| Objective: To isolate and identify the chemical constituents in Lychee seeds.To investigate the inhibitory effect of Lychee seed saponins(LSS)on the inflammatory response of LPS-induced RAW264.7 cells and BALB/c mice,to analyze the potential molecular mechanism of the anti-inflammatory of LSS.The purpose of this study is to provide a theoretical basis for the further study of the active components of Lychee seeds and its pharmacodynamic material basis.Methods: Using the Lychee seeds as raw material,3 kg,was extracted with 70% methanol refluxing and extracting 3 times,1.5 h each time,combined with filtrate,methanol was recovered to tasteless,Lychee seeds extract were obtained and dispersed by adding water.Petroleum ether,ethyl acetate and n-butanol were extracted respectively,and the chemical constituents of n-butanol fraction and ethyl acetate fraction were separated by C18 column,semi-preparative liquid chromatography and silica gel chromatography.The structures of the compounds were identified by physical and chemical identification,NMR and LC-MS.The inflammatory model of RAW264.7 macrophages induced by LPS was established,and the safe administration concentration of LSS on the activity of RAW264.7 cells was determined by MTT method.The expression of inflammatory cytokines in the supernatant of RAW264.7 cells induced by LPS was detected by Griess and ELISA,and the expression of NO was detected by flow cytometry.Western blot was used to detect the expression of COX-2,i NOS proteins,as well asNF-κB,MAPK and Nrf2-keap1 signal pathways inflammation related proteins expression.The expression of p65 was detected by cytoplasmic separation and immunofluorescence staining,and the expression of TLR4 was detected by CETSA.The inflammation model of BALB/c mice induced by LPS was established,and the contents of WBC and Neu in blood were measured by automatic biochemical analyzer.The expression of inflammatory cytokines in serum were detected by ELISA.Western blot was used to detect the expression of inflammation-related proteins in NF-κB and MAPK signal pathways in lung tissue.HE staining was used to observe the protective effects of Lychee seed saponins on lung and kidney tissues induced by LPS in mice.Results: 17 compounds were isolated from the Lychee seeds,and the structure of 13 known compounds were identified.They are4-Hydroxybenzoic acid(1),Protocatechuic acid(2),Protocatechuicaldehyde(3),Gallic acid(4),(+)-Catechin hydrate(5),Procyanidin A1(6),Procyanidin A2(7),Procyanidin B2(8),Rutin(9),Phlorizin(10),Naringin(11),Narirutin(12),Quercetin(13).The results of MTT showed that LSS had no significant effect on the survival rate of RAW264.7 cells in the concentration range of 100,200,300 μg/m L,so the anti-inflammatory activity of LSS was studied at 100,200,300 μg/m L.The results of Griess and flow cytometry showed that the amount of Nitrite and NO secreted by RAW264.7cells stimulated by LPS was significantly increased compared with the normal control group(P<0.001).Compared with the LPS model group,LSS could significantly inhibit the release of Nitrite and NO(P<0.01 or P<0.001).The results of ELISA showed that the secretion of TNF-α and IL-6 by RAW264.7cells stimulated by LPS was significantly increased compared with the normal control group(P<0.001).Compared with the LPS model group,LSS could significantly inhibit the release of inflammatory factors TNF-α and IL-6(P<0.01 or P<0.001).The results of cytoplasmic separation and immunofluorescence showed that compared with the normal control group,the expression of p65 protein in the cytoplasm of RAW264.7 cells stimulated by LPS for 2 hours was significantly decreased(P<0.001),and the expression of p65 protein in the nucleus was significantly increased(P<0.001).Compared with the LPS model group,the expression of p65 protein in the cytoplasm was significantly increased after treatment with LSS(300 μg/m L)(P<0.001).Reduce the expression of p65 protein in nucleus(P<0.001)and inhibit the nuclear translocation of p65 protein.The results of Western blot showed that compared with the normal control group,after LPS stimulated RAW264.7 cells,intracellular i NOS,COX-2,p-IKKα/β,p-p65,p-IκBα,p-JNK,p-ERK and p-p38 proteins were significantly increased(P<0.001).Compared with LPS model group,low-dose,medium-dose and high-dose LSS groups can significantly reduce the expression levels of i NOS,COX-2,p-IKKα/β,p-p65,p-JNK,IκBα,p-JNK,p-ERK and p-p38 proteins(P<0.001).In addition,the expression of Nrf2 and HO-1 proteins were up-regulated and keap1 protein expression was down-regulated(P<0.001).In addition,CETSA results show that LSS can be combined with TLR4(P<0.001).The results of LPS-induced inflammatory model in BALB/c mice showed that compared with the normal control group,the contents of WBC and Neu in blood and the contents of TNF-α,IL-6,IL-1β,BUN and CRE in serum or organization increased significantly in LPS-induced mice.Compared with LPS model group,LSS treatment could significantly reduce the release of pro-inflammatory mediators WBC,Neu,TNF-α,IL-6,IL-1β,BUN and CRE.In addition,the results of Western blot showed that compared with the normal control group,the expression of inflammation-related proteins in i NOS,COX-2,NF-κB and MAPK signal pathways in lung tissues of LPS-induced in mice were significantly increased(P<0.001).Compared with LPS model group,LSS could reduce the expression of inflammation-related proteins in i NOS,COX-2,NF-κB and MAPK signal pathways(P<0.001 or P<0.01 or P<0.05).The results of HE staining showed that LSS could reduce the infiltration of inflammatory cells in lung and kidney induced by LPS and protect the integrity of tissue structure.Conclusion: 13 known compounds were identified from Lychee seeds.In addition,LSS can inhibit LPS-induced inflammation in vivo and in vitro,which is thought to play an anti-inflammatory effect by binding to TLR4 and inhibiting the activation of downstream NF-κB and MAPK signal pathways. |
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