C-藻蓝蛋白纳米微球的制备及体外抗脂多糖联合海水诱导急性肺损伤的作用机制研究  

Preparation of C-phycocyanin nanospheres and the in vitro effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater

作　　者：解佑银 汪鎔金 邵丽林 刘关童 张雷芳 XIE Youyin;WANG Rongjin;SHAO Lilin;LIU Guantong;ZHANG Leifang(School of Food and Pharmacy,Zhejiang Ocean University,Zhejiang Zhoushan 316022,China)

机构地区：[1]浙江海洋大学食品与药学学院,浙江舟山316022

出　　处：《中国药房》2024年第16期1964-1971,共8页China Pharmacy

基　　金：国家自然科学基金项目(No.81903693);浙江省高校基本科研业务费专项(No.2019J00013)。

摘　　要：目的制备C-藻蓝蛋白纳米微球(CPC-NPs)并评价其对脂多糖(LPS)联合海水诱导急性肺损伤的体外作用机制。方法采用离子交联法,以CPC为药物、羧甲基壳聚糖(CMCS)为载体、CaCl2为交联剂制备CPC-NPs,对CPC-NPs进行基础表征。小鼠肺泡Ⅱ型上皮细胞MLE-12和巨噬细胞RAW264.7均分为7组:正常组(Con组),模型组(Mod组),空白NPs组,CPC-NPs 30、60、120、240μg/mL组。除Con组外其余各组均采用10μg/mL LPS和25%海水联合处理6 h,造模后各给药组加相应剂量药物处理24 h。检测MLE-12细胞中丙二醛(MDA)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)水平,B细胞淋巴瘤2(Bcl-2)、Bcl-2关联X蛋白(Bax)、胱天蛋白酶-3(caspase-3)蛋白和mRNA,以及CAT、谷胱甘肽S-转移酶(GST)mRNA表达水平;检测RAW264.7细胞中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、IL-6水平,NOD样受体热蛋白结构域相关蛋白3(NLRP3)、剪切的caspase 1(cleaved-caspase-1)蛋白及IL-1β、TNF-α、IL-6、诱导型一氧化氮合酶(iNOS)mRNA表达水平。结果制备的CPC-NPs粒径为(675.69±64.58)nm,Zeta电位为(-20.11±0.98)mV,多分散系数为0.455±0.010(n=3);包封率为35.60%,载药量为16.13%;形态为规则的球形;其中药物可持续释放30h以上。与Mod组比较,CPC-NPs各浓度组MLE-12细胞中T-AOC、SOD、CAT(除CPC-NPs 30μg/mL组外)、GSH-Px水平和CAT、GST mRNA表达水平以及Bcl-2/Bax蛋白比值和mRNA比值均显著升高,MDA水平和caspase-3蛋白及mRNA表达水平均显著降低(P<0.01或P<0.05)。与Mod组比较,CPC-NPs各浓度组RAW264.7细胞中IL-1β、TNF-α、IL-6水平和NLRP3、cleaved-caspase-1蛋白表达水平以及IL-1β、TNF-α、IL-6、iNOS mRNA表达水平均显著降低(P<0.01或P<0.05)。结论成功制备了具有肺靶向性和缓释性的CPC-NPs。CPC-NPs可以通过抗氧化应激及抑制细胞凋亡和炎症反应来缓解LPS联合海水诱导的急性肺损伤。OBJECTIVE To prepare C-phycocyanin nanoparticles(CPC-NPs)and evaluate the in vitro mechanism of CPC�NPs on acute lung injury induced by lipopolysaccharide(LPS)combined with seawater.METHODS Ion crosslinking method was used to prepare CPC-NPs using CPC as the drug,carboxymethyl chitosan(CMCS)as the carrier,and CaCl2 as the crosslinking agent.The basic characterization of CPC-NPs was carried out.Mouse alveolar typeⅡepithelial cells MLE-12 and macrophages RAW264.7 were divided into 7 groups:normal group(Con group),model group(Mod group),blank NPs group,CPC-NPs 30,60,120 and 240μg/mL groups.Except for the Con group,all other groups were treated with a combination of 10μg/mL LPS and 25%seawater for 6 hours.After modeling,each treatment group was treated with corresponding drugs for 24 hours.The levels of malondialdehyde(MDA),total antioxidant capacity(T-AOC),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)in MLE-12 cells,as well as the expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),caspase-3 protein and mRNA,CAT and glutathione S-transferase(GST)mRNA were determined.The levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and IL-6 in RAW264.7 cells,as well as the expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),cleaved caspase-1 protein,and mRNA expressions of IL-1β,TNF-α,IL-6 and inducible nitric oxide synthase(iNOS)were all detected.RESULTS The prepared CPC-NPs had particle size of(675.69±64.58)nm,Zeta potential of(-20.11±0.98)mV,polydispersity coefficient of 0.455±0.010(n=3);entrapment efficiency of 35.60%,and drug loading of 16.13%;CPC-NPs had regular spherical shapes,where the drug could be sustainably released for more than 30 hours.Compared with Mod group,the levels of T-AOC,SOD,CAT(excluding the 30μg/mL group of CPC-NPs)and GSH-Px,mRNA expressions of CAT and GST,as well as the Bcl-2/Bax protein ratio and mRNA ratio were significantly increased in MLE-12 cells of different concentration gro

关 键 词：C-藻蓝蛋白 纳米微球 急性肺损伤 氧化应激 凋亡 炎症反应 海水 脂多糖 

分 类 号：R965[医药卫生—药理学] R944[医药卫生—药学]