Material Basis and Mechanism of Ganjiang(干姜)in Treatment of Acute Lung Injury
Objective:To optimize the ethanol extraction process of Ganjiang(干姜)by orthogonal design,identify the chemical components,and explore the material basis and mechanism of Ganjiang in the treatment of acute lung injury(ALI)by network pharmacology,molecular doc-king,and animal experiments.Methods:With the content of 6-gingerol and yield as indicators,orthogonal design was employed to optimize the ethanol concentration,solvent volume,extraction time,and extraction times,and the optimized conditions were valiated.Ultra perform-ance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)was adopted to qualitatively analyze the chemical components of Ganjiang in positive and negative ion modes.The identified chemical components were analyzed by network pharma-cology,on the basis of which the core components,key targets,and related pathways of Ganjiang in the treatment of ALI were predicted.Mo-lecular docking was then employed to verify the binding affinity of the core components with target proteins.The mouse model of ALI was es-tablished by lipopolysaccharide(LPS),and the lung injury was assessed based on the lung index and observation of the lung tissue sections.The levels of inflammatory cytokines and oxidative stress indicators in the bronchoalveolar lavage fluid(BALF)of mice were emeasured.Western blotting was employed to determine the protein levels of tumor necrosis factor-α(TNF-α),serine/threonine protein kinase 1(AKT1),and phosphorylated AKT1(p-AKT1)in the lung tissue.Results:The extraction conditions of Ganjiang were optimized as extrac-tion with 10 volumes of 80％ethanol for 3 times(1 h for each time).LC-MS revealed 45 chemical components in Ganjiang extract,from which 31 active components were screened out,and the active components shared 139 common targets with ALI.A total of 154 signaling pathways were obtained by KEGG enrichment,including phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway and Ras signaling pathway,most of which were associated with the regulation of immune inflammation.The molecular docking results verified that the core components had strong binding affinity with the key targets.Hematoxylin-eosin(HE)staining results showed that compared with the normal group,the model group presented disarranged alveolar structure,widened alveolar space,massive infiltration of neutrophils and lym-phocytes,and increased lung index(P＜0.01).Moreover,the model group showcased elevated levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-1β,and myeloperoxidase(MPO)(P＜0.01),lowered superoxide dismutase(SOD)level(P＜0.05),and up-regu-lated protein levels of TNF-α and p-AKT1 in the lung tissue(P＜0.01).Compared with the model group,drug administration alleviated the pathological injuries,reduced the neutrophil and inflammatory cell infiltration,and decreased lung index(P＜0.05).Furthermore,drug ad-ministration lowered the levels of TNF-α,IL-6,IL-1β,MPO,and malondialdehyde(MDA),elevated the SOD level,and down-regulated the protein levels of TNF-α and p-AKT1 in the lung tissue.Conclusion:Ganjiang extract exerts definite effect on ALI by down-regulating the protein levels of TNF-α and p-AKT1 and alleviating inflammation and oxidative stress in the lung tissue of mice.
Ganjiang(干姜)Acute lung injuryUltra performance liquid chromatography-quadrupole time-of-flight tandem mass spec-trometry(UPLC-Q-TOF-MS)Network pharmacologyMolecular dockingExperimental validationOrthogonal testInflammatory cyto-kinesProtein kinase
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