Pharmacodynamic Study Of Copper-Coordinated Nanocrystals For Sepsis Therapy Via Inhibiting NLRP3 Inflammasome

Introduction and ObjectivesSepsis has been defined as life-threatening organ dysfunction resulting from disordered host responses to infection.It is characterized by complex pathology,rapid disease progression and high mortality.Even after successful rescue,there are many lifelong complications,which seriously affect the quality of patients life.Clinically,sepsis treatment is limited to antibiotics and organs supportive care and the cost is high.Therefore,it is urgent to develop effective and low-toxicity drugs for sepsis treatment.Compared with the development of completely new drugs which is time-consuming,costly and high failure rate,“Drug Repurposing”provides a shortcut for the discovery of anti-sepsis drugs.Recently,disulfiram（DSF）,a clinically used antialcoholic drug,has been found to have potential in sepsis therapy by inhibiting pyroptosis.Besides,Cu²⁺supplementation could significantly enhance the efficacy of DSF both in vitro and in vivo,but the underlying mechanism is not clear.Therefore,we carried out research and found that after DSF and Cu²⁺co-treated cells,the formation of Cu ET was detected by LC-MS,and its content was positively correlated with the amount of Cu²⁺addition and anti-pyroptosis effect.Further studies showed that the anti-pyroptosis activity of Cu ET compound was significantly higher than that of DSF and was comparable to that of DSF/Cu²⁺co-treatment.Thus,Cu ET is speculated to be a highly potential anti-sepsis active compound and planned to be developed.However,Cu ET is difficult to dissolve in aqueous solvents and most organic solvents,which poses a challenge for developing pharmaceutical preparations.Therefore,based on the idea of constructing nanodrugs by metal-organic coordination,this project synthesized Cu ET NPs in situ with the aid of stabilizer by mixing Cu Cl₂and DTC,solving the problem of Cu ET insolubility.On the basis of the preparation and characterization of Cu ET NPs,the in vitro/in vivo efficacy and mechanism of Cu ET NPs were systematically studied.Methods and Results（1）Preparation and characterization of Cu ET NPsUsing 0.4%PVP as stabilizer,2 m M Cu Cl₂solution was dropped into 4m M DTC solution at the rate of 50μL/min,and then dialyzed to obtain Cu ET NPs.Cu ET NPs aqueous solution was brown-yellow in color.The hydration diameter of Cu ET NPs was about 90 nm andζpotential was-4.5 m V.The formation of the coordination structure was confirmed by UV-Vis,FTIR,and XRD analysis,and Cu ET NPs was a kind of nanocrystal.The medium stability and storage stability of Cu ET NPs were good.（2）Pharmacodynamic study in vitro of Cu ET NPsUsing macrophages as research model,the biosafety of 2μM Cu ET NPs was proved good.The results of LDH and ELISA experiments showed that Cu ET NPs could significantly inhibit the NLRP3inflammasome-induced pyroptosis,but had no effect on NLRC4,AIM2and Caspase-11 induced pyroptosis.Moreover,the anti-pyroptosis activity of Cu ET NPs was in a concentration-dependent manner.（3）Molecular mechanism study of Cu ET NPsUsing macrophages as research model,the molecular mechanism of Cu ET NPs inhibiting pyroptosis was investigated through Western blot,immunofluorescence and immunoprecipitation.The results showed that after the intervention of Cu ET NPs,the expressions of cleaved-Caspase-1,cleaved-IL-1βand GSDMD-N were decreased,NLRP3 and pro-IL-1βwere not affected,and the formation of ASC polymers was inhibited.In addition,the content of NLRP3 bound to ASC or NEK7 was reduced in IP samples.Therefore,the molecular mechanism of Cu ET NPs was inhibiting the interaction between NLRP3 and NEK7,thus reducing the assembly of NLRP3 inflammasome and the occurrence of pyroptosis.（4）Pharmacodynamic study in vivo of Cu ET NPsTaking LPS-induced sepsis mice as research model,two dose groups were set up,and the in vivo efficacy of Cu ET NPs was evaluated by body temperature monitoring,measurement of inflammatory factor level,and histopathological analysis.The results showed that Cu ET NPs treatment could avoid the obvious drop of body temperature in mice,significantly reduce the level of NLRP3-specific inflammatory factor（IL-1β）,and alleviate the pathological damage of lung and liver of septic mice.ConclusionsIn this project,a kind of copper coordination nanocrystalline（Cu ET NPs）was constructed based on the idea of metal-organic coordination,which solved the druggability problem of Cu ET,a potential anti-sepsis compound.The therapeutic effects of Cu ET NPs on sepsis were verified at the cell and animal levels,of which the underlying mechanism was inhibiting the interaction between NLRP3 and NEK7,thus reducing the assembly of NLRP3 inflammasome and the occurrence of pyroptosis.