NLRP3, caspase-1 and caspase-8 were up-regulated (P<0.05). Compared with the CIRI+Mdivi-1 group, the protein expressions of PINK1, Parkin, LC3 Ⅱ/Ⅰ were up-regulated in the CIRI+MCC950 group and the CIRI+Ac-YVAD-cmk group (P<0.05). The expression levels of NLRP3, caspase-1 and caspase-8 proteins were down-regulated in the CIRI+MCC950 group, and caspase-1 and caspase-8 proteins decreased in the CIRI+Ac-YVAD-cmk group (P<0.05). Conclusion Activation of mitochondrial autophagy or inhibition of NLRP3 inflammasome activation have protective effects on cerebral ischemic injury in neonatal rats.

Key words: mitophagy, NLR family, pyrin domain-containing 3 protein, infant, newborn, diseases, reperfusion injury, symptomatic corpuscles, PTEN induced kinase 1, Parkin

中图分类号:

R722.12

图/表 7

图1 各组神经行为障碍评分统计图 A：Sham组；B：CIRI组；C：CIRI+DMSO组；D：CIRI+Mdivi-1组；E：CIRI+MCC950组；F：CIRI+Ac-YVAD-cmk组；图4同。F=25.610，P<0.05；a与Sham组比较，b与CIRI+DMSO组比较，c与CIRI+Mdivi-1组比较，P<0.05。

Fig.1 Statistical chart of neurobehavioral disorder scores in each group

图2 各组新生大鼠海马CA1区形态学表现（HE染色，×400）

Fig.2 Morphological manifestations of hippocampal CA1 tissue in each group of newborn rats （HE staining, ×400）

表1 各组新生大鼠海马CA1区透射电镜观察结果比较（n=7，个/视野，$\bar{x}±s$）

Tab.1 Comparison of synapses and swelling mitochondrias observed by transmission electron microscopy in the hippocampus CA1 tissue between six groups of neonatal rats

组别	突触数	肿胀线粒体数
Sham组	4.143±0.690	0.857±0.378
CIRI组	2.286±0.756^a	3.714±0.488^a
CIRI+DMSO组	2.286±0.951^a	4.000±0.817^a
CIRI+Mdivi-1组	0.857±0.378^b	6.143±0.900^b
CIRI+MCC950组	5.286±0.756^bc	1.714±0.756^bc
CIRI+Ac-YVAD-cmk组	4.429±0.535^bc	2.857±0.690^bc
F	39.570^＊＊	50.250^＊＊

图3 各组新生大鼠海马CA1区超微结构表现（A1—F1，×8 000；A2—F2，×30 000；A3—F3，×60 000） N：细胞核核仁；P：凋亡神经元细胞；红色箭头为线粒体；蓝色箭头为线粒体自噬；黑色箭头为突触后膜。

Fig.3 Ultrastructure of hippocampal CA1 tissue in each group of newborn rats (A1—F1，×8 000；A2—F2，×30 000；A3—F3，×60 000)

图4 各组新生大鼠海马CA1区线粒体自噬-NLRP3炎症小体蛋白Western blot条带

Fig.4 Western blot bands of mitophagy-NLRP3 inflammasome protein in hippocampal CA1 tissue in each group of newborn rats

表2 各组大鼠新生大鼠海马CA1区线粒体自噬相关蛋白相对表达水平比较（n=3，$\bar{x}±s$）

Tab.2 Comparison of mitophagy protein relative expression levels in hippocampal CA1 tissue of newborn rats between the groups

组别	PINK1	Parkin	TOMM20	LC3Ⅱ/Ⅰ	P62
Sham组	0.583±0.149	0.373±0.056	1.508±0.325	0.177±0.062	1.284±0.238
CIRI组	1.114±0.293^a	0.867±0.125^a	0.861±0.195^a	0.345±0.103^a	0.719±0.037^a
CIRI+DMSO组	1.096±0.283^a	0.864±0.126^a	0.895±0.143^a	0.365±0.128^a	0.723±0.032^a
CIRI+Mdivi-1组	0.581±0.073^b	0.413±0.027^b	1.874±0.275^b	0.183±0.047^b	1.425±0.099^b
CIRI+MCC950组	1.628±0.395^c	1.263±0.256^c	0.746±0.092^c	0.530±0.163^c	0.623±0.067^c
CIRI+Ac-YVAD-cmk组	1.388±0.341^ac	0.903±0.168^c	0.854±0.166^c	0.377±0.081^c	0.685±0.079^c
F	6.860^＊＊	15.740^＊＊	13.670^＊＊	4.830^＊	27.510^＊＊

表3 各组大鼠新生大鼠海马CA1区NLRP3炎症小体蛋白相对表达水平比较（n=3，$\bar{x}±s$）

Tab.3 Comparison of NLRP3 inflammasome protein relative expression levels in hippocampal CA1 tissue of newborn rats between the groups

组别	ASC	NLRP3	caspase-8	caspase-1
Sham组	0.326±0.079	0.233±0.044	0.478±0.067	0.430±0.100
CIRI组	0.869±0.253^a	0.482±0.088^a	1.039±0.081^a	0.835±0.142^a
CIRI+DMSO组	0.826±0.278^a	0.482±0.072^a	1.027±0.137^a	0.794±0.172^a
CIRI+Mdivi-1组	1.117±0.302	0.825±0.189^b	1.443±0.086^b	1.193±0.109^b
CIRI+MCC950组	0.511±0.097^c	0.366±0.087^c	0.516±0.050^bc	0.647±0.148^c
CIRI+Ac-YVAD-cmk组	0.673±0.090	0.488±0.119	0.648±0.051^bc	0.758±0.055^c
F	5.510^＊	9.630^＊＊	60.340^＊＊	11.690^＊＊

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线粒体自噬-NLRP3炎症小体通路在新生大鼠缺血性脑损伤中的作用研究

Research on effect of mitophagy-NLRP3 inflammasome pathway in cerebral ischemia-reperfusion of neonatal rats

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