武汉市第一医院/武汉市中西医结合医院康复医学科,武汉 430022
Received:25 September 2023,
Revised:2024-05-05,
Accepted:13 May 2024,
Published:28 October 2024
移动端阅览
王映云,于利国,曹海燕,等.PERK信号通路介导的线粒体未折叠蛋白反应在低氧缺血性脑损伤中的作用[J].重庆医科大学学报,2024,49(10):1081-1087.
Wang Yingyun,Yu Liguo,Cao Haiyan,et al.Role of PERK signaling pathway-mediated mitochondrial unfolded protein response in hypoxic-ischemic brain injury[J].Journal of Chongqing Medical University,2024,49(10):1081-1087.
王映云,于利国,曹海燕,等.PERK信号通路介导的线粒体未折叠蛋白反应在低氧缺血性脑损伤中的作用[J].重庆医科大学学报,2024,49(10):1081-1087. DOI: 10.13406/j.cnki.cyxb.003608.
Wang Yingyun,Yu Liguo,Cao Haiyan,et al.Role of PERK signaling pathway-mediated mitochondrial unfolded protein response in hypoxic-ischemic brain injury[J].Journal of Chongqing Medical University,2024,49(10):1081-1087. DOI: 10.13406/j.cnki.cyxb.003608.
目的
2
探讨蛋白激酶RNA样ER激酶(protein kinase RNA-like ER kinase,PERK)信号通路介导的线粒体未折叠蛋白反应(mitochondrial unfolded protein response,mtUPR)在缺氧缺血性脑损伤(hypoxic-ischemic brain injury,HIBI)中的作用。
方法
2
将大鼠随机分为假手术(Sham)组和5个HIBI亚组(HIBI后3、6、12、24、48 h)。用于蛋白质印迹检测PERK、转录激活因子4(activating transcription factor 4,ATF4)、热休克蛋白60(heat shock protein 60,HSP60)蛋白的时程表达。将大鼠随机分为Sham组、HIBI组、HIBI + PERK组和HIBI+载体(Vector)组,每组15只。HIBI+PERK组和HIBI+Vector组大鼠在HIBI手术前1 h,将基于腺病毒相关病毒(adeno-associated virus,AAV)的PERK过表达质粒或AAV载体注射到脑室内,用于特异性表达PERK。在HIBI后24 h进行FJC染色分析神经元变性和DHE染色、酶联免疫吸附试验分析氧化应激。将大鼠随机分为Sham组、HIBI组、HIBI+PERK激动剂(CCT020312)组,每组12只。在HIBI手术前1 h,向HIBI+CCT020312组大鼠脑室内注射CCT020312。在HIBI后3周进行开阔场地测试和莫里斯水迷宫测试。
结果
2
与Sham组相比,PERK、ATF4、HSP60在HIBI后3 h开始明显升高,在12 h达到高峰,然后逐渐下降,直到48 h(
F
=60.23、56.72、74.31,均
P
<
0.001)。与HIBI组相比,HIBI+PERK组神经元变性的数量(100.2±3.1 vs
.
582.4±15.7,
P
<
0.001)、活性氧(reactive oxygen species,ROS)(42.4±2.9 vs
.
17.7±2.1,
P
<
0.01)、丙二醛(Malondialdehyde,MDA
)(0.81±0.06 vs
.
0.54±0.04,
P
<
0.001)水平显著降低,和谷胱甘肽过氧化物酶(glutathione peroxidase,GSHPx)(112.4±3.6 vs
.
177.5±6.6,
P
<
0.05)、超氧化物歧化酶(superoxide Dismutase,SOD)活性(46.3±1.9 vs
.
64.2±2.3,
P
<
0.05)活性明显增加。与Sham组相比,HIBI组大鼠海马组织中PERK(1.00±0.03 vs
.
1.66±0.08,
P
<
0.01)、ATF4(1.00±0.04 vs
.
1.53±0.06,
P
<
0.05)、动力蛋白相关蛋白1(dynamin-related protein 1,Drp1)(1.00±0.02 vs
.
1.98±0.07,
P
<
0.01)、HSP60(1.00±0.03 vs
.
1.37±0.04,
P
<
0.05)蛋白表达均明显增加(
P
<
0.05)。与HIBI组相比,HIBI+PERK组大鼠海马组织中PERK(1.66±0.08 vs.
