The Discovery And Mechanism Study Of Natural Products With Anti-inflammatory And Immunomodulatory Activity

Inflammation is a complex biological response of the body against exogenous or endogenous harmful substances or injuries.It is the important step of the body defense,but inflammation may also cause tissue damage,so it is often considered a double-edged sword.Inflammation is involved in the occurrence and development of a variety of diseases,which may delay and aggravate the disease response and harm the body due to its complex pathogenic factors.Natural products have the characteristics of a wide range of sources and rich structure.It is very important to search for anti-inflammatory and immunomodulatory active components in traditional Chinese medicine.As a critical member of innate immune responses,macrophages play a critical role in modulating the course of inflammation.The lipopolysaccharide-induced RAW267.4 macrophage model is a classic inflammation model in vitro.Based on this model,we evaluated 30 crude extracts from medicinal plants such as labiaceae,legumes and composite for NO inhibition activity screening,and found that 19 crude extracts showed good inhibitory activity.In addition,305 compounds from Euphorbia neriifolia L.,Euphorbia kansui,Sophoraflavescens,Sophora alopecuroides L.,Sophora davidii and other plants were screened for their inhibitory activity against NO production.The results showed that 91 compounds significantly inhibited NO production,the main structural types of which were iridoids,giant halberane diterpenes,pseudoelelane diterpenes,neocropane diterpenes,steroids and flavonoids.Further,anti-inflammatory mechanism of some compounds were study.Two ingenane-type diterpenoids,12（Euphorneroid E,Euph E）and 13（Eurifoloid A,Euri A）,isolated from Euphorbia neriifolia L.,inhibited NO production significantly with IC₅₀of 6.37 and 5.78μM,respectively.Moreover,in LPS-activated macrophages,Euph E and Euri A could significantly inhibit the expression of inducible nitric oxide synthase（i NOS）,and the secretion of IL-1βand IL-6 in a dose-dependent manner.Cell signaling pathway studies showed that they inhibited the degradation of IκBαand the translocation of NF-κB/p65 subunit into the nucleus.In addition,TNFα,PGE2,and COX-2 levels were significantly increased under the influence of these compounds,which may be closely related to the increased phosphorylation of protein kinase Cδ（PKCδ）and the activation of mitogen-activated protein kinases（MAPKs）signaling pathway.These results suggested that Euph E and Euri A might play multiple regulatory roles on inflammatory genes through PKCδ/MAPKs and NF-κB signaling pathways,and regulated the function of macrophage immune cells,thus playing a dual role of immune activation and anti-inflammatory immunosuppression.In addition,two iridoid compounds 264（Valeriandoid F,Vale F）and 269（Jatamanvaltrate K,Jata K）isolated from Valeriana jatamansi Jones showed significant anti-inflammatory activity with IC₅₀of 0.88 and 0.62μM,respectively,which could significantly inhibit the secretion of inflammatory mediators NO,IL-1β,IL-6 and TNF-αand the protein expressions of i NOS,COX-2 and NLRP3 induced by LPS.Further results showed that Vale F and Jata K effectively inhibited the activation of NF-κB signaling pathway by inhibiting the phosphorylation of IκBα,and also significantly inhibited the phosphorylation of m TOR and STAT3,which further reduced the secretion of inflammatory mediators and the subsequent feedback effect to achieve anti-inflammatory effect.Compound 109（Rotundic acid-28-O-β-D-glucopyranosyl ester）was a lignan isolated from Mosla chinensis Maxim.Total RNA samples were collected from the blank group,the model group and the compound 109 treatment group for transcriptome sequencing and result analysis.Comparison of gene pools revealed differentially expressed genes.The results of GO analysis showed that the above differentially expressed genes were mainly involved in biological processes such as cellular response to biotic stimulus,immune effector process and innate immune response.KEGG analysis showed that the anti-inflammatory effect of 109 may be involved in the regulation of Toll-like receptor signaling pathway,NF-κB signaling pathway,chemokines and other signaling pathways.Further,ELISA and Western Blot experiments results showed that 109 inhibited the secretion of IL-1βand IL-6 and the expression of i NOS and COX-2 proteins induced by LPS,and down-regulated the phosphorylation level of STAT3,but it has no significant effect on the production of TNF-αand the regulation of the MAPKs signaling pathway,these results are basically the same as the trend of gene up-regulation and down-regulation in the RNA-seq results.