Protective Effects Of Polymethoxyflavones Extract From Citrus Reticulata ‘Chachi' On Acute Lung Injury Induced By Lipopolysaccharide In Mice

The Pericarpium Citri Reticulatae made from the Citrus Reticulata‘Chachi'produced in Xinhui District,Jiangmen City,Guangdong Province,is the most authentic.Studies have shown that Citrus Reticulata‘Chachi'contains a variety of flavonoids.Compared with other flavonoids,polymethoxyflavones(PMFs)have stronger anti-oxidant and free radical scavenging effects.It has a certain protective effect on the heart,lung and endocrine system.Acute lung injury(ALI)is the manifestation of excessive inflammation in the lungs.It is a relatively common and critical illness in clinical practice.The purpose of this study is to explore the protective effects of extracted PMFs and Nobiletin(NOB)on ALI induced by lipopolysaccharide(LPS)in mice,and to explore its mechanism of action at the cytokine and molecular level,and provide evidence for clinical treatment of ALI.At the same time,it provides a theoretical reference for the further effective development and utilization of the functional components in the NOB.In this study,the ultrasonic-assisted extraction method was used to extract the crude PMFs from the Citrus Reticulata‘Chachi',NOB and tangeretin(TAN)purity and structure were identified by D101 macroporous adsorption resin,HSCCC,HPLC,UPLC-MS and ~1H NMR.A mice model of ALI was established by intranasal instillation of LPS.The effects of PMFs and NOB on ALI were explored by measuring the degree of lung tissue damage,the wet/dry mass ratio(W/D),lung injury score;determining the content and activity of lung tissue and serum oxidative damage indicators;The protein expression of pro-inflammatory factors in serum was detected by enzyme-linked immunosorbent assay(ELISA),and the m RNA expression in lung tissue was determined by real-time fluorescent quantitative PCR(q PCR);Finally,western blotting(WB)was used to evaluate the expression of important proteins in the NF-?B and MAPK signaling pathways.Explore the impact of PMFs and NOB on ALI.The main results of this paper are as follows:1.Extraction and structural identification of PMFs from Citrus reticulata‘Chachi'(1)The crude extract of PMFs was extracted by ultrasonic-assisted extraction under the conditions of 100 W,30 min,and 45?.An HPLC method was established for detecting the content of NOB.The results showed that the experimental method adopted was good and feasible.The content of NOB is about 50%,and TAN accounts for about 30%.It shows that NOB is the main ingredient in PMFs of Citrus Reticulata‘Chachi'.(2)Dissolve the PMFs crude extract with 40%ethanol,load the sample flow rate 2BV/h,sample volume 4 BV,43%ethanol eluent,90%ethanol desorbent,desorption volume5 BV for separation of D101 macroporous adsorption resin purification.After purification,the content of Citrus citrinin increased from 7.99±0.32 mg/g to 29.40±1.76 mg/g;the content of tangeretin increased from 5.72±0.53 mg/g to 17.96±1.10 mg/g;increased by3.7 times and 3.1 times,respectively,indicating that this method can enrich PMFs while removing other impurities,and is an effective method for purifying the crude extracted PMFs.(3)The separation and purification of HSCCC was carried out using a solvent system of water:methanol:n-hexane:ethyl acetate(1:1:1:0.8,V/V).The obtained monomer was analyzed by UPLC-MS and ~1H NMR,and the results proved that the method can be obtained.The purity of 97.52%NOB and 92.08%TAN,the masses were 7.12 mg and 5.83mg,respectively.2.Anti-oxidant and anti-inflammatory effects of PMFs and NOB on ALI mice(1)Gavaging the 25mg/kg NOB-low-dose group(NOB-L),50mg/kg NOB-middle-dose group(NOB-M),100mg/kg NOB-high-dose group(NOB-H)and 250mg/kg extracted PMFs group in ALI mice,the alveolar wall and septum in the lung tissue of the model group were thickened after 6 hours by LPS instillation in the nose,with obvious lung tissue damage,neutrophil infiltration,lung injury score and lung wet/dry weight ratio increased significantly,indicating that the model was successful.(2)The content and activity of myeloperoxidase(MPO),malondialdehyde(MDA),and glutathione peroxidase(GSH-PX)in lung tissue and serum are reduced,and the difference between the NOB-M and the PMFs group is extremely different.Significantly(P<0.01),indicating that the NOB and PMFs have a certain anti-oxidant effect on ALI mice.(3)Enzyme-linked immunosorbent assay experiments(ELISA)show that the various doses of NOB and PMFs can significantly inhibit tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and interleukin-1?(IL-1?)in the serum of mice with ALI the production.The results of q PCR on the expression of the three pro-inflammatory factors on the m RNA are consistent with the above.3.Effects of PMFs and NOB on signal pathways related to ALI miceWestern blot(WB)analysis showed that the different dose groups of NOB and the extracted PMFs group can significantly inhibit the inflammation pathway NF-?B and MAPK signaling pathway in ALI mice,inhibtiting degree of phosphorylation of the characteristic proteins P65,P38,ERK and JNK and degradation of I?B.In summary,the PMFs and NOB can improve the appearance,scavenge free radicals,and regulate the main inflammatory signal pathways,thereby inhibiting the excessive secretion of inflammatory factors and protecting mice with ALI.