2.95±0.17,
P
<
0.01)、ATF4(1.53±0.06 vs
.
3.42±0.22,
P
<
0.01)、HSP60(1.37±0.04 vs
.
2.03±0.09,
P
<
0.05)蛋白表达均明显增加(
F
=46.72、30.63、20.64,
P
<
0.001),和Drp1(1.98±0.07 vs
.
1.04±0.05,
P
<
0.05)蛋白表达明显降低(
F
=35.72,
P
<
0.001)。HIBI+CCT020312组的平均逃避潜伏期和平台穿越次数均较HIBI组明显增加(
F
=246.84、113.62,
P
<
0.001)。
结论
2
PERK减轻HIBI模型诱导的氧化应激和神经元凋亡,其机制可能涉及PERK/ATF4信号通路对mtUPR的调节。通过CCT020312给药具有神经保护作用。
Objective
2
To examine the role of mitochondrial unfolded protein response(mtUPR) mediated by the protein kinase RNA-like ER kinase(PERK) signaling pathway in hypoxic-ischemic brain injury(HIBI).
Methods
2
Rats were randomly divided into the sham group and five HIBI groups(3,6,12,24 and 48 h after HIBI) for Western blot analysis of PERK,activating transcription factor 4 (ATF4),and heat shock protein 60(HSP60) protein expression over time. In another experiment,rats were randomly divided into the sham group,HIBI group,HIBI+PERK group,and HIBI+Vector group,with 15 rats in each group. Rats in the HIBI+PERK group and HIBI+Vector group were given intracerebroventricular injection of adeno-associated virus(AAV)-based PERK overexpression plasmid or empty AAV vector 1 h before HIBI to induce specific PERK expression. FJC staining was performed 24 h after HIBI to analyze neuronal degeneration,and oxidative stress was examined by DHE staining and enzyme-linked immunosorbent assay. In a third experiment,rats were randomly divided into the sham group,HIBI group and HIBI+CCT020312(PERK agonist) group,with 12 rats in each group. Rats in the HIBI+CCT020312 group were given intracerebroventricular injection of CCT020312 one hour before HIBI. Open field test and Morris water maze test were performed 3 weeks after HIBI.
Results
2
Compared with the sham group,the HIBI groups showed significantly increased PERK,ATF4,and HSP60 expression at 3 h post-HIBI,which peaked at 12 h and then gradually decreased until 48 h(
F
=60.23,56.72,74.31,all
P
<
0.001). The HIBI+PERK group had significantly lower number of degenerated neurons(100.2±3.1 vs. 582.4±15.7,
P
<
0.001),reactive oxygen species(ROS) level (42.4±2.9 vs.
17.7±2.1,
P
<
0.01),and malondialdehyde(MDA) level(0.81±0.06 vs. 0.54±0.04,
P
<
0.001),but significantly higher glutathione peroxidase(GSH-Px) activity (112.4±3.6 vs. 177.5±6.6,
P
<
0.05) and superoxide dismutase (SOD) activity (46.3±1.9 vs. 64.2±2.3,
P
<
0.05),as compared with the HIBI group. PERK(1.00±0.03 vs. 1.66±0.08,
P
<
0.01),ATF4 (1.00±0.04 vs. 1.53±0.06,
P
<
0.05),dynamin-related protein 1 (Drp1;1.00±0.02 vs. 1.98±0.07,
P
<
0.01),and HSP60 (1.00±0.03 vs. 1.37±0.04,
P
<
0.05) protein expression in the hippocampal tissue were significantly higher in the HIBI group than in the sham group. Furthermore,the HIBI+PERK group demonstrated signif
icantly higher PERK(1.66±0.08 vs. 2.95±0.17,
P
<
0.01),ATF4(1.53±0.06 vs. 3.42±0.22,
P
<
0.01),and HSP60 (1.37±0.04 vs. 2.03±0.09,
P
<
0.05) protein expression(
F
=46.72,30.63,20.64,
P
<
0.001) but significantly lower Drp1(1.98±0.07 vs.
1.04±0.05,
P
<
0.05) protein expression in the hippocampal tissue than the HIBI group(
F
=35.72,
P
<
0.001). Mean escape latency and the number of platform crossings were significantly higher in the HIBI+CCT020312 group than in the HIBI group(
F
=246.84,113.62,
P
<
0.001).
