微乳对葛根素在MDCK-MDR1细胞的转运影响及机制研究
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摘要
目的探讨微乳制剂对葛根素在血脑屏障(BBB)细胞模型MDCK-MDR1的转运影响以及机制。方法采用MTT法评价葛根素微乳与溶液的细胞毒性浓度范围,确定适宜给药质量浓度,考察葛根素溶液及微乳在MDCK-MDR1单层的双侧转运特性,通过免疫组化染色研究细胞紧密连接蛋白的表达,荧光光漂白恢复法测定细胞膜流动性的变化以及阴离子探针结合流式细胞技术研究细胞膜电位的改变,进而阐明微乳对葛根素转运影响的作用机制。结果葛根素溶液质量浓度50~300μg/m L,微乳稀释500倍以上对MDCK-MDR1无显著毒性。溶液中葛根素在MDCK-MDR1单层的吸收方向转运表观渗透系数(Papp)为1.04×10-6 cm/s,外排方向转运Papp值为1.05×10-6 cm/s,微乳中葛根素双侧转运Papp值均较溶液中葛根素显著增加(P<0.05)。微乳作用可以减少MDCK-MDR1紧密连接蛋白Claudin-1、Occludin、ZO-1、F-actin的表达,促进细胞膜流动,降低细胞膜电位。结论微乳制剂可以促进葛根素在BBB细胞模型MDCK-MDR1的双侧转运,其作用机制与打开细胞间紧密连接,增加细胞膜流动性,使细胞去极化,降低膜电位进而增大葛根素细胞旁路渗透有关。
Objective To explore the effect of microemulsion on the transport and mechanism of puerarin in blood brain barrier(BBB) cell model MDCK-MDR1. Methods MTT assay was used to evaluate the cytotoxicity of puerarin microemulsion and solution, and determine the appropriate concentration of administration. The bilateral transport characteristics of puerarin solution-microemulsion was investigated in MDCK-MDR1 monolayer. Immunohistochemical staining was used to study the expression of tight junction proteins, and the changes in cell membrane fluidity was studied by fluorescence bleaching recovery, and the changes of membrane potential was measured by anion probe combined with flow cytometry. The mechanism of the effect of microemulsion on puerarin transport was clarified. Results The MDCK-MDR1 showed no significant toxicity when the mass concentration of puerarin solution ranged from 50 to 300 μg/m L and the microemulsion dilution was over 500 times. The Papp value in absorption direction of puerarin solution on MDCK-MDR1 monolayer was 1.04 × 10-6 cm/s, and the Papp value of excretion direction was 1.05 × 10-6 cm/s. The Pappvalue of puerarin in microemulsion was significantly increased compared with that in solution(P < 0.05). Microemulsification could reduce the expression of Claudin-1, Occludin, ZO-1, and F-actin in MDCK-MDR1, promote cell membrane flow, and decrease cell membrane potential. Conclusion Microemulsion can promote the bilateral transport of puerarin in the BBB cell model MDCK-MDR1. The mechanism is closely connected with the opening of tight junctions, increasing the cell membrane fluidity, making the cell depolarizing and reducing membrane potential, and increasing the permeation of paracellular.
Objective To explore the effect of microemulsion on the transport and mechanism of puerarin in blood brain barrier(BBB) cell model MDCK-MDR1. Methods MTT assay was used to evaluate the cytotoxicity of puerarin microemulsion and solution, and determine the appropriate concentration of administration. The bilateral transport characteristics of puerarin solution-microemulsion was investigated in MDCK-MDR1 monolayer. Immunohistochemical staining was used to study the expression of tight junction proteins, and the changes in cell membrane fluidity was studied by fluorescence bleaching recovery, and the changes of membrane potential was measured by anion probe combined with flow cytometry. The mechanism of the effect of microemulsion on puerarin transport was clarified. Results The MDCK-MDR1 showed no significant toxicity when the mass concentration of puerarin solution ranged from 50 to 300 μg/m L and the microemulsion dilution was over 500 times. The Papp value in absorption direction of puerarin solution on MDCK-MDR1 monolayer was 1.04 × 10-6 cm/s, and the Papp value of excretion direction was 1.05 × 10-6 cm/s. The Pappvalue of puerarin in microemulsion was significantly increased compared with that in solution(P < 0.05). Microemulsification could reduce the expression of Claudin-1, Occludin, ZO-1, and F-actin in MDCK-MDR1, promote cell membrane flow, and decrease cell membrane potential. Conclusion Microemulsion can promote the bilateral transport of puerarin in the BBB cell model MDCK-MDR1. The mechanism is closely connected with the opening of tight junctions, increasing the cell membrane fluidity, making the cell depolarizing and reducing membrane potential, and increasing the permeation of paracellular.
引文
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[4]赵庭,贾运涛,张良珂.载葛根素聚乙烯亚胺/海藻酸钠自组装纳米粒的制备及性能研究[J].中草药,2017,48(17):3523-3528.
