Study On Protective Effect And Anti-inflammatory Mechanism Of Quzhou Fructus Aurantii Extract On Respiratory Inflammation

Acute lung injury（ALI）and its more severe manifestation,acute respiratory distress syndrome（ARDS）,has been recognized as a rapidly onset and life-threatening disease,covering all age groups,with high morbidity and mortality worldwide and lack of appropriate treatment drugs.The etiology of ALI is complex and numerous.The proportion of ALI associated with Gram-negative bacterial infection is large,and endotoxin is the main pathogenic factor.Quzhou Fructus Aurantii（QFA）,recorded in the“Zhejiang Traditional Chinese Medicine Processing Norms（2015）”,was selected in new“Zhejiang 8 Famous Kinds Herbal Medicines”recently,which makes QFA have an official and legitimate medicinal identity and become an authentic herb.QFA is an unripe fruit of Rutaceae Citrus changshan-huyou Y.B.Chang in Changshan County,Quzhou City,Zhejiang Province.QFA has long been used as a folk medicine in China.Its immature fruit is mainly used for digestive system diseases of loss of appetite,nausea and vomiting syndrome,while its fruit is mainly used for respiratory system diseases such as sputum or sputum-free cough.In the study,Quzhou Fructus Aurantii Extract（QFAE）was used as the research object,and three inflammation models,including RAW264.7 cells induced by lipopolysaccharide（LPS）,transgenic neutrophil fluorescent zebrafish induced by copper sulfate pentahydrate（CuSO₄·5H₂O）and ALI mice induced by LPS,were used to elucidate the anti-inflammatory effect and anti-inflammatory mechanism of QFAE.Part 1.The protective effect of QFAE on respiratory inflammation in miceFirstly,naringin was quantified via High Performance Liquid Chromatography（HPLC）which was used as a criterion for the quality control and quantification of QFAE.Next,the anti-inflammatory activity of QFA extract（QFAE）was evaluated on copper sulfate pentahydrate（CuSO₄·5H₂O）-induced transgenic neutrophil fluorescent zebrafish model.QFAE showed a significant effect of anti-inflammation in CuSO₄·5H₂O-induced zebrafish by reducing the neutrophil number in the inflammatory site.We investigated the anti-inflammatory activity of QFAE on lipopolysaccharide（LPS）-induced acute lung injury（ALI）mice models and RAW264.7 cells.QFAE had an anti-inflammatory effect on reducing total cells,neutrophils,and macrophages in BALF and attenuated alveolus collapse,neutrophils infiltration,lung W/D ratio,myeloperoxidase（MPO）protein expression and other pulmonary histological changes in lung tissues,as well as hematological changes.Levels of pro-inflammatory cytokines,including TNF-α,IL-6,IFN-γ,MCP-1,and IL-12p70,were decreased,whereas anti-inflammatory cytokine IL-10 was increased after treatment with QFAE both in vivo and in vitro.In summary,our results suggested that QFAE had apparent anti-inflammatory effects on CuSO₄·5H₂O-induced zebrafish,LPS-induced ALI mice,and RAW 264.7 cells.Part 2.The anti-inflammatory mechanism of QFAE on respiratory inflammation in miceFirstly,components in QFAE,including eriocitrin,narirutin,naringin,neohesperidin,hesperidin and synephrine,were quantified via HPLC,which were used as a criterion for the quality control and quantification of QFAE.Then,the anti-inflammatory mechanism of QFAE on LPS-induced RAW 264.7 cells and ALI mice was studied.QFAE restrained mitogen-activated protein kinase（MAPK）and nuclear factor-kappa B（NF-κB）signaling pathways in LPS-induced RAW 264.7 cells,while AMP-activated protein kinase（AMPK）signaling pathways were activated,as revealed by prominent attenuation of phosphorylation of ERK,JNK,p38,p65,IκBα,RSK and MSK,whereas overt enhancement of phosphorylation of ACC and AMPKα.Levels of pro-inflammatory cytokines TNF-α,IL-6,IL-1βwere suppressed,but anti-inflammatory cytokine IL-10 was raised pretreated with QFAE both in vivo and in vitro.Moreover,QFAE prevented mice from LPS-provoked ALI,bases on alleviating neutrophils,and macrophages in BALF and mitigating pulmonary histological alters,as well as hematological change.MAPK and NF-κB signaling pathways in LPS-stimulated ALI mice were dampened by QFAE pretreatment,while AMPK signaling pathways were accelerated,as testify by significant restraint of phosphorylation of ERK,JNK,p38,p65 and IκBα,while distinct elevation of phosphorylation of ACC and AMPKα.Taken together,the remarkable anti-inflammatory effect of QFAE was associated with suppression of MAPK and NF-κB signaling pathways and motivation of AMPK signaling pathway.The anti-inflammatory effects of QFAE were systematically studied in vitro and in vivo on three inflammatory models,including LPS-induced RAW 264.7 cells,CuSO₄·5H₂O-induced transgenic neutrophil fluorescent zebrafish and LPS-induced ALI mice.The anti-inflammatory mechanism of QFAE was related to LPS-mediated MAPK/NF-κB/AMPK signaling pathway.