Conclusion
2
PERK attenuates oxidative stress and neuronal apoptosis induced by HIBI,possibly through the regulation of mtUPR via the PERK/ATF4 signaling pathway. CCT020312 has neuroprotective effects in HIBI.
You Q , Lan XB , Liu N , et al . Neuroprotective strategies for neonatal hypoxic-ischemic brain damage:current status and challenges [J ] . Eur J Pharmacol , 2023 , 957 : 176003 .
Greco P , Nencini G , Piva I , et al . Pathophysiology of hypoxic-ischemic encephalopathy:a review of the past and a view on the future [J ] . Acta Neurol Belg , 2020 , 120 ( 2 ): 277 - 288 .
Lee BL , Glass HC . Cognitive outcomes in late childhood and adolescence of neonatal hypoxic-ischemic encephalopathy [J ] . Clin Exp Pediatr , 2021 , 64 ( 12 ): 608 - 618 .
Chevin M , Chabrier S , Allard MJ , et al . Necroptosis blockade potentiates the neuroprotective effect of hypothermia in neonatal hypoxic-ischemic encephalopathy [J ] . Biomedicines , 2022 , 10 ( 11 ): 2913 .
Cheng S , Chen C , Wang LL . Gelsemine exerts neuroprotective effects on neonatal mice with hypoxic-ischemic brain injury by suppressing inflammation and oxidative stress via Nrf2/HO-1 pathway [J ] . Neurochem Res , 2023 , 48 ( 5 ): 1305 - 1319 .
Arteaga Cabeza O , Zhang Z , Smith Khoury E , et al . Neuroprotective effects of a dendrimer-based glutamate carboxypeptidase inhibitor on superoxide dismutase transgenic mice after neonatal hypoxic-ischemic brain injury [J ] . Neurobiol Dis , 2021 , 148 : 105201 .
Lu HD , Wang XL , Li M , et al . Mitochondrial unfolded protein response and integrated stress response as promising therapeutic targets for mitochondrial diseases [J ] . Cells , 2022 , 12 ( 1 ): 20 .
Lee Y , Heo JY , Ju XS , et al . General anesthesia activates the mitochondrial unfolded protein response and induces age-dependent,long-lasting changes in mitochondrial function in the developing brain [J ] . Neurotoxicology , 2021 , 82 : 1 - 8 .
Zhao Y , Li HX , Luo Y , et al . Lycopene mitigates DEHP-induced hepatic mitochondrial quality control disorder via regulating SIRT1/PINK1/mitophagy axis and mitochondrial unfolded protein response [J ] . Environ Pollut , 2022 , 292 ( Pt B ): 118390 .
Zhu MX , Ma XF , Niu X , et al . Mitochondrial unfolded protein response in ischemia-reperfusion injury [J ] . Brain Res , 2022 , 1797 : 148116 .
Dastidar SG , Pham MT , Mitchell MB , et al . 4E-BP1 protects neurons from misfolded protein stress and Parkinson’s disease toxicity by inducing the mitochondrial unfolded protein response [J ] . J Neurosci , 2020 , 40 ( 45 ): 8734 - 8745 .
Feng Y , Nouri K , Schimmer AD . Mitochondrial ATP-dependent proteases-biological function and potential anti-cancer targets [J ] . Cancers , 2021 , 13 ( 9 ): 2020 .
Lebeau J , Saunders JM , Moraes VWR , et al . The PERK arm of the unfolded protein response regulates mitochondrial morphology during acute endoplasmic reticulum stress [J ] . Cell Rep , 2018 , 22 ( 11 ): 2827 - 2836 .
Almeida LM , Pinho BR , Duchen MR , et al . The PERKs of mitochondria protection during stress:insights for PERK modulation in neurodegenerative and metabolic diseases [J ] . Biol Rev Camb Philos Soc , 2022 , 97 ( 5 ): 1737 - 1748 .
Leterme S , Michaud M . Mitochondrial membrane biogenesis:a new pathway for lipid transport mediated by PERK/E-Syt1 complex [J ] . J Cell Biol , 2023 , 222 ( 3 ): e202301132 .
