Ellagic Acid Protects Dopamine Neurons via Inhibition of NLRP3 Inflammasome Activation in Microglia

Abstract

Neuroinflammation plays a crucial role in the pathological process of Parkinson's disease (PD). Nod-like receptor protein 3 (NLRP3) inflammasome was highly located in microglia and involved in the process of neuroinflammation. Activation of the NLRP3 inflammasome has been confirmed to contribute to the progression of PD. Thus, inhibition of NLRP3 inflammasome activation could be an important breakthrough point on PD therapy. Ellagic acid (EA) is a natural polyphenol that has been widely found in soft fruits, nuts, and other plant tissues with anti-inflammatory, antioxidant, and neuroprotective properties. However, the mechanisms underlying EA-mediated anti-inflammation and neuroprotection have not been fully elucidated. In this study, a lipopolysaccharide- (LPS-) induced rat dopamine (DA) neuronal damage model was performed to determine the effects of EA on the protection of DA neurons. In addition, the DA neuronal MN9D cell line and microglial BV-2 cell line were employed to explore whether EA-mediated neuroprotection was through an NLRP3-dependent mechanism. Results indicated that EA ameliorated LPS-induced DA neuronal loss in the rat substantia nigra. Further, inhibition of microglial NLRP3 inflammasome signaling activation was involved in EA-generated neuroprotection, as evidenced by the following observations. First, EA reduced NLRP3 inflammasome signaling activation in microglia and subsequent proinflammatory cytokines' excretion. Second, EA-mediated antineuroinflammation and further DA neuroprotection from LPS-induced neurotoxicity were not shown upon microglial NLRP3 siRNA treatment. In conclusion, this study demonstrated that EA has a profound effect on protecting DA neurons against LPS-induced neurotoxicity via the suppression of microglial NLRP3 inflammasome activation.

Figure 1

EA attenuated LPS-induced DA neuronal damage in SN in vivo. Rats were intragastrically given EA (50 mg/kg) for 7 consecutive days. Rat behavior changes were analyzed by the rotarod test (a). TH protein expression in the rat midbrain was tested by western blot assay (b). Brain sections were immunostained with an anti-TH antibody, and the number of TH-positive neurons in SN was counted (c). The “ellipse” presented the area of SN. Scalebar = 200μm. Data were the mean ± SEM from 6 rats. ^∗p < 0.05 compared with the control group; ^#p < 0.05 compared with the LPS group.

Figure 2

EA ameliorated LPS-elicited activation of microglia and NLRP3 inflammasome signaling in vivo. Rat brains were collected and stained by double immunofluorescence with anti-NLRP3 and anti-OX-42 antibodies (green fluorescence represented NLRP3 inflammasome, and red fluorescence represented microglia) (a). The protein expressions of Iba-1 (b); NLRP3, caspase-1, and pro-caspase-1 (c); and TNF-α, IL-1β, and IL-18 (d) in the rat midbrain were determined via western blot assay. Data were the mean ± SEM from 6 rats and expressed as a percentage of the control group. ^∗p < 0.05 compared with the control group; ^#p < 0.05 compared with the LPS group.

Figure 3

EA had no direct neuroprotective effects on DA neurons. MN9D cells were treated with EA (0.1 and 1 μM) for 30 min and then incubated with 6-OHDA (100 μM) for 24 h. Cell viability was determined by MTT assay (a). 6-OHDA-induced MN9D cell damage was evaluated by immunostaining (b) and cell counting (c). Scalebar = 100μm. The protein expression of TH was detected by western blot assay (c). Data were the mean ± SEM from three independent experiments performed in triplicate. ^∗p < 0.05 compared with control cultures; ^#p < 0.05 compared with 6-OHDA-treated cultures.

Figure 4

EA inhibited microglial NLRP3 inflammasome activation in vitro. BV-2 cells were treated with EA (0.1 and 1 μM) for 30 min and then incubated with LPS (100 ng/ml) for 24 h. Microglial activation was evaluated by immunostaining with an anti-OX-42 antibody (a) and quantitated by western blot analysis with an anti-Iba-1 antibody (b). Scalebar = 100μm. The effects of EA on NLRP3 inflammasome signaling activation in BV-2 cells were detected via western blotting (b). The ratios of densitometry values of Iba-1, NLRP3, caspase-1, and pro-caspase-1 with β-actin were analyzed and normalized to each respective control cultures. The release of proinflammatory factors, such as TNF-α, IL-1β, and IL-18, in BV-2 cell culture medium was measured by ELISA (c). Data were the mean ± SEM from three independent experiments performed in triplicate. ^∗p < 0.05 compared with control cultures; ^#p < 0.05 compared with LPS-treated cultures.

Figure 5

NLRP3 inflammasome signaling inactivation was involved in EA-mediated anti-inflammatory properties. BV-2 cells were treated with NLRP3 siRNA (40 nM). After 6 h of transfection, the transfection solution was removed and cells were rinsed with PBS. The silencing efficiency was assessed via NLRP3 protein expression detection (a). Moreover, BV-2 cells were treated with EA (1 μM) in the presence of NLRP3 siRNA and then exposed to LPS for 24 h. The protein expressions of Iba-1, NLRP3, caspase-1, and pro-caspase-1 in BV-2 cells were detected via western blot assay (b). The levels of TNF-α, IL-1β, and IL-18 in the culture medium were measured by ELISA (c). Data were the mean ± SEM from three independent experiments performed in triplicate. ^∗p < 0.05 compared with control cultures; ^#p < 0.05 compared with LPS-treated cultures.

Figure 6

EA targeted microglial NLRP3 inflammasome to produce DA neuroprotection. Microglia-conditioned medium (MCM) prepared from BV-2 cell cultures with administration of EA (MCM (EA)), LPS (MCM (LPS)), LPS+EA (MCM (LPS+EA)), NLRP3 siRNA (MCM (NLRP3 siRNA)), NLRP3 siRNA+EA (MCM (NLRP3 siRNA+EA)), NLRP3 siRNA+LPS (MCM (NLRP3 siRNA+LPS)), and LPS+NLRP3 siRNA+EA (MCM (LPS+NLRP3 siRNA+EA)) was harvested and added to MN9D cells incubated for 24 h. MN9D cell viability was determined by MTT assay (a). TH protein expression was tested by western blot assay (b). Data were the mean ± SEM from three independent experiments performed in triplicate. ^∗p < 0.05 compared with control cultures; ^#p < 0.05 compared with MCM (LPS)-treated cultures.

Figure 7

The mechanisms underlying EA-mediated dopamine neuroprotection.