Effects of transcutaneous auricular vagus nerve stimulation on the hippocampal TLR4/MyD88/NF-κB signaling pathway in rats exposed to chronic unpredictable mild stress☆

经皮耳迷走神经刺激(transcutaneous auricular vagus nerve stimulation, taVNS)作为一种非侵入性神经调控技术, 被证实能有效治疗抑郁症并与调控应激诱导的神经炎症相关, 但其具体的抗抑郁机制尚未完全阐明. 探讨taVNS能否调控海马TLR4/NF-κB信号通路, 抑制神经炎症, 发挥抗抑郁作用. 将36只SD大鼠随机分为四组:对照组,模型组,taVNS组和经皮非耳迷走神经刺激(transcutaneous auricular non-vagus nerve stimulation, tnVNS)组, 每组9只.除对照组外, 其余各组采用慢性不可预知温和应激(chronic unpredictable mild stress, CUMS)联合单笼饲养模拟抑郁模型, 持续35天.自第14天起, 每天在应激前1 h在taVNS组和tnVNS组大鼠双侧耳甲腔/耳缘进行电刺激, 强度2 mA, 频率2/15 Hz, 每次干预30 min, 持续21天.记录造模前及干预后每周各组大鼠体质量增加量及强迫游泳实验静止不动时间.实验结束后取材, 通过Western blot检测各组大鼠海马Toll样受体4(TLR4),髓样分化因子88(MyD88),核因子κB p65(NF-κB p65),白细胞介素1β(IL-1β)和白细胞介素-18(IL-18)的蛋白相对表达量, 免疫荧光法检测海马小胶质细胞(Iba1)中的TLR4表达与共定位. CUMS可诱导大鼠出现抑郁样行为及海马神经炎症, 与对照组相比, 模型组大鼠强迫游泳静止不动时间显著延长( P <0.01), 海马区域TLR4,MyD88,NF-κB p65,IL-1β和IL-18蛋白表达增加( P <0.01).干预21天(第35天), 与模型组相比, taVNS组大鼠体质量显著增加( P <0.01), 强迫游泳静止不动时间显著缩短( P <0.01), 且海马区域TLR4( P <0.01),MyD88( P <0.01),NF-κB p65,IL-1β( P <0.01)和IL-18( P <0.01)表达显著降低, 小胶质细胞中TLR4表达降低. taVNS可显著改善CUMS模型大鼠的抑郁样行为, 其作用机制可能与抑制海马TLR4/ NF-κB信号通路,抑制小胶质细胞介导的神经炎症相关. 抑郁症;经皮耳迷走神经刺激;神经炎症;Toll样受体4(TLR4);核因子κB(NF-κB);慢性不可预知温和应激 Transcutaneous auricular vagus nerve stimulation (taVNS), a non-invasive neuromodulation technique, has been demonstrated to effectively treat depression and is associated with modulating stress-induced neuroinflammation, while its specific antidepressant mechanism remains unclear. To investigate whether taVNS can exert antidepressant effects by regulating the hippocampal TLR4/NF-κB signaling pathway and inhibiting neuroinflammation. A total of 36 SD rats were randomized into 4 groups, a control group, a model group, a taVNS group and a transcutaneous auricular non-vagus nerve stimulation (tnVNS) group, 9 rats in each one. Except the control group, all other groups were subjected to a chronic unpredictable mild stress (CUMS) with single-cage housing to formulate depression model, lasting for 35 days. From Day 14, 1 hour prior to the stress test, the rats in the taVNS group and tnVNS group were subjected to electrical stimulation at the bilateral auricular cavity/auricular rim for 30 min daily at an intensity of 2 mA and a frequency of 2/15 Hz, for 21 days. The body weight gain and the immobility time in forced swimming test (FST) were recorded before and after the intervention. After experiment, the samples were collected and the relative protein expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor κB p65 (NF-κB p65), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in the hippocampus of rats were detected by using Western blot. The expression and co-localization of TLR4 in hippocampal microglia (Iba1) were examined by using immunofluorescence. CUMS could induce depression-like behavior and hippocampal neuroinflammation in rats. Compared with the control group, the model group exhibited a prolonged forced swimming immobility time ( P < 0.01) and increased protein expression of TLR4, MyD88, NF-κB p65, IL-1β and IL-18 in the hippocampus ( P < 0.01). On Day 35 (21 days after intervention), compared with the model group, the taVNS group exhibited an increased body weight ( P < 0.01) and shortened forced swimming immobility time ( P < 0.01). Additionally, the expression of TLR4, MyD88, NF-κB p65, IL-1β and IL-18 in the hippocampus was decreased ( P < 0.01), and the expression of TLR4 also decreased in microglia. TaVNS could alleviate depression-like behaviors in CUMS rats, and its mechanism may be associated with the inhibition of the hippocampal TLR4/NF-κB signaling pathway and the suppression of microglia-mediated neuroinflammation.