Effects of Rhizoma Pinelliae Extract on Macrophage Polarization and Lung Injury in Bronchiolitis Mice by Regulating the TNF-α/NOX2 Pathway
BI Jingxian1, LI Shan1 *, REN Siwei2, XIANG Jiangyu3, GAO Qijia2, WU Jianxiang1
This study was aimed to investigate the effects of Rhizoma Pinelliae extract on macrophage polarization and lung injury in mice with bronchiolitis by regulating the TNF-α/NOX2 (NADPH oxidase 2) pathway. BALB/c mice were intranasal instilled with RSV (respiratory syncytial virus) suspension on both sides to establish a bronchiolitis model, they were randomly separated into model group, Rhizoma Pinelliae extract group, Rhizoma Pinelliae extract+pc-NC group, and Rhizoma Pinelliae extract+pc-TNF-α group, with 10 mice in each group. Additionally, 10 BALB/c mice were intranasal instilled with an equal amount of normal saline as the control group. After intervention treatment with Rhizoma Pinelliae extract and pc-NC, pc-TNF-α plasmids, lung function indicators such as Raw (airway resistance), FVC (forced vital capacity), and inflammatory cell count in BALF (bronchoalveolar lavage fluid) were detected in each group. HE and PAS (periodic acid Schiff) staining were applied to detect the pathological morphology of lung tissue in each group. Flow cytometry experiments were applied to determine macrophage polarization in various groups. ELISA method was applied to detect the levels of inflammation related factors in BALF and serum of mice in each group. RT-qPCR (quantitative Real-time PCR) and immunoblotting were applied to detect the expression of TNF-α/NOX2 and macrophage polarization related proteins in lung tissues of mice in each group. The results showed that, compared with the control group, the lung tissue of mice in the model group exhibited obvious pathological damage, the number of inflammatory cells, Raw, inflammation score, mucus secretion score, M1 macrophage proportion, levels of IL-6 and TNF-α in serum, expressions of TNF-α, NOX2 and iNOS in mouse lung tissue were obviously increased (P<0.05), while FVC, M2 macrophage proportion, and TGF-β, IL-10, ARG-1 expression in mouse lung tissue were obviously decreased (P<0.05). Compared with the model group, the lung tissue pathological damage of mice in the Rhizoma Pinelliae extract group was reduced, the number of inflammatory cells, Raw, inflammation score, mucus secretion score, M1 macrophage proportion, levels of IL-6 and TNF-α in serum, expressions of TNF-α, NOX2 and iNOS in mouse lung tissue were decreased (P<0.05), while FVC, M2 macrophage proportion,while FVC, M2 macrophage proportion, and TGF-β, IL-10, ARG-1 expression in mouse lung tissue were obviously increased (P<0.05); there were no obvious changes in various indicators of mice in the Rhizoma Pinelliae extract+pc-NC group (P>0.05). Compared with the Rhizoma Pinelliae extract group, the mice in the Rhizoma Pinelliae extract+pc-TNF-α group showed aggravated lung tissue pathological damage, the number of inflammatory cells, Raw, inflammation score, mucus secretion score, M1 macrophage proportion, levels of IL-6 and TNF-α in serum, expressions of TNF-α, NOX2 and iNOS in mouse lung tissue were increased (P<0.05), while FVC, M2 macrophage proportion, while FVC, M2 macrophage proportion, and TGF-β, IL-10, ARG-1 expression in mouse lung tissue were obviously decreased (P<0.05). Rhizoma Pinelliae extract can promote M2 polarization of macrophages and inhibit M1 polarization by blocking the activation of the TNF-α/NOX2 signaling pathway, thereby suppressing inflammation in the body and lungs of bronchiolitis mice and ultimately reducing their lung injury.
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