Preparation of C-phycocyanin nanospheres and the <italic style="font-style: italic">in vitro</italic> effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater

XIE Youyin; WANG Rongjin; SHAO Lilin; LIU Guantong; ZHANG Leifang

doi:10.6039/j.issn.1001-0408.2024.16.05

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

更新时间：2024-08-23

- Preparation of C-phycocyanin nanospheres and the in vitro effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater
- ZHONGGUO YAOFANG Vol. 35, Issue 16, Pages: 1964-1971(2024)
- 作者机构：
  
  浙江海洋大学食品与药学学院，浙江舟山　316022
- 作者简介：
- 基金信息：
- DOI：10.6039/j.issn.1001-0408.2024.16.05
  CLC： R965;R944
- Published：30 August 2024，
  
  Received：06 January 2024，
  
  Revised：19 May 2024，
- 稿件说明：
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解佑银,汪鎔金,邵丽林等.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 ^Δ[J].中国药房,2024,35(16):1964-1971.

XIE Youyin,WANG Rongjin,SHAO Lilin,et al.Preparation of C-phycocyanin nanospheres and the in vitro effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater[J].ZHONGGUO YAOFANG,2024,35(16):1964-1971.
解佑银,汪鎔金,邵丽林等.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 ^Δ[J].中国药房,2024,35(16):1964-1971. DOI： 10.6039/j.issn.1001-0408.2024.16.05.

XIE Youyin,WANG Rongjin,SHAO Lilin,et al.Preparation of C-phycocyanin nanospheres and the in vitro effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater[J].ZHONGGUO YAOFANG,2024,35(16):1964-1971. DOI： 10.6039/j.issn.1001-0408.2024.16.05.

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、CaCl

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、240 μg/mL组。除Con组外其余各组均采用10 μg/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（

＝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 30 μg/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（

＜0.01

或

＜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（

＜0.01或

＜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。

Abstract

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 CaCl

as the crosslinking agent. The basic characterization of CPC-NPs was carried out. Mouse alveolar type Ⅱ epithelial 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 240 μg/mL groups. Except for the Con group， all other groups were treated with a combination of 10 μg/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 （

＝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 30 μg/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 groups of CPC-NPs， while MDA levels and caspase-3 protein and mRNA expression were significantly reduced （

＜0.01 or

＜0.05）. Compared with Mod group， the levels of IL-1β， TNF-α and IL-6， NLRP3 and cleaved-caspase-1 protein expressions， as well as the mRNA expressions of IL-1β， TNF-α， IL-6 and iNOS in RAW264.7 cells of different concentration groups of CPC-NPs were significantly reduced （

＜0.01 or

＜0.05）.

CONCLUSIONS

CPC-NPs with lung targeting and sustained release property were prepared successfully， which can alleviate acute lung injury induced by LPS combined with seawater through antioxidant stress， inhibiting cell apoptosis and inflammatory response.

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

Keywords

nanospheresacute lung injuryoxidative stressapoptosisinflammatory responseseawaterlipopolysaccharide

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

DOI：10.6039/j.issn.1001-0408.2024.16.05

摘要

Abstract

关键词

Keywords

references