Regulation Of β-Carboline And Canthinone Alkaloids On Inflammatory Signaling Pathways

Picrasma quassioides（D.don）Bennet（Simaroubaceae）is used extensively in the traditional Chinese medicine for anti-inflammatory treatments.The main active chemical constituents of simaroubaceae were reported to be alkaloids,including:β-carboline alkaloids,canthinone alkaloids and dimeric alkaloids,however,the anti-inflammatory mechanism of this group of natural compounds remains unclear.In this paper,we studied the anti-inflammatory activity of indole alkaloids and its anti-inflammatory molecular mechanism,and provide theoretical explanation for the clinical use of Simaroubaceae plants.Our compound library consists of seventy-five indole alkaloids,which were isolated from Picrasma quassioides and other Simaroubaceae plants or chemical synthesized.In the present article,we aim to screen the anti-inflammatory activity of the compound library,all compounds were firstly assessed their inhibitory activities on the overproduction of NO by the Griess reaction,the cell viability was investigated by MTT assay.The active compound was selected to study inhibition effect on the release of pro-inflammatory cytokines and the regulation of inflammatory protein expression in the inflammatory signaling pathway.Firstly,the inhibitory activity on the overproduction of inflammatory mediator NO in LPS-activated macrophage RAW 264.7 cells were determined to evaluate the anti-inflammatory activity of the compound library.The level of nitrite as an indicator of NO was determined by the Griess reaction,and the cell viability was investigated by MTT assay.Furthermore,the effect of selected active compounds on the release of PGE₂,IL-6and TNF-αwas determined by using ELISA method.Western blot analysis was used to determine the inhibition effect of six selected active compounds on the high expression of iNOS and COX-2 proteins and regulation of the inflammatory NF-κB signaling pathway（IκB-αprotein degradation）and the inflammatory MAPK signaling pathway（JNK,p38and ERK protein phosphorylation）.The compound library of seventy-five alkaloids,including thirty-nineβ-carboline alkaloids（7-45）,thirty canthinone alkaloids（1,3-6 and 46-70）,and six dimeric alkaloids（72-76）.11,12,13,15,21,22,23,27,29,30,31,37,41,42 out of 39β-carboline alkaloids showed NO inhibitory activities（the inhibitory rate on NO production was higher than 80%）,some alkaloids exhibited cytotoxicity.On the other hand,five compounds（46,47,55,57,64）out of 30 canthinone alkaloids showed NO inhibitory activities（the inhibitory rate on NO production was higher than 80%）and no strong cytotoxicity were observed.Among the six dimeric alkaloids,although 71,72 and 76exhibited high inhibitory rate on the NO production,the results of MTT assay also indicated the strong cytotoxicity.When the dose was declined to low concentration,no cytotoxicity was observed but their inhibitory rate on the NO production also disappeared.According to the preliminary screening experiment results,four active compounds were selected,including twoβ-carboline alkaloids:Benzalharman（23）,Kumujian C（27）,and two canthinone alkaloids:Canthin-6-one（46）,9-methoxy-canthin-6-one（57）.The results of Griess and ELISA showed that the four active compounds could inhibit the release of inflammatory cytokines NO,IL-6 and TNF-αin LPS-stimulated RAW 264.7cells.Two canthinone alkaloids 46 and 57 significantly inhibited LPS-induced macrophage RAW 264.7 release of PGE₂,but twoβ-carboline alkaloids 23 and 27 did not inhibit PGE₂ release.Western Blot results showed that twoβ-carboline alkaloids 23 and 27down-regulated iNOS protein expression,but had no inhibitory effect on COX-2 protein expression;two canthinone alkaloids 46 and 57 potently down-regulated the expression of iNOS and COX-2 protein.All four active compounds inhibit the degradation of IκB-αprotein in the NF-κB pathway.46 and 57 inhibited JNK protein phosphorylation in the MAPK pathway,and 57 also down-regulated p38 phosphorylation,but neitherβ-carboline alkaloids phosphorylated JNK,ERK and p38 proteins in the MAPK pathway.In summary,although theβ-carboline alkaloids and canthinone alkaloids have similar alkaloid structures,there are still some differences in the anti-inflammatory molecular mechanisms.β-carboline alkaloids blocked the activation of NF-κB pathway induced by LPS and had no effect on MAPK pathway,but canthinone alkaloids inhibited the activation of NF-κB pathway and MAPK pathway.