Investigation On Novel Di-ART-GPC Liposomes For Anti-inflammatory Effects And Mechanism Of Intervention On Inflammation Induced By Nano-TiO₂

With the rapid development of nanotechnology,metal nanoparticles are widely used in various fields,and nanometer titanium dioxide（nano-TiO₂）is one of the most used metal nanoparticles,annual production capacity of 10000 tons.The large-scale production and application of nano-TiO₂led to its inevitable entry into the environment,and the toxic effects and safety issues caused by nano-TiO₂ on organisms and ecosystems have attracted wide attention as well.This study was to evaluate the anti-inflammatory effect of artesunate phospholipid diploid（Di-ART-GPC）liposomes synthesized by the group,on the basis of investigating the inflammatory effect and mechanism of nano-TiO₂,combined with the concept of chemical prevention of health injury caused by exogenous harmful substances.And then intervened the inflammatory process of nano-TiO₂used Di-ART-GPC liposomes to observe the influence of which on the inflammatory effect induced by nano-TiO₂ and explore the potential of Di-ART-GPC liposome in preventing or reducing nano-TiO₂ health hazard,providing the basis for in-depth research on the toxicity effect and application safety of nano-TiO₂.The specific experimental contents mainly consisted of the three parts:1.Effects and mechanism of nano-TiO₂ on the expression of inflammatory cytokinesObjective:To investigate the inflammatory effect and mechanism of nano-TiO₂,providing basic theory and data support for the studies on the toxicology and safety of nano-TiO₂.Methods:Transmission electron microscope（TEM）was applied to observe the shape of nano-TiO₂ particles,and Dynamic light scattering（DLS）method was used to characterize the average hydration particle size,dispersion coefficient and Zeta potential of nano-TiO₂.CCK8 assay was used to determine the effects of nano-TiO₂ on the proliferation activity of RAW264.7 cells,and ELISA method was used to determine the effects of different concentration of nano-TiO₂ on the expression level of inflammatory factors in RAW264.7 cells.Western Blot method was used to determine the effects of different concentration of nano-TiO₂ on the protein expression of p-ERK,ERK,p-JNK,JNK,p-p38 and p38 in MAPK signaling pathway.Results:The results of TEM observation showed that the nano-TiO₂ particles dispersed in the DMEM complete medium were spherical and some particles could be aggregated.The results of DLS method showed that the average hydrated particle size of nano-TiO₂ particles was 74.10±0.21 nm,the dispersion coefficient（PDI）was 0.14±0.04,and the Zeta potential was-28.87±0.42 m V.CCK8results showed that RAW264.7 respective treated with 5μg/mL,10μg/mL and 20μg/mL nano-TiO₂had no significant effects on cell proliferation（cell proliferation rate>80%）,while the cell proliferation rate was lower than 80%treated with 40μg/mL nano-TiO₂.ELISA results showed that10μg/mL and 20μg/mL nano-TiO₂ used to interfere RAW264.7 cells could increase the expression levels of its inflammatory factors such as TNF-α,IL-1βand IL-6（P<0.05）.Western Blot results showed that the expression of phosphorylated proteins such as p-ERK,p-p38 and p-JNK could be increased by the intervention of 10μg/mL and 20μg/mL nano-TiO₂（P<0.05）,and the protein gray value ratios of p-ERK/ERK,p-JNK/JNK and p-p38/p38 could be increased（P<0.05）.2.Evaluation of anti-inflammatory effects of Di-ART-GPC liposomesObjective:To evaluate the anti-inflammatory effects of the novel Di-ART-GPC liposomes prepared earlier and to verify the effects in the animal inflammatory model preliminarily.Meanwhile,to observe the systemic toxicity of the Di-ART-GPC liposomes,providing experimental basis for the basic research on the application of anti-inflammatory chemoprevention of Di-ART-GPC.Methods:CCK8 method was used to determine the cytotoxicity of Di-ART-GPC liposomes on RAW264.7 cells in vitro,LPS-induced inflammatory cell model was used to study its anti-inflammatory effects in vitro.Then,the anti-inflammatory