LACC1 Aggravated Ischemia Reperfusion by Phosphorylate AMP-activated Protein Kinase/Nucleotide-binding Oligomerization Domain-like Receptor Protein 3 in Cerebral Infarction

doi:10.3969/j.issn.1673-5765.2023.09.006

Abstract

Abstract: Objective To investigate the effect and mechanism of laccase gene LACC1 on ischemic reperfusion after cerebral infarction.
Methods ①Purchase 20 C57BL/6J LACC1 knockout (LACC1-/-) mice and 20 wild-type (WT) mice, and establish middle cerebral artery occlusion/reperfusion (MCAO/R) model with 15 mice each, the volume of cerebral infarction was compared between the two groups, and western blot was performed to detect phosphorylate AMP-activated protein kinase (p-AMPK) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in brain tissue. Microarray analysis of long noncoding RNA (lncRNA) expression profiles in peripheral blood and exploration of possible signal transduction pathways involved. ②Preparation of a mouse microglia oxygen glucose deprivation/reperfusion (OGD/R) model and upregulation and inhibition of LACC1 expression by siRNA transfection technique to clarify the regulatory effects of LACC1 on inflammation and oxidative stress in an in vitro model of cerebral infarction, western blot detection levels of p-AMPK and NLRP3. Serum catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), reactive oxygen species (ROS), IL-1β, IL-6, interferon-γ (INF-γ), TNF-α levels were measured by enzyme linked immunosorbent assay (ELISA).
Results The proportion of cerebral infarction volume in WT mouse group of MCAO/R model was (21.38%±4.06%) and that in LACC1-/- mouse group was (19.07%±2.86%), the difference was statistically significant (P=0.041). Meanwhile, the expression level of p-AMPK protein in brain tissue of LACC1-/- mice was increased. NLRP3 protein expression was inhibited. Down-regulation of LACC1 inhibited the expression of NLRP3 protein and increased the expression of p-AMPK protein in OGD/R cell models. In OGD/R cell model, over expression of LACC1 increased the production of IL-1β, IL-6, INF-γ, TNF-α, MDA and ROS, and decreased the levels of CAT and SOD (P<0.05).
Conclusions LACC1 may exacerbate the inflammatory response after ischemia-reperfusion via the AMPK/NLRP3 pathway, which may provide a new therapeutic option for cerebral infarction or other neurological diseases and their related complications.

Key words: LACC1; Stroke; Inflammation; AMP-activated protein kinase; Nucleotide-binding oligomerization domain-like receptor protein 3

摘要： 目的探讨漆酶基因LACC1对脑梗死后缺血再灌注损伤的影响及其机制。
方法 ①采购C57BL/6J LACC1基因敲除（LACC1-/-）小鼠20只，野生型小鼠20只，建立大脑中动脉闭塞/再灌注（middle cerebral artery occlusion/reperfusion，MCAO/R）模型各15只，比较两组小鼠的脑梗死体积。采用蛋白质免疫印迹实验检测脑组织中磷酸化腺苷酸活化蛋白激酶（phosphorylate AMP-activated protein kinase，p-AMPK）及NOD样受体热蛋白结构域相关蛋白3（nucleotide-binding oligomerization domain-like receptor protein 3，NLRP3）水平，采用微阵列分析外周血中长链非编码RNA（long noncoding RNA，lncRNA）表达谱及探讨可能涉及的信号转导通路。②制备小鼠小胶质细胞氧糖剥夺/再灌注（oxygen glucose deprivation/reoxygenation，OGD/R）模型，并通过小干扰RNA（siRNA）转染技术上调及抑制LACC1的表达，明确LACC1对脑梗死体外模型炎症和氧化应激的调节作用。采用蛋白质免疫印迹实验检测p-AMPK、NLRP3水平，采用酶联免疫吸附测定法检测血清过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、活性氧（reactive oxygen species，ROS）、IL-1β、IL-6、干扰素-γ（interferon-γ，IFN-γ）、TNF-α水平。
结果 MCAO/R模型野生型小鼠组脑梗死体积比例为（21.38%±4.06%），LACC1-/-小鼠组脑梗死体积比例为（19.07%±2.86%），差异有统计学意义（P=0.041），同时LACC1-/-小鼠脑组织中p-AMPK的蛋白表达水平增加，NLRP3蛋白表达水平受到抑制。OGD/R细胞模型中，LACC1的下调抑制了NLRP3蛋白的表达、增加了p-AMPK蛋白的表达。OGD/R细胞模型中，LACC1的过度表达增加了IL-1β、IL-6、INF-γ、TNF-α、MDA和ROS生成，降低了CAT和SOD的水平（P＜0.05）。
结论 LACC1可能通过AMPK/NLRP3途径加重小鼠缺血再灌注后的炎症反应，这可能为脑梗死或其他神经系统疾病及其相关并发症提供一种新的治疗方案。

关键词: LACC1; 脑梗死; 炎症反应; 腺苷酸活化蛋白激酶; NOD样受体热蛋白结构域相关蛋白3

JIAO Junping, BAO Junqiang, SHI Huimin, GAO Chao, TIAN Shujuan. LACC1 Aggravated Ischemia Reperfusion by Phosphorylate AMP-activated Protein Kinase/Nucleotide-binding Oligomerization Domain-like Receptor Protein 3 in Cerebral Infarction[J]. Chinese Journal of Stroke, 2023, 18(9): 1006-1013.

焦俊萍, 鲍军强, 史慧敏, 高超, 田书娟. 漆酶基因LACC1经腺苷酸活化蛋白激酶/NOD样受体热蛋白结构域相关蛋白3加重脑梗死缺血再灌注损伤的机制研究[J]. 中国卒中杂志, 2023, 18(9): 1006-1013.

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