The Discovery And Mechanism Study Of Terpenoid On Anti-inflammation In Vitro

Inflammation is the body’s defense response to the damage factor,the infection of the pathogen and the tissue damage can cause the inflammatory reaction,which can occur in any tissues and organs.Inflammation is a?double edge sword‘for human health,On the one hand,it can remove pathogens and restore tissue damage,which helps to maintain the immune homeostasis of the body;on the other hand,excessive and abnormal inflammation can lead to tissue lesions and two injuries,and also the basis of many diseases.At present,clinical anti-inflammatory drugs show serious adverse reactions.Therefore,it is urgent to find highly effective and low toxic natural inhibitors.Natural terpenoids are compounds derived from methamyl dihydroxy acid.They have many types of structures and numerous biological activities.It is reported that terpenoids have significant anti-inflammatory activity,which provides a new idea for our discovery of new anti-inflammatory drugs.In this paper,the RAW264.7 macrophage inflammation model induced by lipopolysaccharide（LPS）in vitro was used to detect the activity.We detected the NO inhibitory activity of different fractions extracted of the ethyl acetate extracts from the plants of Euphorbia neriifolia L.,Euphorbia Antiquorum,Euphorbia royleana.It was found that 70%and 90%methanol/water fractions of three plants had better inhibitory activity,especially the 90%methanol/water fraction IC₅₀ were 0.4μg/m L,15μg/m L and 6.85μg/m L.We continued to detect NO inhibitory activity of 134 compounds,including 91 terpenoids（iridoid,sesquiterpenes,Ingenane diterpene,Lathyrane type diterpenoid,ent-kaurane diterpenoids,Abietane/pimarane diterpenoid,Atisane diterpenoid,Triterpene）and other 43 compounds.Screening results showed that 17compounds significantly inhibited the generation of NO,including 13 terpenoids and3 isopentenyl flavones and 1 lignans.What‘s more,The discovery of structure-activity relationship analysis that the acyl group on the terpenoid nucleus could improve the activity,and the more acyl number and the longer the acyl carbon chain,the better the activity of the acyl carbon chain,and the type of substituent on the C-ring directly affects its anti-inflammatory activity.Moreover,some abietane/pimarane diterpenoid and ingenane diterpene showed better inhibitory activity,and we will explore the anti-inflammatory activity and the mechanism of these compounds.Five abietane diterpenoids:52,57,54,55 and 56 isolated from T.hypoglaucum,whose structures were similar and all showed the NO inhibitory activity in vitro.Among them,the inhibitory effect of compounds 52（tritophenolide）and 57（triptoquinone）were most obvious with IC₅₀ of 21.4 and 2.58μM.We further confirmed the anti-inflammatory activity of the compounds by ELISA,and explored the mechanism by Western Blot and immunofluorescence.The results showed that the expression of inflammatory factors IL-1β,IL-6,TNF-αand iNOS COX-2 protein was obviously inhibited by two compounds in LPS-activated macrophages.Compound 57may inhibit the activation of two signal pathways of MAPKs（p-p38,p-JNK,p-ERK1/2）and NF-κB（p-IκB）,52 mainly inhibits the activation of MAPKs signaling pathway to achieve anti-inflammatory effects.Another five compounds,extracted from Euphorbia neriifolia L.:27（euphorneroid E）,28（eurifoloid A）,60（antiquorine A）,72（eurifoloid N）,64（sandaracopimaradiene-3）,showed the inhibitory activity of NO in vitro with IC50 of6.31,5.78,22.7,37.1 and 19.0μM,respectively.Further study showed that compound60,71,64 directly showed inhibitory effects on the expression of inflammatory factors IL-1β,IL-6,TNF-αand iNOS,COX-2 protein,and three compounds had no significant effect on the phosphorylation level of IκBαprotein,which showed a certain inhibitory effect on the phosphorylation level of p38MAPK（p<0.05）.It is suggested that the three compounds may exert their anti-inflammatory activity by acting on the MAPKs signaling pathway.Compounds 27 and 28 showed significant inhibitory effects on the expression of IL-1β,IL-6 and iNOS.However,the expression of TNF-αand COX-2 was obviously promoted.In signal transduction pathway,the activation of MAPK/p38 was obviously downregulated,but the phosphorylation level of MAPK/erk was up-regulated.Therefore,the mechanism of compound 27 and 28needs further exploration.