Zhang Biqiong, Zhang Yaodong, Wu Wenning, Xu Tanzhen, Yin Yanyan, Zhang Junyan, Huang Dake, Li Weizu
Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, China.
J Neuroinflammation. 2017 Jul 21;14(1):139. doi: 10.1186/s12974-017-0911-9.
Neuroinflammation mediated by NLRP1 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 1) inflammasome plays an important role in many neurological diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Our previous studies showed that chronic glucocorticoid (GC) exposure increased brain inflammation via NLRP1 inflammasome and induce neurodegeneration. However, little is known about the mechanism of chronic GC exposure on NLRP1 inflammasome activation in hippocampal neurons.
Hippocampal neurons damage was assessed by LDH kit and Hoechst 33258 staining. The expression of microtubule-associated protein 2 (MAP2), inflammasome complex protein (NLRP1, ASC and caspase-1), inflammatory cytokines (IL-1β), and large-conductance Ca and voltage-activated K channel (BK channels) protein was detected by Western blot. The inflammatory cytokines (IL-1β and IL-18) were examined by ELISA kit. The mRNA levels of NLRP1, IL-1β, and BK were detected by real-time PCR. BK channel currents were recorded by whole-cell patch-clamp technology. Measurement of [K] was performed by ion-selective electrode (ISE) technology.
Chronic dexamethasone (DEX) treatment significantly increased LDH release and neuronal apoptosis and decreased expression of MAP2. The mechanistic studies revealed that chronic DEX exposure significantly increased the expression of NLRP1, ASC, caspase-1, IL-1β, L-18, and BK protein and NLRP1, IL-1β and BK mRNA levels in hippocampal neurons. Further studies showed that DEX exposure results in the increase of BK channel currents, with the subsequent K efflux and a low concentration of intracellular K, which involved in activation of NLRP1 inflammasome. Moreover, these effects of chronic DEX exposure could be blocked by specific BK channel inhibitor iberiotoxin (IbTx).
Our findings suggest that chronic GC exposure may increase neuroinflammation via activation of BK-NLRP1 signal pathway and promote hippocampal neurons damage, which may be involved in the development and progression of AD.
由NLRP1（核苷酸结合寡聚化结构域（NOD）样受体蛋白1）炎性小体介导的神经炎症在许多神经疾病如帕金森病（PD）和阿尔茨海默病（AD）中起重要作用。我们之前的研究表明，长期暴露于糖皮质激素（GC）会通过NLRP1炎性小体增加脑部炎症并诱导神经退行性变。然而，关于长期GC暴露对海马神经元中NLRP1炎性小体激活的机制知之甚少。
通过乳酸脱氢酶（LDH）试剂盒和Hoechst 33258染色评估海马神经元损伤。通过蛋白质免疫印迹法检测微管相关蛋白2（MAP2）、炎性小体复合蛋白（NLRP1、凋亡相关斑点样蛋白（ASC）和半胱天冬酶-1）、炎性细胞因子（白细胞介素-1β（IL-1β））以及大电导钙和电压激活钾通道（BK通道）蛋白的表达。通过酶联免疫吸附测定（ELISA）试剂盒检测炎性细胞因子（IL-1β和白细胞介素-18（IL-18））。通过实时聚合酶链反应（PCR）检测NLRP1、IL-1β和BK的信使核糖核酸（mRNA）水平。通过全细胞膜片钳技术记录BK通道电流。通过离子选择性电极（ISE）技术测量钾离子浓度（[K]）。
长期地塞米松（DEX）处理显著增加LDH释放和神经元凋亡，并降低MAP2的表达。机制研究表明，长期DEX暴露显著增加海马神经元中NLRP1、ASC、半胱天冬酶-1、IL-1β、IL-18和BK蛋白的表达以及NLRP1、IL-1β和BK的mRNA水平。进一步研究表明，DEX暴露导致BK通道电流增加，随后钾离子外流和细胞内低钾浓度，这参与了NLRP1炎性小体的激活。此外，长期DEX暴露的这些作用可被特异性BK通道抑制剂iberiotoxin（IbTx）阻断。
我们的研究结果表明，长期GC暴露可能通过激活BK-NLRP1信号通路增加神经炎症并促进海马神经元损伤，这可能与AD的发生和发展有关。
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