金雀异黄素通过下调HDAC6减轻LPS诱导的大鼠背根神经节神经元炎性损伤

Genistein attenuates LPS-induced inflammatory injury of rat dorsal root ganglion neuron via down-regulating HDAC6

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1μg/mL LPS处理24 h;TSA+LPS组、Gen1+LPS组、Gen2+LPS组给予5μmol/L TSA、5μmol/L Gen、10μmol/L Gen预处理0.5 h后加入1μg/mL LPS处理24 h;Gen2+TSA+LPS组给予10μmol/L Gen和5μmol/L TSA共同预处理0.5 h后加入1μg/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10μmol/L Gen预处理0.5 h,再加入1μg/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 receptor4,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。

Objective: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+pc DNA-histone deacetylase 6(HDAC6)+LPS group,and a Gen2+pc DNA3.1+LPS group.The LPS group was treated with 1μg/m L LPS for 24 h;the TSA+LPS group,the Gen1+LPS group,the Gen2+LPS group were treated with 5μmol/L TSA,5μmol/L Gen,10μmol/L Gen respectively for 0.5 h,and then added 1μg/m L LPS for 24 h;the Gen2+TSA+LPS group was treated with 10μmol/L Gen and 5μmol/L TSA for 0.5 h and then added 1μg/m L LPS for 24 h;the Gen2+pc DNA-HDAC6+LPS group and the Gen2+pc DNA3.1+LPS group received 100 nmol/L pc DNA-HDAC6 and pc DNA3.1 plasmids respectively,and 24 h after transfection,10μmol/L Gen was pretreated for 0.5 h,and then added 1μg/m L LPS for 24 h.Real-time RT-PCR was used to detect the HDAC6 m RNA 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-αin DRGn culture supernatant;Western blotting was used to detect the protein expression of HDAC6,Toll-like receptor 4(TLR4),myeloid differentiation factor 88(My D88),and NF-κB p65 in DRGn.Results:Compared with the control group,the expression levels of HDAC6 m RNA and protein,the expression levels of TLR4 and My D88 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-αin the DRGn culture supernatant were significantly increased(all P<0.05).Compared with the LPS group,the expression levels of HDAC6 m RNA and protein,TLR4 and My D88 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-αin 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 m RNA and protein,TLR4 and My D88 protein expression levels in DRGn of the Gen2+pc DNA-HDAC6+LPS group were significantly upregulated,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-αin the DRGn culture supernatant were significantly increased(all P<0.05).Conclusion: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/My D88/NF-κB signaling pathway.