Mechanism Study Of Active Compounds From Nanhaia Speciosa To Treat Pulmonary Edema Secondary Acute Lung Injury And Spray Development

Objectives: To study the effect and mechanism of active compounds from Nanhaia speciosa(Nh S)on pulmonary edema(PE)secondary acute lung injury(ALI),and to research formulation development.Methods: 1.An in vitro cell model of PE secondary ALI was established,and q RT-PCR and WB were used to detect the effects of Nh S on the expression levels of ENa C-α/NKAB1/AQP1 genes and proteins.2.Based on the TCMSP database and literatures,active compounds of Nh S and their related targets were collected,and disease targets of “Acute lung injury” and “pulmonary edema”were collected from disease databases of OMIM,Drug Bank,Gene Cards and TTD.The intersection targets of “drug-disease” were obtained,and PPI was selected to key targets.The intersection targets were analyzed by GO and KEGG.q RT-PCR was used to verify the key targets and related pathway of Nh S to treat PE secondary ALI,and effective targets were docked with related active compounds to select the best active compound.3.q RT-PCR and WB were used to detect the effects of the best active component on the expression of ENa C-α/NKAB1/AQP1 genes and proteins.4.Spray formulation was screened and optimized based on morphology,particle size,potential,encapsulation rate,and drug loading.Quality evaluation of the formulation was evaluated by air dynamic diameter,powder flowability and lung deposition rate.Results: Nh S was able to inhibit TNF-α-induced downregulation of ENa C-α/NKAB1/AQP1 protein and gene levels(p < 0.05),and the drug effects showed concentration-dependency.The low-dose group to ENAC-α had no statistically significant difference in protein expression.Network pharmacology analysis and experimental verification combined with molecular docking results showed that Maackiain,one of active component in Nh S might exert therapeutic effects on PE Secondary ALI by PTGS2 protein in NF-κB pathway.Maackiain can significantly increase the expression levels of NKAB1/AQP1 protein and genes at a dose of 20-40μM(p < 0.05),and its effect was concentration-dependent.The formulation was set to a drug-lipid ratio of 1:8(mol/mol),with egg yolk phospholipids to cholesterol ratio of 5:1(m/m).Maackiain liposomes were prepared by thin film dispersion method with hydration under ultrasonic condition for 30 min,followed by addition of 20% mannitol(m/v)and lyophilize to obtain Maackiain Spray.The encapsulation rate of Maackiain Spray was 92.57%,and the drug loading was 8.67%.The hydrodynamic diameter was(225.37±3.81)nm,the polydispersity index(PDI)was0.283±0.046,and the zeta potential was(-9.24±0.27)m V.The air dynamic diameter was(4.74±0.30)μm,the powder angle of repose was 26.7°,and the lung deposition rate was 33.27%.Conclusions: Maackiain in Nh S may relieve the fluid transport disorder of PE Secondary ALI by upregulating NKAB1/AQP1.Maackiain Spray is easy to obtain,with a simple process and conforming to lung inhalation standards.