Protective Effect Of Platycladi Cacumen Carbonisatum On Lipopolysaccharide-induced Human Umbilical Vein Endothelial Cells Damage And Cell Metabolomics Study

Platycladi cacumen,the dry shoots and leaves of Platycladus orientalis(L.)Franco,had the effects of cooling blood to stanch bleeding.After carbonized,its hemostatic efficacy could be enhanced and it was commonly used as hemostatic medicine in the TCM clinical treatment.However,previous studies mainly focused on the chemical,processing technology and the effect of hemostasis of Platycladi Cacumen Carbonisatum,its mechanism of action and material basis have not been clear elaborated.Vascular endothelial cells were the first barrier between cells and blood when exchange material.Its functional and structural abnormalities could lead to coagulation disorders,causing a series of vascular-related diseases.The Lipopolysaccharide could stimulate endothelial cells to excessively release the inflammatory cytokines,resulting in increased vascular permeability and damaged the blood vessel walls.This paper mainly used LPS to injure HUVECs in vitro to screen the effective method extract,solvent extract,elution fraction and active compounds of Platycladi Cacumen Carbonisatum,analyzed seven flavonoids of different solvent extracts,established Spectrum-effect relationship of different elution fractions,and analyzed cell Metabolomics.The studies in this paper mainly include the following sections:Part one:Literature reviewAfter having reviewed relative domestic and overseas documents,we summarize the progress of Platycladi Cacumen Carbonisatum and the protective of human umbilical vein endothelial cells(HUVECs)by traditional Chinese medicine and recent application process of cell metabonomics to support the new research direction,the feasible experimental program and examine indicators.Part two:Protective effect of HUVECs of different method extracts from Platycladi Cacumen CarbonisatumLPS was adopted to establish the injury model.MTT colorimetric method was used to determine cell activity,the nitrate reductase method was used to detect the content of nitric oxide(NO).The results showed that 100 μg mL-1 of LPS significantly decreased the absorbance values of HUVECs in comparison with the control group;Compared with the model group,the doses of 57.84 μg-mL-1 and 28.92 μg·mL-1,of the composite extracts,57.84 μg ml-1 of alcohol extracts and water extracts of Platycladi Cacumen Carbonisatum could enhance cell activity(P<0.05),significantly inhibit the increase of NO content(P<0.01,P<0.05).Moreover,compared with the ethanol extracts,the same dose of composite extracts showed better protective effect(P<0.01).Thus,the preliminary studies indicated that the composite extract was the most effective extraction method and the extracts have better protection than other extraction methods.Part three:Protective effects of different solvent extracts of Platycladi Cacumen Carbonisatum on Human Umbilical Vein Endothelial Cells injury induced by LPSAccording to the previous experimental results,we chosen the chemical composition of the alcohol extracts and water extracts of Platycladi Cacumen Carbonisatum to prepare the different solvent extracts including the petroleum ether extract,ethyl acetate extract,n-BuOH extract and the water extract.MTT colorimetric method was used to determine cell activity,the xanthine oxidase method was adopted to detect the activity of superoxide dismutases(SOD),the thibabituric acid developing method was adopted to determine the content of malondialdehyde(MDA),the nitrate reductase method was used to detect the content of nitric oxide(NO).The results showed that,compared with the model group,the dose of 100μg·mL-1 of N-butanol extract and the doses of 100 μg·mL-1,50 μg mL-1 of ethyl acetate extract could enhance cell activity(P<0.05)，significantly inhibit the increase of MDA and NO concentrations and prevent the decline of SOD activity(P<0.05).Moreover,the same dose of ethyl acetate extracts showed better protective effects in the index of NO and SOD in comparison with N-butanol extract.Thus,the preliminary studies indicated that the ethyl acetate extract was the most effective solvent extract and the extracts have better protection than other extraction methods.Part four:Analysis on chemical