| Background:In the early stage,our research group conducted a rough extraction of Euonymus fortunei（Turcz.）Hand.-Mazz and preliminary screening of its anti-inflammatory ability.The results showed that in in vitro experiments,the crude extraction of Euonymus fortunei（Turcz.）Hand.-Mazz can inhibit various inflammatory factors and has obvious anti-inflammatory activity;In vivo experiments,it also has a certain inhibitory effect on mouse ear swelling,indicating that the crude extract of Euonymus fortunei（Turcz.）Hand.-Mazz has a certain anti-inflammatory effect.In this paper,on the basis of the previous experimental conclusions,we will further study the activity of Euonymus fortunei（Turcz.）Hand.-Mazz crude extract against LPS-induced acute lung injury,and extract the different polarities of Euonymus fortunei（Turcz.）Hand.-Mazz to explore its anti-acute lung injury active ingredients and mechanism.Objective:This topic aims to evaluate the anti-inflammatory activity of Euonymus fortunei（Turcz.）Hand.-Mazz and its impact on macrophages in vivo and in vitro,and to preliminary explore the potential mechanism of Euonymus fortunei（Turcz.）Hand.-Mazz main effective monomers in regulating macrophages and lung cancer cells.Method:Part Ⅰ:The effect of extracts,monomer components and various extracts of Chinese medicine Euonymus fortunei（Turcz.）Hand.-Mazz on inflammation1.A mouse model of Acute Lung Injury was established to determine the effect of Euonymus fortunei（Turcz.）Hand.-Mazz solution high,medium and low dose groups on LPS-induced ALI in mice.2.The cell viability of Ethyl acetate,n-butanol and Petroleum ether on RAW264.7cells was detected by MTT assay.3.The RAW264.7 cells were exposed to LPS treated with different concentrations of Ethyl acetate,n-butanol and Petroleum ether,then the content of NO in the cell supernatant was detected by Griess assay.4.ELISA method was used to determine the production of IL-6 and TNF-αby LPS-induced RAW264.7 macrophage supernatant by monomeric Ethyl acetate,n-butanol and Petroleum ether.5.HPLC method to detect the main active ingredients contained in the n-butanol part and the content of the main active ingredients contained.Part Ⅱ:The effect and mechanism of Chinese medicine Euonymus fortunei（Turcz.）Hand.-Mazz on LPS-induced RAW264.7 macrophages1.The cell viability of Dulcite,Ethyl acetate,n-butanol and Petroleum ether on RAW264.7 cell was detected by MTT assay.2.The RAW264.7 cells were exposed to LPS treated with different concentrations of Dulcite,Ethyl acetate,n-butanol and Petroleum ether,then the content of NO in the cell supernatant was detected by Griess assay.3.ELISA method was used to determine the production of IL-6 and TNF-αby LPS-induced RAW264.7 macrophage supernatant by monomeric Dulcite,Ethyl acetate,n-butanol and Petroleum ether.4.RT-PCR detection of LPS-induced RAW264.7 macrophages Myd88,TLR4,TRIF,NF-κB p65,iNOS,COX-2,IL-6,TNF-αand IL-1βmRNA expression influences.5.Western-blot detected the effect of Dulcite on the expression of TLR4,Myd88,TRIF and NF-κB p65 proteins induced by LPS in RAW264.7 cells.6.Immunofluorescence method was used to detect the effect of monomeric Dulcite on NF-κB p65 nuclear translocation of RAW264.7 macrophages induced by LPS.Part Ⅲ:The effect and mechanism of Chinese medicine Euonymus fortunei（Turcz.）Hand.-Mazz on LPS-induced A549 cells1.The cell viability of Dulcite,Ethyl acetate,n-butanol and Petroleum ether on A549cells was detected by MTT assay.2.The A549 cells were exposed to LPS treated with different concentrations of Dulcite,Ethyl acetate,n-butanol and Petroleum ether,then the content of NO in the cell supernatant was detected by Griess assay.3.RT-PCR detection of LPS-induced A549 cells Myd88,TLR4,TRIF,NF-κB p65,iNOS,COX-2,IL-6,TNF-αand IL-1βmRNA expression influences.4.Western-blot to detect the effect of Dulcite on the secretion of TLR4,Myd88,TRIF and NF-κB p65 protein expression in A549 cells induced by LPS.5.Immunofluorescence was used to detect the effect of Dulcite on the nuclear translocation of NF-κB p65 in A549 cells induced by LPS.Part Ⅳ:Anti-inflammatory effect and mechanism of Chinese herbal medicine Euonymus fortunei（Turcz.）Hand.