Objective: Activation of nicotinic acetylcholine receptors (nAChRs) results in neuroprotection via a poorly understood molecular mechanism. In this study, we aimed to investigate the effect of nAChR stimulation with nicotine on the regulation of microRNA (miRNA) expression and identify the molecular pathway involved in neuroprotection.

Methods: We conducted miRNA expression profiling using a microarray to identify the miRNAs regulated by nicotine. miR-132-5p expression was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Cells were treated with nicotine, a miR-132-5p mimic or its inhibitor, and the cell viability was assessed. CREB, Bcl-2, Bax, cleaved caspase-3, and α-tubulin protein expression levels were determined by Western blotting analysis.

Results: Using a rodent miRNA microarray, we identified 37 miRNAs regulated by nicotine. The microarray and RT-qPCR results showed 1.67-fold and 1.5-fold increases in miR-132-5p, respectively, upon nicotine treatment. Immunoblotting revealed a >2-fold increase in phosphorylation of CREB with nicotine, peaking at 4 h. Nicotine treatment of cells increased viability from 35% to 54%, and Bcl-2 immunoreactivity increased by 1.4-fold. Overexpression of miR-132-5p increased cell viability from 38% to 70% and increased Bcl-2 expression by 3.9-fold. Inhibition of miR-132-5p decreased cell viability to 25%, whereas no change was observed in Bcl-2. Bax expression remained unchanged following treatment with a miR-132-5p mimic or its inhibitor.

Discussion: Our results suggest that nAChR activation facilitates cell survival by upregulating miR-132-5p, which upregulates the anti-apoptotic protein Bcl-2. These results present miR-132-5p as a potential novel therapeutic target to achieve neuroprotection via stimulation of nAChRs.

Abbreviations: CCK-8: Cell counting kit-8; nAChR: Nicotinic acetylcholine receptor; NGF: Nerve growth factor; WST-8: [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]

目的：烟碱化乙酰胆碱受体（nAChRs）的激活通过一个尚不了解的分子机制导致神经保护作用。在这项研究中，我们旨在研究尼古丁对nAChR刺激对microRNA（miRNA）表达调节的影响，并确定参与神经保护的分子途径。

方法：我们使用微阵列芯片进行了miRNA表达谱分析，以鉴定尼古丁调节的miRNA。通过逆转录-定量聚合酶链反应（RT-qPCR）分析验证了miR-132-5p的表达。用烟碱，miR-132-5p模拟物或其抑制剂处理细胞，并评估细胞活力。通过蛋白质印迹分析确定CREB，Bcl-2，Bax，裂解的caspase-3和α-微管蛋白的蛋白表达水平。

结果：使用啮齿动物miRNA芯片，我们鉴定了37种受烟碱调节的miRNA。微阵列和RT-qPCR结果显示，尼古丁治疗后miR-132-5p分别增加1.67倍和1.5倍。免疫印迹显示CREB的尼古丁磷酸化增加> 2倍，在4小时达到峰值。尼古丁对细胞的治疗将生存力从35％提高到54％，Bcl-2免疫反应性提高了1.4倍。miR-132-5p的过表达将细胞活力从38％增加到70％，并使Bcl-2表达增加3.9倍。对miR-132-5p的抑制将细胞活力降至25％，而在Bcl-2中未观察到变化。用miR-132-5p模拟物或其抑制剂处理后，Bax表达保持不变。

讨论：我们的结果表明，nAChR激活可通过上调miR-132-5p（上调抗凋亡蛋白Bcl-2）来促进细胞存活。这些结果表明，miR-132-5p是潜在的新型治疗靶标，可通过刺激nAChRs实现神经保护。

缩写：CCK-8：细胞计数试剂盒8；nAChR：烟碱乙酰胆碱受体；NGF：神经生长因子；WST-8：[2-（2-甲氧基-4-硝基苯基）-3-（4-硝基苯基）-5-（2,4-二磺基苯基）-2H-四唑鎓，单钠盐]