β-淀粉样蛋白通过 TLR4 在小鼠小胶质细胞中激活 NLRP3 炎性体。

Beta-amyloid activates NLRP3 inflammasome via TLR4 in mouse microglia.

机构信息

Department of Neurology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China; Institute of Neurological Diseases, Xuzhou Medical University, Xuzhou, China.

Institute of Neurological Diseases, Xuzhou Medical University, Xuzhou, China; Department of Neurosurgery, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China.

出版信息

Neurosci Lett. 2020 Sep 25;736:135279. doi: 10.1016/j.neulet.2020.135279. Epub 2020 Jul 26.

DOI:10.1016/j.neulet.2020.135279

PMID:32726591

Abstract

Beta-amyloid(Aβ)-induced inflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD). Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) inflammasome is involved in the Aβ-induced inflammation. However, the mechanisms by which extracellular Aβ activates cytoplasmic NLRP3 inflammasome are poorly understood. Toll-like receptor 4(TLR4) acts as a sensor of Aβ and performs a key role in neuroinflammation. TLR4 is involved in activating the NLRP3 inﬂammasome in several diseases. In this study, the interaction between TLR4 and NLRP3 inflammasome in Aβ-induced neuroinflammation was investigated. BV-2 microglia and primary microglia were primed with lipopolysaccharide (LPS) and then pretreated with TLR4 inhibitor CLI-095, followed by stimulation with Aβ. The protein expression of NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 p 10 was detected by western blotting and immunostaining. The mRNA expression of inflammatory factors was measured by real-time PCR. The protein level of pro IL-1β and IL-1β was examined by ELISA. Activated microglia were examined by immunoﬂuorescence staining for ionized calcium-binding adapter molecule-1 (Iba-1). Conditioned medium of BV-2 cells was collected to challenge HT-22 neurons. Cell viability was assessed with MTT assay. Assessment of HT-22 cell apoptosis was performed by Annexin V/PI staining and western blotting to detect the protein level of cleaved caspase 3. The results showed that Aβ activated and up-regulated the expression of NLRP3 inflammasome in BV-2 microglia, as indicated by increased activation of caspase-1 and secretion of IL-1β. Pharmacological inhibition of TLR4 by CLI-095 abolished Aβ-induced NLRP3 inflammasome activation, which curbed the development of inflammation and exerted protective effect on HT-22 neurons. Furthermore, the inhibitory effects of CLI-095 on Aβ-induced inflammation were reversed by NLRP3 activator ATP. Overall, our findings suggested TLR4 mediated Aβ-induced NLRP3 inflammasome activation in mouse microglia. TLR4/NLRP3 pathway plays a critical role in Aβ-induced neuroinflammation.

摘要

β-淀粉样蛋白（Aβ）诱导的炎症在阿尔茨海默病（AD）的发病机制中起着关键作用。核苷酸结合寡聚结构域样受体含富含亮氨酸重复序列蛋白 3（NLRP3）炎性小体参与 Aβ诱导的炎症。然而，细胞外 Aβ激活细胞质 NLRP3 炎性小体的机制尚不清楚。Toll 样受体 4（TLR4）作为 Aβ的传感器，在神经炎症中起关键作用。TLR4 参与了几种疾病中 NLRP3 炎性小体的激活。在这项研究中，研究了 TLR4 与 Aβ 诱导的神经炎症中的 NLRP3 炎性小体之间的相互作用。用脂多糖（LPS）预刺激 BV-2 小胶质细胞和原代小胶质细胞，然后用 TLR4 抑制剂 CLI-095 预处理，再用 Aβ 刺激。通过 Western blot 和免疫染色检测 NLRP3、衔接蛋白凋亡相关斑点样蛋白（ASC）和半胱天冬酶-1 p10 的蛋白表达。通过实时 PCR 测量炎症因子的 mRNA 表达。通过 ELISA 检测前 IL-1β 和 IL-1β 的蛋白水平。通过离子钙结合接头分子-1（Iba-1）的免疫荧光染色检测活化的小胶质细胞。收集 BV-2 细胞的条件培养基以挑战 HT-22 神经元。通过 MTT 测定评估细胞活力。通过 Annexin V/PI 染色和 Western blot 检测裂解 caspase 3 的蛋白水平评估 HT-22 细胞凋亡。结果表明，Aβ 激活并上调了 BV-2 小胶质细胞中 NLRP3 炎性小体的表达，表现为半胱天冬酶-1 的激活增加和 IL-1β 的分泌。CLI-095 通过 TLR4 的药理学抑制消除了 Aβ 诱导的 NLRP3 炎性小体的激活，从而抑制了炎症的发展，并对 HT-22 神经元发挥了保护作用。此外，NLRP3 激活剂 ATP 逆转了 CLI-095 对 Aβ 诱导的炎症的抑制作用。总之，我们的研究结果表明 TLR4 介导了 Aβ 诱导的小鼠小胶质细胞中的 NLRP3 炎性小体激活。TLR4/NLRP3 通路在 Aβ 诱导的神经炎症中起着关键作用。