-Mazz effective main ingredient Dulcite on LPS-induced acute lung injury（ALI）in mice1.ELISA method was used to detect the effect of Dulcite on the content of IL-1β,IL-6 and TNF-αin the mice with acute lung injury induced by LPS.2.The lung wet mass/dry mass（W/D）ratio was used to detect the effect of Dulcite on the lung tissue dry-wet ratio of acute lung injury induced by LPS in mice.3.BCA and cell counting methods were used to detect the effect of Dulcite on the total protein concentration and cell exudation number in BALF induced by LPS in mice with acute lung injury.4.HE staining was used to detect the effect of the compound Dulcite on the lung inflammation in mice with acute lung injury induced by LPS.5.Western-blot method was used to detect the effect of Dulcite on the expression of TLR4,Myd88,TRIF and NF-κB p65 protein in lung tissue cells of mice with acute lung injury induced by LPS.Result:1.The extracts of Chinese medicine Euonymus fortunei（Turcz.）Hand.-Mazz can significantly reduce the TNF-α,IL-6 and IL-1βsecreted by acute lung injury in mice caused by LPS;It can significantly reduce the secretion of NO in the supernatant of RAW264.7cells caused by LPS;it can significantly reduce the secretion of IL-6 in RAW264.7 cells caused by LPS.2.The monomer Dulcite,ethyl acetate part,n-butanol part and petroleum ether part of the traditional Chinese medicine Euonymus fortunei（Turcz.）Hand.-Mazz have no obvious toxicity to RAW264.7 cells at a concentration of 0-50μmol·L-1（P﹤0.05）;Dulcite is no significant toxicity to A549 cells at a concentration of 1～25μmol·L-1（P﹤0.05）.The doses selected for RAW264.7 cells in the subsequent experiments are 10μmol·L-1,25μmol·L-1and 50μmol·L-1;the doses selected for A549 cells is 1μmol·L-1,10μmol·L-1and 25μmol·L-1.3.The ethyl acetate extraction part,the n-butanol extraction part and the petroleum ether extraction part of Euonymus fortunei（Turcz.）Hand.-Mazz had a significant inhibitory effect on the secretion of NO,IL-6 and TNF-αin RAW264.7 cells induced by LPS.4.HPLC showed that the content of Dulcite in Euonymus fortunei（Turcz.）Hand.-Mazzwas 2.915%,the content of Dulcite in ethyl acetate was 2.6%,and the content in n-butanol was 4.6%.The content is the highest in the n-butanol site,which corresponds to the most significant effect of the n-butanol site against LPS-induced acute lung injury.It is speculated that Dulcite is one of the main active components.5.Dulcite inhibits the secretion of NO in RAW264.7 and A549 cells induced by LPS.6.Dulcite inhibited LPS-induced expression of TLR4,Myd88,TRIF and NF-κB p65protein in RAW264.7 cells.7.From the level of gene transcription,Dulcite inhibits LPS-induced iNOS,COX-2,TNF-α,IL-6,IL-1β,TRIF,TLR4,Myd88 and NF-κB p65 mRNA expression in RAW264.7cells.8.Dulcite inhibits the expression of iNOS,COX-2,TNF-α,IL-6,IL-1β,TRIF,TLR4,Myd88 and NF-κB p65 mRNA in A549 cells induced by LPS;at the same time,sweet alcohol inhibits the expression of TLR4,Myd88 in cells,TRIF and NF-κB p65 protein expression.9.The main effective component of Dulcite in Euonymus fortunei（Turcz.）Hand.-Mazz,significantly improves the inflammatory infiltration of lung tissue in mice with acute lung injury induced by LPS from a pathological point of view,significantly reduces the congestion of lung tissue in mice,and can also reduce visible bleeding in the alveolar cavity.And the exudation even reduced the visible red mutation area,suggesting that the lung tissue showed a decrease in acute inflammation,and Dulcite played a significant role in improving acute lung injury in mice.Conclusion:1.The high,medium and low doses of Euonymus fortunei（Turcz.）Hand.-Mazz crude extract have a significant inhibitory effect on the inflammatory response in vitro;the ethyl acetate part,n-butanol part and petroleum ether part of Euonymus fortunei（Turcz.）Hand.-Mazz have a significant inhibitory effect on the inflammatory reaction,and the n-butanol site has the highest activity.2.The extracted parts all contain Dulcite,and n-butanol has the highest Dulcite content,which proves that Dulcite is one of the main anti-inflammatory active ingredients in Euonymus fortunei（Turcz.）Hand.-Mazz.3.Dulcite has a significant inhibitory effect on the inflammatory response of RAW264.7 and A549 cells induced by LPS.4.Dulcite inhibits the TLR4-NF-κB p65 signaling pathway in RAW264.7 and A549cells.5.The main active ingredient of Dulcite from Euonymus fortunei（Turcz.）Hand.-Mazz,exerts an anti-inflammatory effect by inhibiting the TLR4-NF-κB p65pathway,thereby protecting mice with LPS-induced acute lung injury. |
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