Malaysian Carrageenophyte Kappapphycus species inhibit Lipopolysaccharides-Induced Neuroinflammation by suppressing the AKT/NFxB and ERK signaling pathways in BV2 Microglia AKT/NFkB and ERK signaling pathways in BV2 Microglia.

Abstract

Neuroinflammation is normally caused by the stimulation of microglia and astrocytes with the production of proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO) and cyclooxygenase-2 (COX-2), as well as the generation of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Unregulated activities and overproduction of proinflammatory mediators and cytokines will cause neuronal death, which will trigger the development of neurodegenerative diseases. Kappaphycus alvarezii and Kappaphycus malesianus are red algae cultivated in Semporna, Sabah, East Malaysia. They are remarkable with their k-carrageenans which have been widely used as a thickening agent in food, cosmetics, and nutraceutical industry. Kappaphycus spp. have been reported with several pharmacological effects, including antioxidant, anti-viral and anti-cancer effects. However, limited research was focusing on their neurological effects. Therefore, it is our interest to explore and compare the bioactivity of K. alvarezii and K. malesianus from the neuroprotective aspect. This study aimed to investigate the anti-neuroinflammatory activity in K. alvarezii and K. malesianus crude extracts (ethyl acetate, ethanol, and methanol extracts) on lipopolysaccharides (LPS)-induced BV2 microglial cell line and their underlying mechanisms. Cell viability assay (MTT assay) was used to examine the percentage of viable BV2 microglial cell line after treating with respective K. alvarezii and K. malesianus crude extracts. Nitric oxide inhibition assay (Griess assay) was used to investigate the nitric oxide inhibitory activity of all crude extracts, while the crude extract with the highest nitric oxide inhibitory activity was used for western blot and ELISA analyses to investigate the proinflammatory mediators (iNOS and COX-2) and cytokines (TNF-⍺, IL-6 and IL-1β) expression in LPS-induced BV2 microglial cell line. The potential compound(s) in the extract with the highest promising anti-neuroinflammatory effect were identified using the liquid chromatography-mass spectrometry (LC-MS) analysis. Our results indicated that all extracts of both seaweeds had maintained cell viability above 80% at the concentration of 2.5 mg/mL, except for K.malesianus ethanol extract (69.7% ± 0.31 mg/mL). K. alvarezii ethyl acetate extract and K. malesianus methanol extract indicated the highest nitric oxide inhibitory activity without showing significant cytotoxicity towards BV2 microglial cell line. Thus, K. alvarezii ethyl acetate and K. malesianus methanol extract was selected to further evaluate their proinflammatory mediators and proinflammatory cytokines expressions and signaling pathways. The results disclosed that K. malesianus methanol extract demonstrated higher inhibitory activity in the formation of proinflammatory mediators and cytokines via AKT/NF-kB and ERK pathways. According to the LC-MS analysis for K. malesianus methanol extract, three known bioactive compounds were identified with anti-neuroinflammatory activities, namely, 2,6-nonadien-1-ol, prosopinine and eplerenone. In conclusion, all K. alvarezii and K. malesianus extracts showed inhibitory activity on nitric oxide and proinflammatory cytokines production, which may contribute to the anti-neuroinflammatory activity. Moreover, western blot results explained that K. alvarezii and K. malesianus extracts had contributed to anti-neuroinflammatory activity through inhibiting iNOS and COX-2 production via AKT/NF-kB, and ERK pathways. K. malesianus methanol extract is the most potent extract and may acts as a potent anti-neuroinflammatory therapy in the prevention of neurodegenerative disorders