Abstract：
BACKGROUND: Psychological stress is a major trigger for visceral hypersensitivity (VH) in irritable bowel syndrome. The zinc finger protein ZBTB20 (ZBTB20) is implicated in somatic nociception via modulating transient receptor potential (TRP) channels, but its role in the development of VH is unclear. This study aimed to investigate the role of ZBTB20/TRP channel axis in stress-induced VH.
METHODS: Rats were subjected to water avoidance stress (WAS) for 10 consecutive days. Small interfering RNA (siRNA) targeting ZBTB20 was intrathecally administered. Inhibitors of TRP channels, stress hormone receptors, and nuclear factor kappa-B (NF-κB) were administered. Visceromotor response to colorectal distension was recorded. Dorsal root ganglia (DRGs) were dissected for Western blot, coimmunoprecipitation, and chromatin immunoprecipitation. The DRG-derived neuron cell line was applied for specific research.
RESULTS: WAS-induced VH was suppressed by the inhibitor of TRPV1, TRPA1, or TRPM8, with enhanced expression of these channels in L6-S2 DRGs. The inhibitor of glucocorticoid receptor or β2-adrenergic receptor counteracted WAS-induced VH and TRP channel expression. Concurrently, WAS-induced stress hormone-dependent ZBTB20 expression and NF-κB activation in DRGs. Intrathecally injected ZBTB20 siRNA or an NF-κB inhibitor repressed WAS-caused effect. In cultured DRG-derived neurons, stress hormones promoted nuclear translocation of ZBTB20, which preceded p65 nuclear translocation. And, ZBTB20 siRNA suppressed stress hormone-caused NF-κB activation. Finally, WAS enhanced p65 binding to the promoter of TRPV1, TRPA1, or TRPM8 in rat DRGs.
CONCLUSIONS: ZBTB20 mediates stress-induced VH via activating NF-κB/TRP channel pathway in nociceptive sensory neurons.
METHODS: Rats were subjected to water avoidance stress (WAS) for 10 consecutive days. Small interfering RNA (siRNA) targeting ZBTB20 was intrathecally administered. Inhibitors of TRP channels, stress hormone receptors, and nuclear factor kappa-B (NF-κB) were administered. Visceromotor response to colorectal distension was recorded. Dorsal root ganglia (DRGs) were dissected for Western blot, coimmunoprecipitation, and chromatin immunoprecipitation. The DRG-derived neuron cell line was applied for specific research.
RESULTS: WAS-induced VH was suppressed by the inhibitor of TRPV1, TRPA1, or TRPM8, with enhanced expression of these channels in L6-S2 DRGs. The inhibitor of glucocorticoid receptor or β2-adrenergic receptor counteracted WAS-induced VH and TRP channel expression. Concurrently, WAS-induced stress hormone-dependent ZBTB20 expression and NF-κB activation in DRGs. Intrathecally injected ZBTB20 siRNA or an NF-κB inhibitor repressed WAS-caused effect. In cultured DRG-derived neurons, stress hormones promoted nuclear translocation of ZBTB20, which preceded p65 nuclear translocation. And, ZBTB20 siRNA suppressed stress hormone-caused NF-κB activation. Finally, WAS enhanced p65 binding to the promoter of TRPV1, TRPA1, or TRPM8 in rat DRGs.
CONCLUSIONS: ZBTB20 mediates stress-induced VH via activating NF-κB/TRP channel pathway in nociceptive sensory neurons.
摘要：
背景：心理应激是肠易激综合征内脏高敏感性（VH）的主要诱因。锌指蛋白ZBTB20（ZBTB20）通过调节瞬时受体电位（TRP）通道参与体细胞伤害感受，但其在VH发展中的作用尚不清楚。本研究旨在探讨ZBTB20/TRP通道轴在应激诱导的VH中的作用。
方法：对大鼠进行连续10天的避水应激（WAS）。鞘内施用靶向ZBTB20的小干扰RNA(siRNA)。TRP通道抑制剂，应激激素受体，并给予核因子κB（NF-κB）。记录结肠直肠扩张的内脏运动反应。对背根神经节(DRGs)进行蛋白质印迹,共免疫沉淀，和染色质免疫沉淀。将DRG衍生的神经元细胞系用于特定研究。
结果：WAS诱导的VH被TRPV1，TRPA1或TRPM8的抑制剂抑制，这些通道在L6-S2DRG中的表达增强。糖皮质激素受体或β2-肾上腺素能受体的抑制剂可抵抗WAS诱导的VH和TRP通道表达。同时,WAS在DRGs中诱导应激激素依赖性ZBTB20表达和NF-κB激活。鞘内注射ZBTB20siRNA或NF-κB抑制剂抑制WAS引起的作用。在培养的DRG衍生的神经元中，应激激素促进ZBTB20的核易位，先于p65核易位。And,ZBTB20siRNA抑制应激激素引起的NF-κB活化。最后,WAS增强了大鼠DRGs中p65与TRPV1，TRPA1或TRPM8启动子的结合。
结论：ZBTB20通过激活伤害性感觉神经元的NF-κB/TRP通道通路介导应激诱导的VH。
方法：对大鼠进行连续10天的避水应激（WAS）。鞘内施用靶向ZBTB20的小干扰RNA(siRNA)。TRP通道抑制剂，应激激素受体，并给予核因子κB（NF-κB）。记录结肠直肠扩张的内脏运动反应。对背根神经节(DRGs)进行蛋白质印迹,共免疫沉淀，和染色质免疫沉淀。将DRG衍生的神经元细胞系用于特定研究。
结果：WAS诱导的VH被TRPV1，TRPA1或TRPM8的抑制剂抑制，这些通道在L6-S2DRG中的表达增强。糖皮质激素受体或β2-肾上腺素能受体的抑制剂可抵抗WAS诱导的VH和TRP通道表达。同时,WAS在DRGs中诱导应激激素依赖性ZBTB20表达和NF-κB激活。鞘内注射ZBTB20siRNA或NF-κB抑制剂抑制WAS引起的作用。在培养的DRG衍生的神经元中，应激激素促进ZBTB20的核易位，先于p65核易位。And,ZBTB20siRNA抑制应激激素引起的NF-κB活化。最后,WAS增强了大鼠DRGs中p65与TRPV1，TRPA1或TRPM8启动子的结合。
结论：ZBTB20通过激活伤害性感觉神经元的NF-κB/TRP通道通路介导应激诱导的VH。
