Objectives: Neuropathic pain (NP) is a chronic pain caused by somatosensory neuropathy or disease, and genistein (Gen) might be a potential drug for the treatment of NP. Therefore, this study aims to investigate the effect of Gen on lipopolysaccharide (LPS)-induced inflammatory injury of dorsal root ganglion neuron (DRGn) in rats and the possible molecular mechanism.
Methods: The DRGn of 1-day-old juvenile rats were taken for isolation and culture. The DRGn in logarithmic growth phase were divided into a control group, a LPS group, a tubastatin hydrochloride (TSA)+LPS group, a Gen1+LPS group, a Gen2+LPS group, a Gen2+LPS+TSA group, a Gen2+pcDNA-histone deacetylase 6 (HDAC6)+LPS group, and a Gen2+pcDNA3.1+LPS group. The LPS group was treated with 1 μg/mL LPS for 24 h; the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group were treated with 5 μmol/L TSA, 5 μmol/L Gen, 10 μmol/L Gen respectively for 0.5 h, and then added 1 μg/mL LPS for 24 h; the Gen2+TSA+LPS group was treated with 10 μmol/L Gen and 5 μmol/L TSA for 0.5 h and then added 1 μg/mL LPS for 24 h; the Gen2+pcDNA-HDAC6+LPS group and the Gen2+pcDNA3.1+LPS group received 100 nmol/L pcDNA-HDAC6 and pcDNA3.1 plasmids respectively, and 24 h after transfection, 10 μmol/L Gen was pretreated for 0.5 h, and then added 1 μg/mL LPS for 24 h. Real-time RT-PCR was used to detect the HDAC6 mRNA expression in DRGn; CCK-8 method was used to detect cell viability of DRGn; flow cytometry was used to detect cell apoptosis of DRGn; ELISA was used to detect the levels of IL-1β, IL-6, and TNF-α in DRGn culture supernatant; Western blotting was used to detect the protein expression of HDAC6, Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and NF-κB p65 in DRGn.
Results: Compared with the control group, the expression levels of HDAC6 mRNA and protein, the expression levels of TLR4 and MyD88 protein in DRGn of LPS group rats were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, and the activity of DRGn was significantly decreased, the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05). Compared with the LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the TSA+LPS group, the Gen1+LPS group, the Gen2+LPS group and the Gen2+TSA+LPS group were significantly down-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly decreased, the activity of DRGn was significantly increased, the apoptosis rate was significantly decreased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly decreased (all P<0.05), and the above changes were most obvious in the Gen2+TSA+LPS group. Compared with the Gen2+LPS group, the expression levels of HDAC6 mRNA and protein, TLR4 and MyD88 protein expression levels in DRGn of the Gen2+pcDNA-HDAC6+LPS group were significantly up-regulated, the ratio of p-NF-κB p65/NF-κB p65 was significantly increased, the activity of DRGn was significantly decreased, and the apoptosis rate was significantly increased, and the levels of IL-1β, IL-6 and TNF-α in the DRGn culture supernatant were significantly increased (all P<0.05).
Conclusions: Gen can alleviate LPS-induced DRGn inflammatory injury in rats, which might be related to down-regulating the expression of HDAC6 and further inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway.
目的: 神经病理性疼痛(neuropathic pain，NP)是一种由躯体感觉神经病变或疾病引起的慢性疼痛，金雀异黄素(genistein，Gen)可能是治疗NP的潜在药物。因此，本研究旨在探讨Gen对脂多糖(lipopolysaccharide，LPS)诱导大鼠背根神经节神经元(dorsal root ganglion neuron，DRGn)炎性损伤的作用及其可能的分子机制。方法: 取出生 1 d的幼年大鼠进行DRGn的分离和培养。取对数生长期的DRGn，分为8组：对照组、LPS组、Tubastatin A盐酸盐(tubastatin A hydrochloride，TSA)+LPS组、Gen1+LPS组、Gen2+LPS组、Gen2+TSA+LPS组、Gen2+pcDNA-组蛋白脱乙酰化酶6(histone deacetylase 6，HDAC6)+LPS组、Gen2+pcDNA3.1+LPS组。LPS组给予1 μg/mL LPS处理24 h；TSA+LPS组、Gen1+LPS组、Gen2+LPS组给予5 μmol/L TSA、5 μmol/L Gen、10 μmol/L Gen预处理0.5 h后加入1 μg/mL LPS处理24 h；Gen2+TSA+LPS组给予10 μmol/L Gen和5 μmol/L TSA共同预处理0.5 h后加入1 μg/mL LPS处理24 h；Gen2+pcDNA-HDAC6+LPS组、Gen2+pcDNA3.1+LPS组将100 nmol/L pcDNA-HDAC6、pcDNA3.1质粒分别转染至DRGn，转染24 h后，给予10 μmol/L Gen预处理0.5 h，再加入1 μg/mL LPS处理24 h。采用real-time RT-PCR检测DRGn中HDAC6 mRNA的表达水平，CCK-8法检测DRGn活力，流式细胞术检测DRGn凋亡情况，ELISA检测DRGn培养上清液中IL-1β、IL-6、TNF-α水平；蛋白质印迹法检测DRGn中HDAC6、Toll样受体4(Toll-like receptor 4，TLR4)、髓样分化因子88(myeloid differentiation factor 88，MyD88)、NF-κB p65的蛋白质表达水平。结果: 与对照组相比，LPS组大鼠DRGn中HDAC6 mRNA和蛋白质的表达水平，TLR4和MyD88蛋白质表达水平均显著上调，p-NF-κB p65/NF-κB p65比值显著增加，DRGn活性显著降低且凋亡率显著升高，DRGn培养上清液中IL-1β、IL-6、TNF-α水平均显著升高(均P<0.05)。与LPS组相比，TSA+LPS组、Gen1+LPS组、Gen2+LPS组和Gen2+TSA+LPS组大鼠DRGn中HDAC6 mRNA和蛋白质的表达水平，TLR4和MyD88蛋白质表达水平均显著下调，p-NF-κB p65/NF-κB p65比值显著降低，DRGn活性显著升高且凋亡率显著降低，DRGn培养上清液中IL-1β、IL-6、TNF-α水平均显著降低(均P<0.05)，且Gen2+TSA+LPS组上述变化最明显。与Gen2+LPS组相比，Gen2+pcDNA-HDAC6+LPS组大鼠DRGn中HDAC6 mRNA和蛋白质的表达水平，TLR4和MyD88蛋白质表达水平均显著上调，p-NF-κB p65/NF-κB p65比值显著增加，DRGn活性显著降低且凋亡率显著升高，DRGn培养上清液中IL-1β、IL-6、TNF-α水平均显著升高(均P<0.05)。结论: Gen可减轻LPS诱导的大鼠DRGn炎性损伤，其作用机制可能与下调HDAC6的表达，进一步抑制TLR4/MyD88/NF-κB信号通路的激活有关。.
Keywords: dorsal root ganglion neuron; genistein; histone deacetylase 6; lipopolysaccharide; neuropathic pain.