Network Pharmacology and Experimental Validation to Explore the Mechanism of Qing-Jin-Hua-Tan-Decoction Against Acute Lung Injury

Abstract

Qing-Jin-Hua-Tan-Decoction (QJHTD), a classic famous Chinese ancient prescription, has been used for treatment of pulmonary diseases since Ming Dynasty. A total of 22 prototype compounds of QJHTD absorbed into rat blood were chosen as candidates for the pharmacological network analysis and molecular docking. The targets from the intersection of compound target and ALI disease targets were used for GO and KEGG enrichment analyses. Molecular docking was adopted to further verify the interactions between 22 components and the top 20 targets with higher degree values in the component–target–pathway network. In vitro experiments were performed to verify the results of network pharmacology using SPR experiments, Western blot experiments, and the PMA-induced neutrophils to produce neutrophil extracellular trap (NET) model. The compound–target–pathway network includes 176 targets and 20 signaling pathways in which the degree of MAPK14, CDK2, EGFR, F2, SRC, and AKT1 is higher than that of other targets and which may be potential disease targets. The biological processes in QJHTD for ALI mainly included protein phosphorylation, response to wounding, response to bacterium, regulation of inflammatory response, and so on. KEGG enrichment analyses revealed multiple signaling pathways, including lipid and atherosclerosis, HIF-1 signaling pathway, renin–angiotensin system, and neutrophil extracellular trap formation. The molecular docking results showed that baicalin, oroxylin A-7-glucuronide, hispidulin-7-O-β-D-glucuronide, wogonoside, baicalein, wogonin, tianshic acid, and mangiferin can be combined with most of the targets, which might be the core components of QJHTD in treatment of ALI. Direct binding ability of baicalein, wogonin, and baicalin to thrombin protein was all micromolar, and their KD values were 11.92 μM, 1.303 μM, and 1.146 μM, respectively, revealed by SPR experiments, and QJHTD could inhibit Src phosphorylation in LPS-activated neutrophils by Western blot experiments. The experimental results of PMA-induced neutrophils to produce NETs indicated that QJHTD could inhibit the production of NETs. This study revealed the active compounds, effective targets, and potential pharmacological mechanisms of QJHTD acting on ALI.

Qingjin Huatan decoction attenuates lipopolysaccharide-induced acute lung injury in mice by controlling platelet-associated formation of neutrophil extracellular traps

“…[21,38] Qingjin Huatan decoction could inhibit PMA-induced production of NETs and the results of network pharmacology suggested that NETs may be the mechanism of QJHTD in treating ALI. [15] The previous research results and transcriptomic data suggested that QJHTD might attenuate ALI by inhibiting the formation of NETs.…”

“…The KEGG pathway enrichment results showed that QJHTD treated ALI via osteoclast differentiation, TNF, NET formation, and IL-17 signaling pathways. Our previous network pharmacology-based study also revealed that the mechanism of QJHTD in treating ALI involved the formation of NETs, [15] indicating that QJHTD might play a protective role against ALI by inhibiting NETs. The recent studies have shown that a large number of NETs are produced during the progress of ALI.…”

“…Male Sprague Dawley rats (220–240 g) (SCXK 2016-0011) were obtained from the Beijing Vital River Laboratory Animal Technology Co, Ltd (China) and housed at 25°C–28°C and humidity of 45%–55%, with free access to food and water for 3 days before use. Neutrophils were isolated from the whole blood of healthy rats by gradient centrifugation with Percoll according to the method described in the study by Xiao et al [15] Briefly, blood was collected from the abdominal aorta of rats and anticoagulated with 109 mM sodium citrate (1:9 blood vol/vol). Diluted blood was smeared on the interface of the two layers of 82% Percoll and 69% Percoll, and centrifugation was carried out at 710 g for 30 min.…”

“…Our previous work showed that QJHTD inhibited phorbol 12-myristate 13-acetate (PMA)-induced formation of neutrophil extracellular traps (NETs). [15] NETs composed of DNA, histones, and granular proteins such as neutrophil elastase and myeloperoxidase and capable of killing bacteria extracellularly were first identified in 2004. [16] NETs have dual effects.…”

Pharmacological effects of baicalin in lung diseases

“…Pharmacological studies have shown suppression of TLR4-mediated NF-κB activation ( Zhang et al, 2016 ; Feng et al, 2018 ), downregulation of MMP9 expression ( Zhang et al, 2016 ), inhibition of p-Src in LPS-activated neutrophils and formation of neutrophil extracellular traps (NETs) in Phorbol 12-myristate 13-acetate (PMA)-induced neutrophils ( Xiao et al, 2022 ) in response to traditional Chinese medications containing baicalin.…”