Two sesquiterpene lactones, arnicolide B and arnicolide C, isolated from Centipeda minima, exert anti-inflammatory effects in LPS stimulated RAW 264.7 macrophages via inactivation of the MAPK pathway

Abstract Arnicolide B and arnicolide C are two sesquiterpene lactones isolated and identified from Centipeda minima, but the anti-inflammatory effects and mechanisms of these two compounds have not been reported. In this study, LPS was used to establish RAW 264.7 macrophages inflammatory response model. Griess, ELISA, Western blot were used to investigate the anti-inflammatory effects in vitro and the molecular mechanisms of these two active compounds. The results showed that arnicolide B and arnicolide C could not only inhibit the production of inflammatory mediators NO, PGE2, TNF-α and IL-6, but also down-regulate the high expression of inflammatory proteins iNOS and COX-2. Furthermore, arnicolide B and arnicolide C inhibited the phosphorylation of ERK, JNK, p38 proteins in the MAPK signaling pathway, but had no effect on the degradation of IκB-α protein and the activation of the NF-κB pathway. As conclusion, these two compounds exert anti-inflammatory effects by inactivation of the MAPK pathway. Graphical Abstract


Introduction
Centipeda minima is the whole plant of Centipeda minima (L.) A. Br. et Aschers., which is widely distributed in China, Australia and Southeast Asia (Wu et al. 2012). The whole plant is used in traditional Chinese folk medicine for rhinitis, pain relief, swelling reduction and cancer treatment (Huang et al. 2014). The chemical constituents of this plant were identified as sesquiterpenoids, volatile oils, triterpenes, flavones, phenolics, and organic acids (Chan et al. 2016;Nguyen et al. 2017). Previous research showed that Centipeda minima has anti-cancer, anti-inflammatory, anti-bacterial, and anti-allergic properties (Linh et al. 2021).
In previous study of our research group focused on the chemical constituents of Centipeda minima, 43 compounds have been isolated and identified from the ethanolic extract. The in vitro anti-inflammatory activities of isolated forty-three compounds have been evaluated by determination the inhibitory effect on LPS-induced release of nitric oxide (NO). Among them, 16 sesquiterpenoid lactones showed significant antiinflammatory activities, especially the activities of arnicolide B and arnicolide C are very potent. However, the anti-inflammatory effects and mechanisms of these two compounds have not been reported. In the present study, LPS was used to establish macrophages RAW 264.7 cell inflammatory response model, and the anti-inflammatory effect and mechanism of arnicolide B and arnicolide C representing sesquiterpene lactones in Centipeda minima were studied and reported.

Results and discussion
In our previous study, arnicolide B and arnicolide C showed significant anti-inflammatory activity in the preliminary screening and evaluation of in vitro activity. 1 H-NMR and 13 C-NMR spectroscopic data of arnicolide B and arnicolide C are summarised in Table S1. Their chemical structures are shown in Figure 1. Then, in-depth studies on arnicolide B and arnicolide C were carried out to further clarify their anti-inflammatory regulatory mechanism on relative inflammatory signaling pathways. MTT method was used to detect the cytotoxicity of arnicolide B and arnicolide C to macrophages RAW 264.7. As shown in Figure S1, arnicolide B and arnicolide C showed no cytotoxicity when the concentrations were lower than 6.25 lM and 25 lM, respectively. The inhibitory effects of arnicolide B and arnicolide C on the release of NO, PGE 2, TNF-a, and IL-6 induced by LPS were evaluated. Hydrocortisone succinate sodium (HSS) was used as positive control. As shown in Figure S2(a-d), arnicolide B and arnicolide C could significantly inhibit the overproduction of NO, PGE 2 , TNF-a and IL-6 in a dose-dependent manner. These results indicated that these two compounds have ideal anti-inflammatory activities and may have potential for anti-inflammatory application. iNOS and COX-2, as downstream proteins in the inflammatory pathway, can be overexpressed under LPS induction. As shown in Figure S3(a, b), arnicolide B and arnicolide C could down-regulate the high expression of iNOS and COX-2 in a dose-dependent manner. The degree of degradation of IjB-a protein can be regarded as an important indicator of NF-jB activation. As shown in Figure S4(a, b), arnicolide B and arnicolide C had almost no significant inhibition on the degradation of IjB-a protein. The anti-inflammatory mechanism of these sesquiterpene lactones may not be related to NF-jB pathway. As one of the inflammation signaling pathways, MAPKs pathway exists in the vast majority of cells (Gao et al. 2022). The activation of MAPKs pathway always starts from the phosphorylation of ERK, JNK and p38 proteins. As shown in Figure S5(a-f), arnicolide B had no significant inhibitory effect on LPS-induced phosphorylation of ERK. Arnicolide C had a dose-dependent inhibitory activity on ERK phosphorylation. The differences in structure between arnicolide B and arnicolide C are mainly manifested in the difference of ester side chains connected at C-6 position. Arnicolide B is connected with isovalyl group, while arnicolide C is connected with isobutanyl group. Therefore, the difference in the effect of the two compounds on the ERK protein in the MAPK pathway may be due to the difference in the C-6-linked ester side chain. Both arnicolide B and arnicolide C had a certain inhibitory effect on JNK phosphorylation and p38 phosphorylation in a dose-dependent manner. Pac¸o et al reviewed that most sesquiterpene lactones exert anti-inflammatory effects mainly through NF-jB pathway (Paço et al.2022). Our results suggested that arnicolide B and arnicolide C exert anti-inflammatory effects mainly through the MAPK pathway, and these two compounds differ slightly from most known sesquiterpene lactones in not affecting NF-jB nor its inhibitor.

Conclusion
In conclusion, our present findings demonstrated that arnicolide B and arnicolide C, two sesquiterpene lactones isolated and identified from Centipeda minima, have significant anti-inflammatory potential with low cytotoxicity. Elucidation on their mechanism suggested that they exert anti-inflammatory effects mainly through inactivation of the MAPK pathway ( Figure S6).

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Natural Science Foundation of Shandong Province (ZR2019ZD24), the Graduate Innovation Foundation of Yantai University (GIFYTU).