Protective Effect Of Fagopyrum Dibotrys （D.Don） Hara Extract On LPS-induced Acute Lung Injury In Mice

Objective: To take Fagopyrum dibotrys（D.Don）Hara as the research object,screen its anti-inflammatory active site and carry out chemical constituents analysis and content determination.To observe the effects of Fagopyrum dibotrys（D.Don）Hara extract on lung pathological changes,inflammatory factors,TLR4/NLRP3 signaling pathway by establishing an LPS-induced acute lung injury model in mice and explore the preventive effect and mechanism of Fagopyrum dibotrys（D.Don）Hara extract on acute lung injury.Methods: 1 By establishing the LPS-induced inflammatory model of RAW264.7 cells,the expression level of inflammatory factors in the supernatant of cells in each treatment group was detected,and the anti-inflammatory active site of Fagopyrum dibotrys（D.Don）Hara was screened.2 Rapid identification method of chemical constituents in Fagopyrum dibotrys（D.Don）Hara extract was established by UPLC-Q Exactive-orbitrap HRMS,and determination method of its main active constituents was established by HPLC.3 Using the model of acute lung injury induced by LPS,the mice were randomly divided into control group,model group,low,medium and high dose groups of Fagopyrum dibotrys（D.Don）Hara extract and dexamethasone positive group.The preventive effect of Fagopyrum dibotrys（D.Don）Hara extract on acute lung injury in mice was evaluated by observing the pathological morphology of lung tissue,pathological score,wet/dry weight ratio of lung tissue,expression of inflammatory factors（TNF-α,IL-6,IL-1β）in bronchoalveolar lavage fluid,number of WBC and activity of MPO in lung tissue.4 The levels of TLR4,NLRP3,ASC,Caspase-1,IL-18 and IL-1β m RNA in TLR4/NLRP3 signaling pathway in lung tissue of mice with acute lung injury were detected by q PCR to explore the mechanism of preventive effect of Fagopyrum dibotrys（D.Don）Hara extract on acute lung injury in mice.Results: 1 Compared with the control group,the expression levels of NO,IL-1β,and IL-6 in the cells culture supernatant of the model group were significantly increased（p<0.01）.After the intervention of the total extract,petroleum ether,chloroform,ethyl acetate,n-butanol and water layer extract（40 μg/m L）of Fagopyrum dibotrys（D.Don）Hara,the expression of inflammatory factors decreased in all groups except in the water layer group,and the effect of ethyl acetate group was the best.2 The ethyl acetate extract of Fagopyrum dibotrys（D.Don）Hara mainly contains flavonoids and phenolic acids,including Epicatechin,Hyperin,Proanthocyanidin B2,Gallic acid,Protocatechuic acid,and nucleoside adenosine and alkaloid berberine.Among them,the main active ingredients Gallic acid,Proanthocyanidin B2 and Epicatechin content were0.0563%,0.3707%,0.3868%.3 Compared with the control group,the pathological score of lung tissue increased significantly（p<0.01）,the wet/dry weight ratio of lung tissue increased significantly（p<0.05）,the expression levels of IL-1β,TNF-α and IL-6 in bronchoalveolar lavage fluid increased significantly（p<0.01）,the number of White blood cell increased significantly（p<0.01）,and the activity of MPO in lung tissue increased significantly（p<0.01）in the model group.After drug intervention,all the indicators of mice in the treatment group were improved in varying degrees.4 Compared with the control group,the levels of TLR4,NLRP3,ASC,Caspase-1,IL-18 and IL-1β m RNA in the TLR4/NLRP3 signaling pathway in the model group increased significantly（p<0.01）.After drug intervention,all the indicators of mice in the treatment group were improved in varying degrees.Conclusions: 1 The anti-inflammatory effect of ethyl acetate extract of Fagopyrum dibotrys（D.Don）Hara is the best,which may be the result of the joint action of several active components.2 The analytical method established in this study can quickly identify the chemical constituents in the extract of Fagopyrum dibotrys（D.Don）Hara,the established content determination method is stable and reliable,and can accurately determine the content of the active constituents,which lays a foundation for the study of its pharmacological effects.3 Ethyl acetate extract of Fagopyrum dibotrys（D.Don）Hara can prevent LPS-induced inflammation in ALI mice by alleviating the inflammatory lesions in lung tissue,improving the degree of pulmonary edema,inhibiting the expression of inflammatory factors such as IL-6 and TNF-α and the number of inflammatory cells such as WBC in bronchoalveolar lavage fluid,and inhibiting the activity of MPO in lung tissue.4 Ethyl acetate extract of Fagopyrum dibotrys（D.Don）Hara can inhibit the activation of TLR4/NLRP3 signaling pathway,reduce the damage of inflammatory factors to lung tissue,inhibit the further expansion of inflammatory reaction,and play an anti-pneumonia effect.