6-O-angeloylplenolin exerts neuroprotection against lipopolysaccharide-induced neuroinflammation in vitro and in vivo

Abstract

Neuroinflammation is one of the critical events in neurodegenerative diseases, whereas microglia play an important role in the pathogenesis of neuroinflammation. In this study, we investigated the effects of a natural sesquiterpene lactone, 6-O-angeloylplenolin (6-OAP), isolated from the traditional Chinese medicine Centipeda minima (L.) A.Br., on neuroinflammation and the underlying mechanisms. We showed that treatment with lipopolysaccharide (LPS) caused activation of BV2 and primary microglial cells and development of neuroinflammation in vitro, evidenced by increased production of inflammatory cytokines TNF-α and IL-1β, the phosphorylation and nuclear translocation of NF-κB, and the transcriptional upregulation of COX-2 and iNOS, leading to increased production of proinflammatory factors NO and PGE₂. Moreover, LPS treatment induced oxidative stress through increasing the expression levels of NOX2 and NOX4. Pretreatment with 6-OAP (0.5-4 μM) dose-dependently attenuated LPS-induced NF-κB activation and oxidative stress, thus suppressed neuroinflammation in the cells. In a mouse model of LPS-induced neuroinflammation, 6-OAP (5-20 mg·kg^-1·d^-1, ip, for 7 days before LPS injection) dose-dependently inhibited the production of inflammatory cytokines, the activation of the NF-κB signaling pathway, and the expression of inflammatory enzymes in brain tissues. 6-OAP pretreatment significantly ameliorated the activation of microglia and astrocytes in the brains. 6-OAP at a high dose caused a much stronger antineuroinflammatory effect than dexamethansone (DEX). Furthermore, we demonstrated that 6-OAP pretreatment could inhibit LPS-induced neurite and synaptic loss in vitro and in vivo. In conclusion, our results demonstrate that 6-OAP exerts antineuroinflammatory effects and can be considered a novel drug candidate for the treatment of neuroinflammatory diseases.

Keywords: 6-O-angeloylplenolin; LPS; NF-κB; Traditional Chinese medicine; dexamethansone; microglia; neuroinflammation; oxidative stress.

Fig. 1

6-OAP inhibits the activation of microglial cells. a Chemical structure of 6-OAP. b, c The percentage of cell viability for 24 h in BV2 cells was determined by a MTT assay. d, e The relative mRNA levels of TNF-α and IL-1β in BV2 cells were detected by real-time PCR analysis. f–i The production of TNF-α and IL-1β in BV2 (f, g) and primary microglial cells (h, i) were examined by ELISA. j Primary microglia was stained using Iba1 antibody (green) and DAPI (blue), purity of primary microglia can reach 95%. k Morphological changes of primary microglial cells (upper panal) and BV2 cells (lower panal) were observed under bright field of microscope, and representative pictures have been shown for comparison. Scale bar, 20 µm. All data are presented as the means ± SEM from three separate experiments. ^##P < 0.01, ^###P < 0.001 versus control cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS alone

Fig. 2

6-OAP show inhibitory effects on NF-κB signaling pathway. a–d The protein levels of NF-κB p65, phospho-NF-κB p65, IκB-α, and phospho-IκB-α were analyzed by Western blotting, β-actin was used as a loading control. e, f The nuclear part of NF-κB was detected by western blotting, lamin B1 was used as a loading control. g Nuclear localization of NF-κB of primary microglial cells (upper panal) and BV2 cells (lower panal) was determined by immunostaining with NF-κB subunit p65 (red), and counterstained with Hoechst 33342 (blue), the images were obtained by confocal microscopy. h The percentage of nuclear NF-κB-positive cells among cultured microglial cells was quantified. Scale bar, 20 µm. Relative protein levels were quantified by densitometry analysis using ImageJ software. Data are presented as the means ± SEM from three separate experiments. ^##P < 0.01, ^###P < 0.001 versus control cells. *P < 0.05, **P < 0.01, ***P < 0.01 versus LPS alone. p-NF-κB: phospho-NF-κB p65, p-IκB-α: phosphor-IκB-α. PC: primary microglial cells

