Interventional Effect And Mechanism Of Apigenin C-glycosides Of Microcos Paniculata During LPS-induced Acute Lung Injury In Mouse Model

Microcos paniculata is an edible and medicinal plant of Tiliaceae family.As one of the main raw materials,it can be used for the preparation of traditional Chinese medicine and herbal teas for keeping daily health care.According to the reports of chemical constituents contained in M.paniculata,flavonoids are the main bioactive components,of which apigenin C-glycosides(ACGs)are various and stable,which can be treated as the active fraction of M.paniculata to carry out further studies.Presently,the researches about main components and pharmacological mechanism have not been reported in ACGs of M.paniculata.The current paper aimed to extract the ACGs fraction from M.paniculata firstly,followed by processing identification and quantification of main compounds in ACGs fraction of M.paniculata.Then to investigate the effects and possible molecular mechanisms of ACGs using the LPS-induced acute lung injury(ALI)in mouse model.The specific contents and results were as follows:(1)The methods of the extraction and isolation of ACGs fractionfrom M.paniculata were combined with column chromatography and semi-prep HPLC,followed by using LC-MS for the qualitative analysis to identify 7 kinds of ACGs,including vicenin-2,isoschaftoside,schaftoside,vicenin-1,vitexin,isovitexin and isoviolanthin,which were then quantitatively analyzed by HPLC to show a percentage of 60.83% in ACGs fraction.(2)Using the classical model(hot plate method in mice model,auricle swelling induced by xylene in mice,as well as acetic acid induced writhing in mice)to investigate analgesic and anti-inflammatory effects of ACGs conveniently and rapidly.The results showed that the thermal threshold of mice could not be prolonged by ACGs,while the numbers of writhing induced by acetic acid and the degree of auricle swelling induced by xylene in mice could be significantly decreased in the treatment of ACGs,suggesting the effects of ACGs in peripheral analgesia and anti-acute nonspecific inflammation.(3)Basing on a mouse model of ALI induced by LPS to observe the lung tissue morphological and common indicators of increasing pulmonary vascular permeability,such as dry wet weight ratio of lung,the number of neutrophils in bronchoalveolar lavage fluid(BALF)and so on.The final results showed that ACGs significantly inhibited the increase of pulmonary vascular permeability induced by LPS and release of inflammatory factors,indicating a good protective effect on LPSinduced ALI in mice.In addition,the protective effects of ACGs on LPS-induced ALI in mice showed a certain dose dependence.(4)Based on the effects of ACGs on LPS-induced ALI in mouse model,the serum of mice were processed by the analysis of targeted lipidomics and non-targeted metabonomics.With the multivariate statistical analysis,30 of differential metabolites were selected out and put to analyze the potential mechanisms on Ingenuity Pathway Analysis(IPA),which then revealed that ACGs protecting LPS-induced ALI in mice were markedly linked with mitogen-activated protein kinase(MAPKs)signaling and the mitochondrial apoptosis pathway.(5)The key proteins in MAPKs signaling and mitochondrial apoptosis pathway were detected by western blot,showing the levels of ERK and JNK phosphorylation as well as the expressions of TLR4 receptors were all significantly inhibited by ACGs(40 mg/kg).The contents of Bax and cleaved-caspase-3 involved in the mitochondrial apoptosis pathway were both significantly reduced in the treatment of ACGs(40 mg/kg),which also increased the content of Bcl-2 protease.Herein,we isolated and identified 7 kinds of main compounds in ACGs,then using the mouse ALI model induced by LPS to investigate the protecting effect of ACGs,followed by revealing the potential mechanisms of anti-inflammatory effects of ACGs by metabonomics analysis methods.The further verification of key proteins involved inpotential pathways were processed in western blot technology.The work provided experimental research and data support for the protecting mechanism of ACGs on ALI mice induced by LPS,which laid the theoretical foundation for the pharmaceutical development and clinical application of ACGs.