effects of Di-ART-GPC liposomes was further verified by constructing the FCA-induced rheumatoid arthritis rat model.Finally,the systemic toxicity of Di-ART-GPC liposomes was evaluated by rabbit vascular stimulation test and guinea pig systemic active allergy test.Results:CCK8 results showed that the proliferation rate of RAW264.7 cells treated with Di-ART-GPC liposomes was higher than that of ART-treated cells at concentrations of 0.625,1.25,2.5,5,10 and 20μg/mL for 6h,12h and 24h respectively（P<0.05）.ELISA results showed that the upregulation of LPS-induced inflammatory factors such as TNF-α,IL-1βand IL-6 could be inhibited by 1.25μg/mL Di-ART-GPC liposomes（P<0.05）,and the inhibition was better than that of the precursor drug ART of the same concentration.The results of arthritis rats model in vivo confirmed that treatments with 3.0mg/kg and 6.0mg/kg Di-ART-GPC liposomes could reduce ankle swelling in rats（P<0.05）,down-regulate the expression of inflammatory cytokines TNF-αand IL-6 in serum of rats（P<0.05）,and the anti-inflammatory effects of 6.0mg/kg dose was better than that of 2.4mg/kg precursor drug ART.The results of vascular irritation test in rabbits and systemic active allergy test in guinea pigs showed that the 6.0 mg/kg Di-ART-GPC liposomes had no irritation to blood vessels in rabbits and no allergy to guinea pigs.3.Effects of MAPK signaling pathway in the influence of Di-ART-GPC liposomes on the inflammatory process reduced by nano-TiO₂Objective:To observe the effects and mechanism of Di-ART-GPC liposomes on the inflammatory process reduced by nano-TiO₂,and to explore the potential application of Di-ART-GPC liposomes in inhibiting the inflammatory damage of nano-TiO₂.Methods:ELISA was used to determine the effects of different concentrations of Di-ART-GPC liposomes on the secretion of RAW264.7 inflammatory cytokines induced by nano-TiO₂;Western Blot method was used to determine the effect of different concentrations of Di-ART-GPC liposomes on the expression of p-ERK,ERK,p-JNK,JNK,p-p38 and p38 proteins in MAPK signaling pathway affected by nano-TiO₂.Results:ELISA results showed that 1.25μg/mL and 2.5μg/mL Di-ART-GPC liposomes could inhibit the expression of inflammatory factors induced by nano-TiO₂（P<0.05）.Western Blot results showed that the protein gray value ratio p-JNK/JNK decreased after 1.25μg/mL Di-ART-GPC liposomes intervention compared with the nano-TiO₂ treatment group（P<0.05）.After the intervention of 2.5μg/mL Di-ART-GPC liposomes,the protein gray value ratios of p-ERK/ERK,p-JNK/JNK,and p-p38/p38 all decreased compared with the nano-TiO₂ treatment group（P<0.05）.4.Conclusions1.The cytotoxicity of nano-TiO₂ was relatively low,and the proliferation activity of RAW264.7 cells was not significantly affected by 20μg/mL or below concentration of nano-TiO₂.Nano-TiO₂ could increase the expression of TNF-α,IL-1β,IL-6 at the concentration of 10μg/mL and 20μg/mL,exposed nano-TiO₂ increased protein grey value ratio of p-ERK/ERK,p-JNK/JNK and p-p38/p38.Triggering inflammatory injury through ERK/JNK/p38 MAPK signaling pathways,which induced the production of inflammatory factors including TNF-α,IL-1β,IL-6 might be one of the inflammatory mechanisms of nano-TiO₂ particles.2.Di-ART-GPC liposomes,as a novel artemisinin derivative,had relatively lower cytotoxicity at the same dose as artesunate in clinical use,and showed good anti-inflammatory effects in both the LPS-induced cellular inflammatory model and the FCA-induced rat arthritis model.It was not irritant to rabbit blood vessel and was not allergic to guinea pig,which indicated its good biosecurity as a potential anti-inflammatory compound.3.Di-ART-GPC liposomes intervention could inhibit the up-regulation of the expression of inflammatory factors induced by nano-TiO₂,and the up-regulation of phosphorylated proteins such as p-ERK,p-p38 and p-JNK in nano-TiO₂ induced cells.Di-ART-GPC liposomes might play an inhibitory role on the inflammatory injury induced by nano-TiO₂ with inhibiting the activation of ERK/JNK/p38 MAPK signaling pathway.