Effects of H2S in paraventricular nucleus on salt-sensitive hypertensive rats via TLR4/MyD88/NF-κB signaling pathway
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目的 研究室旁核(PVN)硫化氢(H2S)对高盐诱导的高血压大鼠Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核转录因子κB(NF-κB)信号通路的影响,探讨H2S降压的作用机制.方法 选择40只成年雄性Dahl盐敏感性大鼠,鼠龄8周,体质量260～280 g.随机把大鼠分成正常对照组(Control组)、正常给药组(Control+GYY组)、高盐模型组(Model组)、高盐给药组(Model+GYY组),每组10只.于第4周末,利用微型渗透泵向Control+GYY组和Model+GYY组大鼠双侧PVN区域持续注射H2S的缓释供体GYY4137,其余组大鼠全部注射等量人工脑脊液.连续注射6周后进行取材,通过酶联免疫吸附分析(ELISA)法来检测PVN H2S及血浆去甲肾上腺素(NE)的水平,免疫荧光法检测TLR4、MyD88的表达,免疫组化法检测磷酸化核转录因子κB(p-NF-κB p65)、磷酸化κB抑制蛋白激酶β(p-IKKβ)的表达,通过Western blot法检测TLR4、MyD88、p-NF-κB p65/NF-κB p65的蛋白表达.结果 Model组平均动脉压(MAP)、血浆NE较 Control 组升高(t=10.204、24.155、23.967、25.657、22.069、31.036、23.054、28.189,P＜0.05),PVN 中 H2S 水平降低(t=14.348,P＜0.05),Model+GYY 组与 Model 组相比 MAP、NE 水平降低(t=8.295、12.931、10.992、16.064、12.228、16.017,P＜0.05),PVN中H2S水平升高(t=7.889,P＜0.05).免疫荧光或免疫组化结果显示,Model组PVN中TLR4、MyD88、p-NF-κB p65、p-IKKβ 因子的表达水平较 Control 组均升高(t=8.844、10.344、6.813、3.909,P＜0.05),Model+GYY 组与 Model 组相比 PVN 中 TLR4、MyD88、p-NF-κB p65、p-IKKβ 因子的表达水平均降低(t=4.719、4.313、3.234、4.955,P＜0.05).Western blot 结果表明,与 Control 组相比,Model 组大鼠 PVN 中 TLR4、MyD88、p-NF-κB p65/NF-κB p65 蛋白表达水平升高(t=15.951、6.537、7.394,P＜0.05);与 Model 组大鼠相比,Model+GYY 组 TLR4、MyD88、p-NF-κB p65/NF-κB p65蛋白表达水平下降(t=9.415、4.901、8.997,P＜0.05).结论 室旁核H2S可明显降低高盐诱导的高血压大鼠的血压、降低外周交感神经活动,其机制可能与抑制TLR4/MyD88/NF-κB信号通路有关.
Objective To investigate the influence of paraventricular nucleus(PVN)hydrogen sulfide(H2S)in high salt induced hypertensive rats on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-kappa B(NF-κB)sig-naling pathway,and explore the mechanism of H2S antihypertensive effect.Methods Forty aged 8-week adult male Dahl salt-sensitive rats with body mass of 260-280 g were selected,and the rats were randomly divided into normal control group(Control group),normal administration group(Control+GYY group),high-salt model group(Model group)and high-salt administration group(Model+GYY group),with 10 rats in each group.At the end of 4th week,the sustained-release donor GYY4137 of H2S was continuously injected into bilateral PVN of rats in Control+GYY group and Model+GYY group by micro osmotic pump,and rats in other groups were injected with the same amount of artificial cerebrospinal fluid.After 6 weeks continuous injection,the levels of PVN H2S and plasma norepinephrine(NE)were detected by enzyme-linked immunosorbent assay(ELISA),and expression of TLR4 and MyD88 was detected by immunofluorescence,the expression of phosphorylated nuclear transcription factor κB(p-NF-κB p65)and phosphorylated κB inhibitory protein kinase β(p-IKKβ)was detected by immunohistochemistry,and the expression of TLR4,MyD88,p-NF-κB p65/NF-κB p65 was detected by Western blot.Results The mean arterial pressure(MAP)and plasma NE in Model group were higher than those in Control group(t=10.204,24.155,23.967,25.657,22.069,31.036,23.054,28.189,P＜0.05),level of H2S in PVN decreased(t=14.348,P＜0.05),levels of MAP and NE in Model+GYY group were lower than those in Model group(t=8.295,12.931,10.992,16.064,12.228,16.017,P＜0.05),level of H2S in PVN increased(t=7.889,P＜0.05).The results of immunofluorescence or immunohis-tochemistry showed that the expression levels of TLR4,MyD88,p-NF-κB p65 and p-IKKβ in PVN of Model group were higher than those in Control group(t=8.844,10.344,6.813,3.909,P＜0.05).Compared with the Model group,the expression levels of TLR4,MyD88,p-NF-κB p65 and p-IKKβ in PVN of Model+GYY group were decreased(t=4.719,4.313,3.234,4.955,P＜0.05).Western blot results showed that compared with Control group,the expression levels of TLR4,MyD88 and p-NF-κB p65/NF-κB p65 protein in PVN of Model group were increased(t=15.951,6.537,7.394,P＜0.05).Compared with Model group,the expression levels of TLR4,MyD88,p-NF-κB p65/NF-κB p65 protein in Model+GYY group were decreased(t=9.415,4.901,8.997,P＜0.05).Conclusion It is demonstrated that H2S in PVN can significantly reduce blood pressure and peripheral sympathetic nerve activity in high-salt-induced hypertensive rats,and its mechanism may be related to inhibition of TLR4/MyD88/NF-κB signaling pathway.
hypertensionparaventricular nucleushydrogen sulfideToll like receptor 4myeloid differentiation factor 88nuclear factor-kappa B
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