Fig. 3

6-OAP suppresses the LPS-induced expression of iNOS and COX-2. a–c The protein levels of iNOS and COX-2 in BV2 and primary microglial cells after 24 h treatment were analyzed by Western blotting, β-actin was used as an internal control. d, e The relative mRNA levels of iNOS and COX-2 in BV2 cells were detected by real-time PCR analysis. f–i Culture supernatants of BV2 and primary microglial cells were harvested after 24 h treatment, the concentration of Nitrite oxide (f, g) and PGE₂ (h, i) were determined and quantified using the Griess reagent and competitive ELISA, respectively. Celecoxib was used as a positive control for COX-2 inhibition. Relative protein levels were quantified by densitometry analysis using ImageJ software. The quantification from three independent experiments was analyzed and expressed as means ± SEM. ^###P < 0.001 versus control group, *P < 0.05, **P < 0.01, and ***P < 0.001 versus LPS group. PC: primary microglial cells

Fig. 4

6-OAP attenuates inflammation-induced oxidative stress. a, b The expression levels of NOX-2 and NOX-4 were examined by Western blotting, β-actin was used as an internal control, relative protein levels were quantified by densitometry analysis using ImageJ software. c The intracellular level of ROS was determined by using the fluorescent probe DCFH-DA, and representative pictures were shown. d The fluorescence intensity of DCFH-DA was quantified using fluorimetric plate reader, and the relative fluorescence was normalized to control group, and expressed as the means ± SEM from three independent experiments. ^##P < 0.01, ^###P < 0.001 versus control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells. PC: primary microglial cells

Fig. 5

6-OAP exerts neuroprotective effects against inflammatory injury. a, b The percentage of cell viability of HT22 cells treated with indicated drugs (a) or cultured under conditioned medium (b) for 24 h was determined by MTT assay. c Morphological changes of HT22 cells (upper panel) and primary cortical neurons (lower panel) were observed, and representative pictures have been taken for comparison. d Neurite lengths of primary cortical neurons were measured by NeuronJ, the relative average neurite length is normalized to control group. Scale bar, 20 µm. All data are normalized to control cells and presented as the mean ± SEM of three independent experiments. ^#P < 0.05 versus control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS-stimulated cells. N.S.: non-statistical significant

Fig. 6

6-OAP ameliorates neuroinflammation in LPS-treated mice. a The expression levels of TNF-α and IL-1β in brain tissues were analyzed by real-time PCR. b The production of TNF-α and IL-1β in brain tissues was examined by ELISA. c The protein levels of iNOS, COX-2, phospho-NF-κB p65, NF-κB p65, phospho-IκB-α, and IκB-α in brain tissues were detected by Western blotting, the results from two group of mice have been shown. d–h The densitometry of indicated immunoblot bands was determined by ImageJ software. All data were expressed as the means ± SEM from five mice of each group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus control group. *P < 0.05, **P < 0.01, ***P < 0.001 versus LPS treated alone. p-NF-κB: phospho-NF-κB p65, p-IκB-α: phosphor-IκB-α

Fig. 7

6-OAP inhibits the activation of microglia and astrocyte in LPS-treated mouse brain. a, b Coronal sections of mouse brain were incubated with the primary antibody against Iba1 (green) and TNF-α (red) (a) or GFAP (green) and TNF-α (red) (b) in the hippocampal DG region, DAPI was used to counter stain cell nuclei. Scale bar, 20 µm. c Bar graphs representing the quantification of the average cell number per field of TNF-α-positive microglia and astrocyte in the DG region of hippocampus. d The protein levels of SYN, Iba1, and GFAP in the hippocampal tissues were detected by Western blotting. e The densitometry of indicated immunoblot bands was determined by ImageJ software. All data were expressed as the means ± SEM from five mice of each group. ^##P < 0.01, ^###P < 0.001 versus control group. *P < 0.05, **P < 0.01 versus LPS treated group