Anti-inflammatory Activity Of Sesquiterpenoids From Roots Of Daphne Genkwa And Their Regulatory Mechanism On Inflammatory Signaling Pathways

Daphne genkwa is a plant belonging to the Thymelaeaceae genus and has been used as a traditional Chinese folk medicine for more than 2,000 years.The dictionary of traditional Chinese medicine also records that the roots of D.genkwa has the effect of detoxification,and has good curative effect on edema,rheumatic arthralgia,scabies,etc.It is often used clinically to treat acute mastitis and rhinitis.The sesquiterpenes in the root of D.genkwa are the main components for its anti-inflammatory activity,but the anti-inflammatory molecular mechanism of this kind of natural compounds is still unclear.The purpose of this article is to use molecular docking and Griess method to select sesquiterpenes extracted from the roots of D.genkwa,and conduct research on its anti-inflammatory activity in vitro and to explore the molecular mechanism of its anti-inflammatory effect,which is contribute to the rational development and utilization of medicinal resources of D.genkwa.The molecular docking was used to score the 10 sesquiterpenes that have been isolated and identified,and the Griess method was used to detect the inhibitory activity of the 10 sesquiterpenes on LPS-induced macrophages to release inflammatory mediator nitric oxide（NO）,and the MTT method was used to detect the cytotoxicity of the compounds on RAW 264.7 cells;according to the results of molecular docking,NO inhibitory activity and cytotoxicity,active compounds with high efficiency and low toxicity were selected and the active compounds were studied in depth.The ELISA method was used to detect the inhibitory effect of the active compounds on the inflammatory mediator prostaglandin E₂（PGE₂）and the inflammatory factor TNF-αand IL-6 excessive release;Western blot method was used to detect the high expression of the inflammatory protein iNOS and COX-2,the degradation of IκB-αprotein in the NF-κB signaling pathway and the regulation of ERK,JNK,and p38 protein phosphorylation in the MAPK pathway,molecular docking was used to analyze the active sites and main forces of the amino acid residues binding between compounds to target proteins（iNOS,COX-2,JNK,ERK,p38）.The molecular docking results showed that compounds（1,2,4,and 7）were ranked higher.10 sesquiterpenoids were screened for their inhibitory activity on LPS-induced macrophage RAW264.7 release of inflammatory mediator nitric oxide（NO）at a concentration of 100μM.compounds（1,2,4,and 7）showed significant inhibition activity;MTT experiment results showed that the compounds had no cytotoxicity on RAW264.7 cells.According to the preliminary screening test results,two guaiacolane sesquiterpenes（2 and 4）and acorolane sesquiterpene（7）were selected for further research.The results of Griess and ELISA showed that all three active compounds can inhibit the release of inflammatory cytokines NO,PGE₂ and TNF-α.Guaiacolane sesquiterpene（2）had a significant inhibitory effect on IL-6 induced by LPS at 6.25μM～25μM;acorolane sesquiterpene（7）significantly inhibited LPS-induced macrophage RAW264.7 release of IL-6 at 12.5μM～25μM,while guaiacolane sesquiterpene（4）inhibited LPS-induced macrophage RAW264.7 release of IL-6 only at 50μM.Western Blot results showed that compounds（2,4,and 7）can significantly down-regulate the high expression of iNOS and COX-2 proteins;Compound（2）can inhibit the phosphorylation of p38 and JNK proteins in the MAPK signaling pathway;compound（7）also inhibited ERK protein and JNK protein phosphorylation in the MAPK signaling pathway,while compound（4）only inhibits the phosphorylation of JNK protein in the MAPK signaling pathway;the three compounds have no ffects on the degradation of IκB-αprotein in the NF-κB signaling pathway.According to the results of Western Blot,compounds that can regulate the high expression of inflammatory proteins were selected,and molecular docking methods was used to search for the active sites and main forces of the amino acid residues binding between guaiacolane sesquiterpenes and acorolane sesquiterpene to target proteins（iNOS,COX-2,JNK,ERK,p38）.In summary,guaiacolane sesquiterpene（2 and 4）and acorolane sesquiterpene 7inhibited the activation of MAPK signaling pathway and down-regulateed the expression of inflammatory proteins iNOS,COX-2 and the production of inflammatory factors but had no effect on the activation of NF-κB signaling pathway.Molecular docking results showed that Arg388,Trp188 and Asp376 were the main active sites of amino acid residues when the compounds interacted with iNOS protein.Arg120 and Ser530 were the main active sites of the compounds acting on COX-2 protein.The main amino acid residue sites where compounds interacted with JNK protein were Met111,Asn194,Asn152,Gln75,Gln37.When the compounds interacted with ERK protein the main amino acid active sites were Met108,Glu50 and Ser170.The main active sites of amino acid residues when the compound interacted with p38 protein are Lys53.