Xue H , Zhang YH , Gao QS , et al . Sevoflurane post-conditioning ameliorates neuronal deficits and axon demyelination after neonatal hypoxic ischemic brain injury:role of microglia/macrophage [J ] . Cell Mol Neurobiol , 2021 , 41 ( 8 ): 1801 - 1816 .
Ji XL , Zhou YY , Gao QQ , et al . Functional reconstruction of the basal Ganglia neural circuit by human striatal neurons in hypoxic-ischaemic injured brain [J ] . Brain , 2023 , 146 ( 2 ): 612 - 628 .
Jiang Y , Li D , Du ZQ , et al . Perampanel stimulates mitochondrial biogenesis in neuronal cells through activation of the SIRT1/PGC-1α signaling pathway [J ] . ACS Chem Neurosci , 2021 , 12 ( 2 ): 323 - 329 .
Meng H , Sun LK , Su J , et al . Serine protease HtrA2/Omi regulates adaptive mitochondrial reprogramming in the brain cortex after ischemia/reperfusion injury via UCP2-SIRT3-PGC1 axis [J ] . Hum Cell , 2022 , 35 ( 1 ): 63 - 82 .
Al Rahim M , Thatipamula S , Pasinetti GM , et al . Neuronal pentraxin 1 promotes hypoxic-ischemic neuronal injury by impairing mitochondrial biogenesis via interactions with active bax[6A7]and mitochondrial hexokinase II [J ] . ASN Neuro , 2021 , 13 : 17590914211012888 .
Lee MJ , Jang Y , Zhu JB , et al . Auraptene enhances junction assembly in cerebrovascular endothelial cells by promoting resilience to mitochondrial stress through activation of antioxidant enzymes and mtUPR [J ] . Antioxidants , 2021 , 10 ( 3 ): 475 .
Yuan Y , Tian Y , Jiang H , et al . Mechanism of PGC-1α-mediated mitochondrial biogenesis in cerebral ischemia-reperfusion injury [J ] . Front Mol Neurosci , 2023 , 16 : 1224964 .
Sasaki K , Uchiumi T , Toshima T , et al . Mitochondrial translation inhibition triggers ATF4 activation,leading to integrated stress response but not to mitochondrial unfolded protein response [J ] . Biosci Rep , 2020 , 40 ( 11 ): BSR20201289 .
Chen YB , Mi YJ , Zhang XF , et al . Dihydroartemisinin-induced unfolded protein response feedback attenuates ferroptosis via PERK/ATF4/HSPA5 pathway in glioma cells [J ] . J Exp Clin Cancer Res , 2019 , 38 ( 1 ): 402 .
Yang Z , Shi XY , Ren JL , et al . Mitochondrial homeostasis is involved in inhibiting hippocampus neuronal apoptosis during ZSWF ameliorate the cognitive dysfunction of SAMP8 mice [J ] . J Funct Foods , 2022 , 91 : 105010 .
Fan P , Jordan VC . PERK,beyond an unfolded protein response sensor in estrogen-induced apoptosis in endocrine-resistant breast cancer [J ] . Mol Cancer Res , 2022 , 20 ( 2 ): 193 - 201 .
Yu XM , Wang XY , Xie YY , et al . Activating transcription factor 4-mediated mitochondrial unfolded protein response alleviates hippocampal neuronal damage in an in vitro model of epileptiform discharges [J ] . Neurochem Res , 2023 , 48 ( 7 ): 2253 - 2264 .
Luo BY , Zhou HS , Sun YF , et al . The fate and prospects of stem cell therapy in the treatment of hypoxic-ischemic encephalopathy [J ] . Eur J Neurosci , 2023 , 58 ( 1 ): 2384 - 2405 .
Zhong L , Wang JJ , Wang P , et al . Neural stem cell-derived exosomes and regeneration:cell-free therapeutic strategies for traumatic brain injury [J ] . Stem Cell Res Ther , 2023 , 14 ( 1 ): 198 .
Chen XD , Malaeb SN , Pan J , et al . Editorial:Perinatal hypoxic-ischemic brain injury:mechanisms,pathogenesis,and potential therapeutic strategies [J ] . Front Cell Neurosci , 2022 , 16 : 1086692 .
0
Views
9
下载量
0
CSCD
Publicity Resources
Related Articles
Related Author
Related Institution
京公网安备11010802024621
