摘要
目的研究室旁核(PVN)硫化氢(H_(2)S)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H_(2)S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H_(2)S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 H_(2)S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H_(2)S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H_(2)S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、pNF-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H_(2)S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(H_(2)S)in high salt induced hypertensive rats on Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-kappa B(NF-κB)signaling pathway,and explore the mechanism of H_(2)S 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 H_(2)S 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 6weeks continuous injection,the levels of PVN H_(2)S 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 factorκB(p-NF-κB p65)and phosphorylatedκB inhibitory protein kinaseβ(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 H_(2)S 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 H_(2)S in PVN increased(t=7.889,P<0.05).The results of immunofluorescence or immunohistochemistry showed that the expression levels of TLR4,MyD88,p-NF-κB p65 and p-IKKβin 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-IKKβin 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 H_(2)S 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.
作者
梁衍锋
陈淑月
任永波
郑咏太
苏嘉璐
秦驁
王轩
袁博洋
张东东
LIANG Yan-feng;CHEN Shu-yue;REN Yong-bo;ZHENG Yong-tai;SU Jia-lu;QIN Ao;WANG Xua;YUAN Bo-yang;ZHANG Dong-dong(School of Basic Medical Sciences,Jiamusi University,Jiamusi 154007,He longjiang,China;School of Clinical Medicine,Jiamusi University,Jiamusi 154002,Heilongjiang,China)
出处
《生物医学工程与临床》
CAS
2024年第4期457-465,共9页
Biomedical Engineering and Clinical Medicine
基金
黑龙江省自然科学基金资助项目(LH2021H103)
黑龙江省省属高等学校基本科研业务费资助项目(2021-KYYWF-0595)
黑龙江省大学生创新创业训练计划项目(S202310222008)
佳木斯大学国家自然科学基金培育项目(JMSUGPZR2023-015)。
关键词
高血压
室旁核
硫化氢
TOLL样受体4
髓样分化因子88
核转录因子ΚB
hypertension
paraventricular nucleus
hydrogen sulfide
Toll like receptor 4
myeloid differentiation factor 88
nuclearfactor-kappa B
