摘要
目的:探讨SPE及其单体对嘌呤霉素氨基核苷(PAN)诱导的损伤足细胞中Toll样受体4(TLR4)通路表达的影响。方法:足细胞与含有甘西鼠尾草总酚酸提取物(SPE)、迷迭香酸(RA)、丹酚酸B(SalB)、RA+SalB混合及他克莫司的完全培养液预先孵育30 min,再加入PAN(100μg/ml)继续作用24 h,观察足细胞骨架结构F-actin变化,western blot及半定量RT-PCR检测足细胞中TLR4、myd88、phosphor-NF-κB(pp65)在蛋白及mRNA水平的表达量变化。结果:(1)PAN作用24 h后,足细胞骨架蛋白F-actin明显损伤,微丝解聚、胞体回缩,少数细胞尚存排列紊乱的丝状结构,而提前给予保护药物SPE、SalB、RA、SalB+RA混合药物及他克莫司组的足细胞损伤明显减弱。(2)western blot数据显示,PAN诱导的损伤足细胞中TLR4、myd88及pp65蛋白的表达与正常组相比均有明显上调(P<0.05);保护药物组:TLR4蛋白表达较模型组有明显下降(P<0.05);My D88蛋白表达除SalB高剂量组及SalB+RA低剂量组较模型组无显著下降外,其余均能明显下调其表达量(P<0.05);pp65在保护药物组中的表达均较模型对照组低(P<0.05)。(3)半定量RT-PCR结果显示,模型对照组中TLR4、My D88及NF-κB的mRNA表达量明显高于正常对照组;保护药物组中:TLR4 mRNA表达量在各药物处理组中均明显低于PAN组(P<0.05),其中的SPE高、低剂量组,RA高、低剂量组,SalB+RA高剂量组与正常组相接近(P>0.05);My D88 mRNA表达量在SPE及其单体处理后均有明显下调(P<0.05);NF-κB mRNA表达量除SalB高剂量组中无明显下调外(P>0.05),其余各保护药物组均下调(P<0.05)。结论:SPE及其活性单体对PAN诱导损伤足细胞的保护作用机制可能部分通过抑制TLR4信号相关蛋白表达来实现;甘西鼠尾草总酚酸提取物中的单体迷迭香酸对TLR4及其下游信号分子My D88、NF-κB的抑制作用优于丹酚酸B和混合给药组。
Objective： To definite the possible protection mechanisms of Salvia Przewalskii extract of total phenolic acids（ SPE） and its active monomer（ Salvianolic acid B and Rosmarinic acid） on the damaged podocytes through TLR4 actived by PAN in vitro.Methods： The conditionally immortalized mouse podocytes were incubation with complete medium which contains the SPE,Salvianolic acid B（ SalB）,Rosmarinic acid（ RA）,SalB ＋ RA or Tacrolimus for 30 min,and then PAN（ 100μ100 th was added for another 24 h culture.The F-actin was observed by fluorescence microscopy to assess the cytoskeleton distribution of podocyte.Western blot and semi-quantitatives RT-PCR were used to detect the quantity change of TLR4 signal pathway relative protein and mRNA levels.Results：（ 1） The F-actin of podocytes was almost completely disrupted after PAN treatment for 24 h,and the damage of podocytes in protection groups was decreased significantly.（ 2） Through western blot analysis,we found that the protein levels of TLR4、My D88、pp65 in PAN groups were significantly higher than that of normal control（ P〈0.05）,while the expressin of protein of TLR4 and pp65 were reduced in the podocytes when pretreat with SPE 、SalB、RA or admixture in different degree（ P〈0.05）; the protein level of My D88 was reduced in all protection groups except for high-dose SalB and low-dose admixture groups;（ 3） The mRNA levels of TLR4、My D88、NF-k B in PAN groups were also significantly higher than that of normal control（ P〈0.05）.The mRNA levels of TLR4、My D88 were reduced in all protection groups（ P〈0.05）; and the mRNA levels of NF-k B was reduced in all protection groups（ P〈0.05） except for high-dose SalB group（ P〉0.05）.Conclusion： The protection mechanisms of SPE on injured podocytes induced by PAN which may be partly through inhibiting TLR4 signal pathway; among the monomers of SPE,the effects of RA was better than those of SalB and admixture.
出处
《中国中西医结合肾病杂志》
2017年第8期672-675,I0002,共5页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
南京军区医学创新重大课题(No.15DX008)
