Wang Yitong, Ge Xiangyu, Yu Shu, Cheng Qiong
Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, NMPA Key Laboratory for Research and Evaluation of Tissue Engineering Technology Products, Nantong University, Nantong, China.
Ann Transl Med. 2021 Oct;9(20):1522. doi: 10.21037/atm-21-4027.
polypeptide fraction k (ABPPk) has been shown to protect ischemic stroke and Parkinson's disease, and can inhibit neuroinflammation in lipopolysaccharide (LPS)-activated BV2 microglia. However, the effect of ABPPk responsible for alleviating microglial neurotoxicity remains unknown.
Primary microglia were cultured to investigate the effect of ABPPk on LPS-induced neuroinflammation. Microglia conditioned medium (MCM) was collected to stimulate primary cortical neurons and then the neuronal viability, lactate dehydrogenase (LDH) release, intracellular calcium influx, mitochondria membrane potential (MMP) were assessed, respectively. Postnatal day 5 Sprague-Dawley rat pups were intracerebral injected with LPS to establish an LPS-induced brain injury model. Double immunohistochemical staining for NeuN and Iba1 was performed to evaluate the effects of ABPPk on LPS-induced neuronal damage and microglial activation. TUNEL assay was conducted to detect cell apoptosis in LPS-injected brain. The effect of ABPPk on LPS-induced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production as well as the phosphorylation of protein kinase B (Akt) was detected. Moreover, LY294002 (a specific PI3K inhibitor) and SC79 (a specific Akt activator) were used to further reveal the underlying mechanism.
ABPPk pretreatment inhibited LPS-induced NLRP3 and cleaved caspase 1 expressions as well as the mRNA levels of IL-1β and IL-18. Moreover, ABPPk inhibited glutamate release from LPS-activated microglia in a concentration-dependent manner. MCM stimulation resulted in characteristic neuronal toxicity including neuronal viability decrease, LDH release increase, calcium overload, and MMP drop. However, ABPPk pretreatment on microglia reduced the neurotoxicity of MCM. LPS intracerebral injection led to neuronal damage, microglial activation and cell apoptosis in the brain, while ABPPk preadministration significantly inhibited LPS-induced microglial activation and alleviated the brain injury. ABPPk pretreatment inhibited NOX2 expression and ROS production in LPS-activated primary microglia. Signaling pathway analysis showed that ABPPk promoted the phosphorylation of Akt in microglia and inhibited LPS-upregulated NOX2 expression, ROS production, and glutamate release, which can be eliminated by pharmacological inhibition of PI3K. Specific Akt activator could inhibit LPS-induced NOX2 expression, ROS production and glutamate release.
The present results suggested that ABPPk could alleviate neurotoxicity of LPS-activated microglia via PI3K/Akt dependent NOX2/ROS pathway.
多肽组分k（ABPPk）已被证明可保护缺血性中风和帕金森病，并且能够抑制脂多糖（LPS）激活的BV2小胶质细胞中的神经炎症。然而，ABPPk减轻小胶质细胞神经毒性的作用机制仍不清楚。
培养原代小胶质细胞以研究ABPPk对LPS诱导的神经炎症的影响。收集小胶质细胞条件培养基（MCM）以刺激原代皮质神经元，然后分别评估神经元活力、乳酸脱氢酶（LDH）释放、细胞内钙内流、线粒体膜电位（MMP）。对出生后第5天的Sprague-Dawley大鼠幼崽进行脑内注射LPS以建立LPS诱导的脑损伤模型。进行NeuN和Iba1的双重免疫组织化学染色以评估ABPPk对LPS诱导的神经元损伤和小胶质细胞激活的影响。进行TUNEL测定以检测LPS注射脑内的细胞凋亡。检测ABPPk对LPS诱导的NADPH氧化酶2（NOX2）表达、活性氧（ROS）产生以及蛋白激酶B（Akt）磷酸化的影响。此外，使用LY294002（一种特异性PI3K抑制剂）和SC79（一种特异性Akt激活剂）来进一步揭示潜在机制。
ABPPk预处理抑制了LPS诱导的NLRP3和裂解的caspase 1表达以及IL-1β和IL-18的mRNA水平。此外，ABPPk以浓度依赖性方式抑制LPS激活的小胶质细胞释放谷氨酸。MCM刺激导致特征性的神经元毒性，包括神经元活力降低、LDH释放增加、钙超载和MMP下降。然而，对小胶质细胞进行ABPPk预处理降低了MCM的神经毒性。脑内注射LPS导致脑内神经元损伤、小胶质细胞激活和细胞凋亡，而预先给予ABPPk可显著抑制LPS诱导的小胶质细胞激活并减轻脑损伤。ABPPk预处理抑制了LPS激活的原代小胶质细胞中NOX2的表达和ROS的产生。信号通路分析表明，ABPPk促进小胶质细胞中Akt的磷酸化，并抑制LPS上调的NOX2表达、ROS产生和谷氨酸释放，而PI3K的药理学抑制可消除这些作用。特异性Akt激活剂可抑制LPS诱导的NOX2表达、ROS产生和谷氨酸释放。
目前的结果表明，ABPPk可通过PI3K/Akt依赖性NOX2/ROS途径减轻LPS激活的小胶质细胞的神经毒性。
