Study On Anti-inflammatory Mechanism And Pharmacodynamic Material Basis Of Crepis Crocea

Objective:Crepis crocea,as traditional Chinese medicine,is used to treat bronchitis,tuberculosis,diabetes,asthmatic chronic bronchitis and unknown swelling and other diseases.However,studies on anti-inflammatory mechanism of Crepis crocea and its active components have not been reported.In this study,the anti-inflammatory effective parts of Crepis crocea were screened based on pharmacological indexes,and the protective mechanism of Huanyangshen on inflammation was discussed.Besides,Clarifythe material basis of Crepis crocea and based on the spectral-effect analysis method.It can provide scientific basis for the research development and the guidance of clinical medication of Crepis turczaninowii.Methods:1.The rat inflammatory model was established by tail vein injection of5mg/Kg LPS.The water extract,the Ethyl acetate part,the N-butanol part and the water part of Crepis crocea were administered intragastrically according to the dosage of 2.5g/Kg to observe the anti-inflammatory effect index and screen the effective parts of anti-inflammatory.2.Enzyme-linked immunosorbent assay(Elisa)was used to determine the content of inflammatory factors in rats,and Western blot(WB)was used to determine the relative expression level of NF-?B signal pathway in lung of rats in each group,and flow cytometry was used to detect the contents of Th1 and Th2 in the whole blood of rats in each group,exploring the anti-inflammatory mechanism of effective fraction of Panax quinquefolium.3.At the same time,based on the technique of ~1H-NMR metabonomics,the serum of rats in each group was detected.According to the value of S-plot and its VIP,combined with the result of t-test,the endogenous different metabolites related to the inflammation model were found.To further explore the anti-inflammatory mechanism of Crepis crocea,bivariate correlation analysis was used to analyze the correlation between differential metabolites and inflammatory factors,Th1/Th2 and NF-?B signaling pathway related proteins.4.Ultra-high performance liquid chromatography(UPLC)was used to establish the fingerprint of different polar parts of Panax quinquefolium.The common peaks of different polar parts were calibrated and the common peak areas were obtained.Using the method of bivariate correlation analysis,we find the chromatographic peaks that have a closer correlation with the anti-inflammatory effect,and the anti-inflammatory substance basis of Huanyangshen was preliminarily discussed.Result:1.Screening of anti-inflammatory effective fractions of Crepis croceaThe anti-inflammatory active part of Crepis crocea was screened and finally determined as the water extract of Crepis crocea on the number of inflammatory cells,spleen and thymus index.2.Study on anti-inflammatory mechanism of effective fraction of Crepis crocea Compared with the blank group,the inflammatory cells,inflammatory factors were significantly increased,indicating that the model was successfully replicated.2.1 Compared with the model group,the water extract of Crepis crocea can significantiy decrease the amount of TNF-?,IL-6 and IL-1?(P<0.01).2.2 Compared with the model group,the water extract of Crepis crocea can regulate the drift of Th1/Th2 and relieve the inflammatory symptoms(P<0.01).2.3 Compared with the model group,the water extract of Crepis crocea can significantiy decrease the expression of p65 and p-I?B?in NF-?B signal pathway(P<0.01).3.Studies on the anti-inflammatory mechanism of Crepis crocea based on ~1H-NMR metabonomicsCombined with the results of ~1H-NMR metabonomics,15 biomarkers were screened,includeing lipid fragments,lipid fragment,?-OH-butyric acid,methionine,glutamine,acetone,succinic acid,dimethylamine,creatine,glycerophosphorus choline,phospha-tidylcholine,trimethylamine,betaine,methyl phosphate,glycine and glucose.The correlation between differential metabolites and inflammatory factors,Th1/Th2 and NF-?B signaling pathway proteins was analyzed.Succinic acid,glycine,creatine and methionine were found to have great correlation with inflammatory indexes,which had biological significance.It can be used as a biomarker of LPS induced inflammation.the metabolic pathways involved are tricarboxylic acid cycle,glycine,serine and threonine metabolism,creatine metabolism.4.Study on the spectral-effect analysis of Anti-inflammatory effects of different Polar parts of Crepis crocea4.1 The results of UPLC fingerprint of different polar parts of Crepis croceaThere were 24 common peaks in polar part of Crepis crocea,23 common peaks in the water extract headquarters,9 common peaks in the Ethyl acetate site,12 common peaks in the N-butanol site,and 16 common peaks in the water site.4.2 Correlation analysis of spectral-effect of anti-inflammatory effect of Crepis croceaBivariate correlation analysis showed that there was a significant negative correlation between the No.18 and No.21 peak content and TNF-?(P<0.05,P<0.01)),between the No.9 peak content with IL-6(P<0.05),between No.12 peak content with IL-1?(P<0.01),between the No.6,No.9,No.20 and No.23 peaks content with IL-1?(P<0.05).It shows that the higher the content of the components represented by these chromatographic peaks,the lower of the contents of TNF-?,IL-6 and IL-1?,and the better effect of anti-inflammatory.Conclusion:The water extract of Crepis crocea,as the effective part of anti-inflammatory effect,cansignificantiy decrease the amount of TNF-?,IL-6 and IL-1?and theexpression of p65 and p-I?B?in NF-?B signal pathway,regulate the drift of Th1/Th2.Based on ~1H-NMR metabonomics,the anti-inflammatory effect of Crepis crocea may be achieved by regulating the metabolism of glycine,serine and threonine and of alanine,aspartate and glutamic acid.By spectral correlation analysis,the chromatographic peaks closely related to inflammation were 6,9,12,18,20,21 and 23,among which peak 9 was chlorogenic acid.Other chromatographic peaks should be further confirmed by liquid chromatography-mass spectrometry(LC-MS)and nuclear magnetic resonance(NMR).The above research laid a foundation for the further development of Crepis crocea,and also provided experimental data for its clinical application.