小胶质细胞淀粉样蛋白受体在介导β淀粉样蛋白（Aβ）诱导的炎症中的作用。

BACKGROUND: Neuroinflammation in the brain consequent to activation of microglia is viewed as an important component of Alzheimer's disease (AD) pathology. Amyloid beta (Aβ) protein is known to activate microglia and unleash an inflammatory cascade that eventually results in neuronal dysfunction and death. In this study, we sought to identify the presence of amylin receptors on human fetal and murine microglia and determine whether Aβ activation of the inflammasome complex and subsequent release of cytokines is mediated through these receptors. METHODS: The presence of dimeric components of the amylin receptor (calcitonin receptor and receptor activity modifying protein 3) were first immunohistochemically identified on microglia. Purified human fetal microglial (HFM) cultures were incubated with an in vivo microglial marker, DyLight 594-conjugated tomato lectin, and loaded with the membrane-permeant green fluorescent dye, Fluo-8L-AM for measurements of intracellular calcium [Ca]i. HFM and BV-2 cells were primed with lipopolysaccharide and then exposed to either human amylin or soluble oligomeric Aβ prior to treatment with and without the amylin receptor antagonist, AC253. Changes in the inflammasome complex, NLRP3 and caspase-1, were examined in treated cell cultures with Western blot and fluorometric assays. RT-PCR measurements were performed to assess cytokine release. Finally, in vivo studies were performed in transgenic mouse model of AD (5xFAD) to examine the effects of systemic administration of AC253 on markers of neuroinflammation in the brain. RESULTS: Acute applications of human amylin or Aβ resulted in an increase in [Ca]i that could be blocked by the amylin receptor antagonist, AC253. Activation of the NLRP3 and caspase-1 and subsequent release of cytokines, TNFα and IL-1β, was diminished by AC253 pretreatment of HFMs and BV2 cells. In vivo, intraperitoneal administration of AC253 resulted in a reduction in microglial markers (Iba-1 and CD68), caspase-1, TNFα, and IL-1β. These reductions in inflammatory markers were accompanied by reduction in amyloid plaque and size in the brains of 5xFAD mice compared to controls. CONCLUSION: Microglial amylin receptors mediate Aβ-evoked inflammation, and amylin receptor antagonists therefore offer an attractive therapeutic target for intervention in AD.

背景：大脑中因小胶质细胞激活而导致的神经炎症被认为是阿尔茨海默病（AD）病理学的一个重要组成部分。已知淀粉样蛋白β（Aβ）蛋白可激活小胶质细胞，并引发炎症级联反应，最终导致神经元功能障碍和死亡。在这项研究中，我们试图确定人胎和鼠小胶质细胞上是否存在胰岛淀粉样肽受体，并确定 Aβ 是否通过这些受体激活炎症小体复合物并随后释放细胞因子。

方法：首先通过免疫组织化学方法鉴定胰岛淀粉样肽受体（降钙素受体和受体活性修饰蛋白 3）的二聚体成分在小胶质细胞上的存在。用体内小胶质细胞标志物 DyLight 594 缀合的番茄凝集素孵育纯化的人胎小胶质细胞（HFM）培养物，并加载膜通透性绿色荧光染料 Fluo-8L-AM，以测量细胞内钙 [Ca]i。用脂多糖对 HFM 和 BV-2 细胞进行预刺激，然后在有或没有胰岛淀粉样肽受体拮抗剂 AC253 的情况下，用人类胰岛淀粉样肽或可溶性寡聚 Aβ 处理细胞培养物。用 Western blot 和荧光测定法检查处理后细胞培养物中炎症小体复合物、NLRP3 和 caspase-1 的变化。进行 RT-PCR 测量以评估细胞因子的释放。最后，在 AD 的转基因小鼠模型（5xFAD）中进行体内研究，以检查全身给予 AC253 对大脑中神经炎症标志物的影响。

结果：人胰岛淀粉样肽或 Aβ 的急性应用可导致 [Ca]i 增加，该增加可被胰岛淀粉样肽受体拮抗剂 AC253 阻断。AC253 预处理 HFMs 和 BV2 细胞可减弱 NLRP3 和 caspase-1 的激活以及随后细胞因子 TNFα 和 IL-1β 的释放。在体内，腹腔内给予 AC253 可导致小胶质细胞标志物（Iba-1 和 CD68）、caspase-1、TNFα 和 IL-1β 的减少。与对照组相比，5xFAD 小鼠大脑中的炎症标志物减少伴随着淀粉样斑块和大小的减少。

结论：小胶质细胞胰岛淀粉样肽受体介导 Aβ 引起的炎症，因此胰岛淀粉样肽受体拮抗剂为 AD 的干预提供了一个有吸引力的治疗靶点。