components of the effective extract from Platycladi Cacumen Carbonisatum by ultraperformance liquid chromatography/quadrupole-time-of-fligh mass spectrometryEleven compounds of the effective extract from PCC were identified by analyzing positive and negative ion mass spectra information and element composition and comparing controls with data from relevant literature.This part of experiment could trace to the source of effect compounds group in Platycladi Cacumen Carbonisatum for future research.Part five:Protective effects of different elution fractions of Platycladi Cacumen Carbonisatum on Human Umbilical Vein Endothelial Cells injury induced by LPSAccording to the previous experimental results,we used the polyamide column chromatography to separate the ethyl acetate extract of Platycladi Cacumen Carbonisatum.Water,30%ethanol,50%ethanol,75%ethanol,95%ethanol were used respectively to elute and five elution fractions were obtained,named CB-1,CB-2,CB-3,CB-4,CB-5,respectively.The injury model was established by LPS,and the cell activity,NO,SOD,MDA were measured through the appropriate kit,respectively.The results showed that,compared with the model group,the doses of 125 μg-mL-1，62.5 μg mL-1 of ethyl acetate extracts and CB-4 could enhance cell activity(P<0.05)，significantly inhibit the increase of MDA and NO contents and prevent the decline of SOD activity(P<0.05).Moreover,compared with ethyl acetate extract,the same dose of CB-4 showed better protective effects.Thus,the preliminary study showed that CB-4 was the most effective elution fraction of PCC.Decrease the production of NO and alleviate the lipid peroxidation of HUVECs may be the possible protective mechanisms of CB-4 of Platycladi Cacumen Carbonisatum.Part six:Spectrum-effect relationship in effect of protecting HUVECs of different elution fractions of Platycladi Cacumen CarbonisatumMTT,NO,SOD and MDA as active indicators were observed in the HUVECs after treatment of these different elution fractions of Platycladi Cacumen Carbonisatum.The pharmacology data and peak data in UPLC fingerprint were correlation analyzed by SPSS and the basis of the could be obtained.According to the correlations of fingerprints and pharmacology,peaks 5,6,9,10,11,12 were positively correlated with the MTT,peaks 5,6,9 were negatively correlated with the NO,peak 5,9 were negatively correlated with the MDA,peak 4,5,9 were positively correlated with the SOD,suggested that peak 4(Myricetin),pdak 5(Quercetin),peak 6(Apigenin),peak 9(Amentoflavone),peak 11(Chinokoflavone),peak 10 and 12 from CB-4 might be the active substance for protecting HUVECs.Part seven:Protective effects of compounds of Platycladi Cacumen Carbonisatum on Human Umbilical Vein Endothelial Cells injury induced by LPSSeven compounds of Platycladi Cacumen Carbonisatum were selected to test for their activities by MTT assays.The results showed that all of them can enhance cell activity,including four flavanoids and three terpenoids.Part eight:Metabolomics study of Platycladi Cacumen Carbonisatum on Human Umbilical Vein Endothelial Cells injury induced by LPSLPS was adopted to establish the indury model.The NO,SOD,MDA were measured,respectively.The result showed that,compared with the model group,the doses of 4.647～18.587 μmol L-1 of Amentoflavone can significantly inhibit the increase of MDA and NO contents and prevent the decline of SOD activity(P<0.05)indicating that Amentoflavone had direct protection on HUVECs,decreasing the production of NO and alleviating the lipid peroxidation of HUVECs may be the possible mechanismsHUVECs were randomly divided into 3 groups as follows:control group,model group and Amentoflavone group.The samples of cell were detected by ultraperformance liquid chromatography/quadrupole-time of flight mass spectrometry(UPLC/Q-TOF-MS)using electrospray positive/negative monitoring mode,pattern recognition methods including PCA,OPLS-DA were applied to analyze multivariate data.10 biomarkers were identified in cell,which were recoveryed in Amentoflavone group.By IPA,We find six HUVECs damage metabolic pathways,namely Sphingolipid metabolism,Glycerolipid metabolism,Glutathione metabolism,Glycerophospholipid metabolism,Arginine and proline metabolism and Purine metabolism,which may be related to the protective of Amentoflavone.