三七总皂苷玉米脂蛋白纳米粒对大鼠脑缺血再灌注损伤的药效学研究

Pharmacodynamics study of Panax notoginseng saponins zein lipoprotein nanoparticles for cerebral ischemia reperfusion injury in rats

对三七总皂苷(Panax notoginseng saponins,PNS)与卵磷脂、β-谷甾醇、玉米醇溶蛋白共组装制备的三七总皂苷玉米脂蛋白纳米粒(PNS-lipid-zein nanoparticles,PLZ-NPs)进行了体外细胞实验,以及大鼠脑缺血再灌注损伤的药效学研究。采用反溶剂沉淀法制备了PLZ-NPs,运用激光粒度仪和透射电镜显微镜对纳米粒进行表征,MTT法评价了纳米粒的细胞毒性,激光共聚焦和流式细胞术分析了Caco-2细胞对纳米粒的摄取情况。大脑中动脉闭塞(MCAO)法构建了大鼠脑缺血再灌注损伤模型,连续3 d灌胃给药后,取脑组织进行2,3,5-氯化三苯基四氮唑(TTC)染色,并检测脂质过氧化物丙二醛(MDA)、炎症因子IL-1β、TNF-α、凋亡相关蛋白Bax、Bcl-2的水平。结果显示,PLZ-NPs平均粒径为(116.4±0.81)nm,PDI为0.048,Zeta电位为-(31.5±0.31)mV。MTT实验表明,玉米脂蛋白载体的生物安全性良好,且能显著提高Caco-2细胞对纳米粒的摄取(纳米粒组在4 h时细胞摄取量提高至游离组的1.76倍)。TTC染色中,PNS和PLZ-NPs组的梗死面积比模型组均有减少,另外MDA、IL-1β、TNF-α、Bax的含量明显下降,Bcl-2含量增加,而且PLZ-NPs组对脑缺血再灌注损伤的保护作用显著优于PNS组(P<0.05)。实验结果表明,本实验制备的玉米脂蛋白纳米粒具有良好的稳定性、药物负载能力和生物安全性,能显著提高肠上皮细胞对药物的摄取,有效改善脑缺血再灌注对大鼠造成的损伤。

PLZ-NPs(PNS-lipid-zein nanoparticles)prepared by co-assembly of Panax notoginseng saponins,lecithin,β-sitosterol and zein were applied for in vitro cell experiment and oral gavage to study the protectiveeffect of cerebral ischemia-reperfusion rats.PLZ-NPs were characterized by Malvin-particle size analyzer andtransmission electron microscope(TEM),respectively.The toxicity of PLZ-NPs and free carrier were evaluatedby MTT,and the uptake of nanoparticles in Caco-2 cells was analyzed by laser confocal and flow cytometry.Thecerebral ischemia reperfusion rat model was established by MCAO method and then be given samples by gavagefor 3 days.The brain tissues were taken to stain by 2,3,5-triphenyltetrazole chloride(TTC)and the biochemical indicators of MDA,inflammatory cytokines IL-1βand TNF-α,apoptosis-related proteins Bax and Bcl-2 from theharvested brain tissues were detected to evaluate the protective effect of PNS in PLZ-NPs on cerebral ischemiareperfusion.The particle size,PDI,and zeta potential of formed PLZ-NPs were(116.4±0.81)nm,0.048 and-(31.5±0.31)m V,respectively.The results of MTT showed that the zein lipoprotein carrier was non-toxic toCaco-2 cells.The results of laser confocal and flow cytometry showed that FITC uptake of nanoparticles could besignificantly improved in Caco-2 cells.The uptake from the nanoparticles at 4 h was 1.76 times of that of the freeFITC group.Compared with the model group,the TTC staining images of free drug PNS group and PLZ-NPsgroup showed certain reduction in the white infarct area.The contents of MDA,IL-1β,TNF-αand Bax weresignificantly decreased,while the content of Bcl-2 was significantly increased.Furthermore,all parameters ofPLZ-NPs group showed better results than those of PNS group,and there was a significant difference(P<0.05).All results indicated that the prepared PLZ-NPs had good stability and biological safety,and could significantlyincrease the uptake in intestinal epithelial cells,and effectively protect against the damage caused by cerebralischemia reperfusion in rats.