Herbacetin attenuates lipopolysaccharide-induced inflammatory lung injury in mice by inhibiting Fth1hi neutrophil aggregation and accelerating its clearance

Description

The aim of this study was to systematically investigate the protective effect of Herbacetin (Her) against lipopolysaccharide (LPS)-induced inflammatory lung injury and its related mechanisms. Firstly, a mouse model of inflammatory lung injury was successfully constructed by intratracheal drip injection of LPS, and its protective effect on mice with inflammatory lung injury was evaluated by observing the overall lung tissue morphology changes, lung histopathological injury, lung wet-to-dry weight ratio, lung organ coefficients, total proteins, and inflammatory factor contents. The results showed that administration of Her treatment improved LPS-induced lung histopathological injury, reduced lung tissue edema, decreased the content of pro-inflammatory factors TNF-α, IL-6 and IL-1β, and reduced the number of inflammatory cells. Meanwhile, our results also showed that two neutrophil populations (Fth1hi Neu and Prok2hi Neu) in two different locations existed in the lung tissues of mice with LPS-induced inflammatory lung injury, and that LPS stimulation promoted the aggregation of Fth1hi Neu and the release of pro-inflammatory factors in the lung tissues, which was accompanied by a decrease in the content of the anti-inflammatory factor IL-10. Administration of Her treatment increased the content of the anti-inflammatory factor IL-10 in lung tissues, promote apoptosis and clearance of Fth1hi Neu, reduce the aggregation of Fth1hi Neu in lung tissues, and ameliorate the inflammatory injury of lung tissues. In conclusion, Her had a significant protective effect on LPS-induced inflammatory lung injury in mice, and its mechanism of action was related to elevating IL-10 levels and reducing Fth1hi Neu accumulation.

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Herbacetin (Her, J12GB151628, purity ≥ 98%) and dexamethasone (DEX, S12HS194411) were purchased from (Yuanye, Shanghai, China). Lipopolysaccharide (LPS, 0000155607) was purchased from (Sigma Aldrich, Shanghai, China). Phosphate buffer saline (PBS, 2401003) was purchased from (VivaCell, Shanghai, China). LPS-Induced Inflammatory lung injury mouse model. Mice were equally divided into negative control, model (LPS), DEX (5 mg/kg) and Her (5 mg/kg, 15 mg/kg, 45 mg/kg) administration groups according to the body weight gradient sampling method. Inflammatory lung injury mouse model was constructed by dropping 50 µl of LPS (5 mg/kg) into the trachea in all groups except the NC group, and an equal amount of saline was dropped into the trachea of the NC group. After 1 h of modeling, the drug was injected intraperitoneally (i.p.) at a dose of 0.1 ml/10 g into the DEX and Her dosing groups, and the same volume of solvent was injected intraperitoneally (i.p.) into the NC group. Samples were taken 24 h after modeling, and the rest of the experimental conditions were the same. The lung tissues of mice were collected, rinsed with saline, and then the blood on the outer surface of the lung tissues was blotted out with filter paper, and photographs were taken to observe the morphological changes of the whole lung tissues. The upper lobe of the left lung was immersed in 4% paraformaldehyde for 24 h at room temperature, paraffin-embedded, and sectioned to a thickness of 4 µm, and then stained with Hematoxylin and Eosin Staining Kit (No.20220720, Solarbio, Beijing, China) for 5 min at room temperature, and then viewed under light microscope for pathological analysis, and the lesions were defined as follows: (a) alveolar neutrophils, (b) interstitial neutrophils, (c) hyaline membranes, (d) proteinaceous debris filling the airspaces, (e) alveolar septal thickening, (f) blood point. According to a previous study, the severity of inflammation was graded between 0 and 4 as follows: 1, <25% lung involvement; 2, 25-49% lung involvement; 3, 50-75% lung involvement; and 4, >75% lung involvement. Lung injury was scored by 3 pathologists blinded to the treatments. A lagging needle was inserted into the trachea and the lung tissue was aspirated back and forth with pre-cooled PBS solution, 0.8 ml at a time, and repeated 3 times to obtain BALF. Centrifuge at 1500 × g at 4 °C for 10 min. The supernatant was collected and stored at -80 °C for further experiments. The cells were resuspended with 300 µl of PBS and stained with Wright-Giemsa staining solution (No.20221019, Solarbio, Beijing, China) for 10 min at room temperature, and then total cells, macrophages and neutrophils were counted under the microscope.

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