Preparation of C-phycocyanin nanospheres and the in vitro effect mechanism on acute lung injury induced by lipopolysaccharide combined with seawater
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 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(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 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(P＜0.01 or P＜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(P＜0.01 or P＜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.
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