[5]Yang B,Du S,Lu Y,et al.Influence of paeoniflorin and menthol on puerarin transport across MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model[J].J Pharm Pharmacol,2018,70(3):349-360.
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[13]Allander S V,Larsson C,Ehrenborg E,et al.Characterization of the chromosomal gene andpromoter for human insulin 1ike growth factor binding protein-5[J].Biol Chem,1994,269(8):10891-10898.
[14]Shimasaki S,Shimonaka M,Ling N.Identification of five different insulin-like growth factorbinding proteins(IGFBPs)from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human[J].Biol Chem,1991,266(5):10646-10653.
[15]Leitner D M,Brown F L H,Wilson K R.Regulation of protein mobility in cell membranes:A dynamic corral model[J].Biophys J,2000,78(1):125-135.
[16]Chen L,Zhu J,Li Y,et al.Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in Chitosan/DNA Complexes[J].PLo S One,2013,8(8):e71953.
[17]Zbytovska J,Raudenkolb S,Wartewig S.Phase behavior of transkarbam 12[J].Chem Phys Lipids,2004,129(1),97-109.
[18]Thanou M M,Florea B I,Langemeyer M W,et al.N-trimethylated chitosan chloride(TMC)improves the intestinal permeation of the peptide drug buserelin in vitro(Caco-2)and in vivo(rats)[J].Pharm Res,2000,17(1):27-31.
[2]Bicker J,Alves G,Fortuna A,et al.Blood-brain barrier models and their relevance for a successful development of CNS drug delivery systems:A review[J].Eur J PharmBiopharm,2014,87(3):409-432.
[3]万小敏,丁宇翔,赵兵杰,等.载葛根素的PEG修饰介孔硅纳米粒的制备及其对急性心肌缺血大鼠的保护作用[J].中草药,2018,49(8):1789-1795.
[4]赵庭,贾运涛,张良珂.载葛根素聚乙烯亚胺/海藻酸钠自组装纳米粒的制备及性能研究[J].中草药,2017,48(17):3523-3528.
[5]Yang B,Du S,Lu Y,et al.Influence of paeoniflorin and menthol on puerarin transport across MDCK and MDCK-MDR1 cells as blood-brain barrier in vitro model[J].J Pharm Pharmacol,2018,70(3):349-360.
[6]Yang B,Du S,Lu Y,et al.Nose-to-brain delivery:Puerarin uptake on olfactoryensheathing cells and transport on Calu-3 cells[J].中华中医药杂志,2018,33(5):2161-2167.
[7]Dominique D,Gilles P,Drug develop[J].Indus Pharmacy,1993,19(1):101-122.
[8]Gabriele V,Ronnet L,Hester D,et al.Primary culture of neonatal rat olfactory neurons[J].J Neurosci,1991,11(5):1243-1255.
[9]Gluckman P,Klempt N,Guan J,et al.A role for IGF-1 in the rescue of CNS neurons following hypoxic-ischemic injury[J].Biochem Biophys Res Commun,1992,182(31):593-599.
[10]Cunningham A M,Manis P B,Reed R R,et al.Olfactory receptor neurons exist as distinct subclasses of immature and mature cells in primary culture[J].Neuroscience,1999,93(4):1301-1312.
[11]Liu N,Shields C B,Roisen F J.Primary culture of adult mouse olfactory receptor neurons[J].Exp Neurol,1998,151(2):173-183.
[12]Kou K,Mittanck D W,Fu C,et al.Structure and function of the mouse insulin‘like growth factor binding protein 5gene promoter[J].DNA Cell Biol,1994,241(14):241-249.
[13]Allander S V,Larsson C,Ehrenborg E,et al.Characterization of the chromosomal gene andpromoter for human insulin 1ike growth factor binding protein-5[J].Biol Chem,1994,269(8):10891-10898.
[14]Shimasaki S,Shimonaka M,Ling N.Identification of five different insulin-like growth factorbinding proteins(IGFBPs)from adult rat serum and molecular cloning of a novel IGFBP-5 in rat and human[J].Biol Chem,1991,266(5):10646-10653.
[15]Leitner D M,Brown F L H,Wilson K R.Regulation of protein mobility in cell membranes:A dynamic corral model[J].Biophys J,2000,78(1):125-135.
[16]Chen L,Zhu J,Li Y,et al.Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in Chitosan/DNA Complexes[J].PLo S One,2013,8(8):e71953.
[17]Zbytovska J,Raudenkolb S,Wartewig S.Phase behavior of transkarbam 12[J].Chem Phys Lipids,2004,129(1),97-109.
[18]Thanou M M,Florea B I,Langemeyer M W,et al.N-trimethylated chitosan chloride(TMC)improves the intestinal permeation of the peptide drug buserelin in vitro(Caco-2)and in vivo(rats)[J].Pharm Res,2000,17(1):27-31.