| Objective: Chemical composition analysis of Huangqin Qingfei Decoction was carried out by liquid-mass combination technology,the fingerprint of Huangqin Qingfei Decoction was established,the difference markers were screened by chemical pattern analysis,and the contents of main components were determined to explore the protective effect of Huangqin Qingfei Decoction on rats with acute lung injury,which laid the foundation for the clinical application of Huangqin Qingfei Decoction.Methods: 1.The chemical components of Huangqin Qingfei Decoction were qualitatively analyzed by using the UPLC-MS method and the reference products and literature review,and the software of Xalibur 4.0,Origin 2019 b,Graph Pad Prism,and ISotope Distribution Calculator.2.HPLC fingerprint of Huangqin Qingfei Decoction was established,and similarity evaluation System of TCM Chromatographic Fingerprint(2012 Edition)was used to evaluate the similarity.SPSS 21.0 software was used for cluster analysis,SIMCA 14.1software was used for partial least squares discriminant analysis,and the different components of Huangqin Qingfei Decoction were screened.The common peak components of Radix Huangqin Qingfei Decoction were quantitatively analyzed.3.Through network pharmacology,51 active ingredients were obtained from Huangqin Qingfei Decoction.Gardenia =2.4: 1,200 active targets,1890 disease targets from acute lung injury,123 common targets and 2396 association lines were obtained by intersection.The core targets were VEGFA,TP53,CASP3,AKT1,IL-1β,IL-6,TNF,HIF1 A,PTGS2,MMP9,NFKBIA.GO was enriched in 3509 items,and biological processes were enriched in lipopolysaccharide reaction,regulation of active oxygen metabolism,biological functions were enriched in early nuclear receptor activity binding with DNA transcription factors,KEGG was enriched in 231 pathways,which were involved in metabolic change,signal transduction,cell growth and death,development and regeneration.Among them,Apoptosis,TNF signaling pathway and MAPK signaling pathway overlap more with core targets.4.LPS air infusion rat model was established,and the rats were randomly divided into blank control group(C),model group(M),Huangqin Qingfei Decoction low-dose group(HZL),Huangqin Qingfei decoction medium-dose group(HZM),Huangqin Qingfei decoction high-dose group(HZH),and positive drug group(DEX).Among them,HZL was given 0.41g/kg,HZM was given 0.81g/kg,HZH was given 1.62g/kg,and positive group was given 2mg/kg dexamethasone intraperitoneal injection for 5 consecutive days,respectively.After the experiment,arterial oxygen partial pressure of rats in each group was evaluated by blood gas analysis,dry and wet lung tissue was weighed to obtain dry and wet mass fraction,lung tissue structural damage was observed by HE staining,collagen fiber accumulation was observed by Masson staining,and protein concentration in alveolar lavage fluid was detected by BCA kit.Neutrophil granulocytes and macrophages in alveolar lavage fluid were observed by Hehua-Gim staining,and MDA,CAT and SOD kits were used to detect MDA content and CAT and SOD activity in lung tissues of rats in each group.MPO activity,VEGA expression and contents of IL-6,TNF-α and IL-1β in alveolar lavage fluid of rats in each group were detected by MPO,IL-6,TNF-α and IL-1β kit.The expressions of ColⅠ,α-SMA,Caspase-3,BAX,BCL-2,IBK-α,P-P65 and P-P38 in lung tissues of rats in each group were detected by immunohistochemistry.Results: 1.40 chemical components of Huangqin Qingfei Decoction were identified by UPLC-MS;26 components were attributed to scutellaria baicalensis and 14 components to gardenia fructus.2.The fingerprint of Huangqin Qingfei Decoction was established,and 24 common peaks were determined,with the similarity greater than 0.98.Eight common peaks were identified,which were Gentioside,gardenia side,crocin I,baicalin,baicalin,baicalin,baicalin and papillaleuca A,and the contents of the eight constituents were determined.3.Network pharmacology: There were 51 active ingredients in Huangqin Qingfei Decoction,scutellaria: gardenia =2.4: 1,200 active targets,1890 disease targets were obtained from acute lung injury,123 common targets and 2396 association lines were obtained by intersection.The core targets were VEGFA,TP53,CASP3,AKT1,IL-1β,IL-6,TNF,HIF1 A,PTGS2,MMP9 and NFKBIA.GO was enriched in 3509 items,and biological processes were enriched in lipopolysaccharide reaction,regulation of active oxygen metabolism,biological functions were enriched in early nuclear receptor activity binding with DNA transcription factors,KEGG was enriched in 231 pathways,which were involved in metabolic change,signal transduction,cell growth and death,development and regeneration.Among them,Apoptosis,TNF signaling pathway and MAPK signaling pathway overlap more with core targets.4.The results of blood gas analysis indicated that the arterial partial pressure of oxygen decreased and the arterial partial pressure of carbon dioxide increased in rats with acute lung injury,and the situation was improved after the intervention of Huangqin Qingfei Decoction.HE results showed that the alveolar wall of acute lung injury rats was thickened,accompanied by diffuse lymphocyte and neutrophilic granulocyte infiltration,eosinophilic mucus secretion in bronchial lumen,local bleeding,and eosinophilic serous exudation around blood vessels.After the intervention of Huangqin Qingfei Decoction,the alveolar wall thickening,rupture and alveolar cavity dilation were rare,the infiltration of lymphocytes and neutrophils were reduced,and the interstitial connective tissue abnormalities were improved.Masson results showed that there was collagen fiber accumulation in the lung tissue of rats with acute lung injury.After the intervention of Huangqin Qingfei Decoction,collagen fiber accumulation was reduced,collagen volume fraction and surface density decreased.Dry and wet mass fraction,total protein concentration of alveolar lavage fluid,number of neutrophils and macrophages were used to judge the extravasation of intravascular high-protein tissue and pulmonary edema in rats with acute lung injury.Compared with model group,different concentrations of Huangqin Qingfei decoction could reduce the dry and wet mass fraction,total protein concentration of alveolar lavage fluid and total cell number,especially neutrophils and macrophages.The expression of inflammatory factors and the activity of myeloperoxidase in alveolar lavage fluid showed that the lungs of rats with acute lung injury were infiltrated by neutrophils,and the inflammatory response continued.Compared with model group,the activity expressions of IL-6,IL-1β,TNF-α and MPO were inhibited after the intervention of Huangqin Qingfei Decoction.The detection results of oxidative stress-related factors in lung tissue showed that,compared with model group,Huangqin Qingfei decoction inhibited SOD and CAT expressions and increased MDA content,and the intervention effect of medium-dose group and high-dose group was better.Immunohistochemical results showed that Huangqin Qingfei decoction could promote the expressions of inflammatory factor IKBα,apoptosis-related factors BCL2,collagen fiber-related proteins ColⅠ and α-SMA,and inhibit the expressions of oxidative stress related proteins P-P38,inflammatory related proteins P-P65 and apoptosis-related cells BAX and Caspase-3.Conclusion: 1.The chemical components of Huangqin Qingfei Decoction are mainly divided into flavonoids,iridoids,terpenoids and organic acids.2.Huangqin Qingfei Decoction can improve lung tissue injury after acute lung injury.3.Huangqin Qingfei Decoction can improve lung tissue injury in rats with acute lung injury by inhibiting inflammation,anti-oxidative stress and anti-apoptosis. |
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