Ellagic acid produces neuroprotection against LPS-induced dopamine neurotoxicity via the inhibition of microglial NLRP3 inflammasome activation

Background: 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 NLRP3 inflammasome has been confirmed to contribute to the progression of PD. Thus, inhibition of NLRP3 inflammasomes activation could be an important breakthrough point in PD drug therapy. Ellagic acid (EA) is a natural polyphenol that has been widely found in soft fruits, nuts and other plant tissues with various anti-inflammatory and anti-oxidant properties. However, the mechanisms underlying EA-mediated anti-inflammatory and neuroprotection have not been fully elucidated. Methods: In this study, lipopolysaccharide (LPS)-induced rat dopamine (DA) neuronal damage model was performed to determine the effects of EA on the protection of DA neurons. Furthermore, DA neuron MN9D cell line and microglia BV-2 cell line were employed to explore whether EA-mediated neuroprotection was through an NLRP3-dependent mechanism. Results: EA ameliorated LPS-induced DA neuronal loss in rat substantia nigra. Furtherly, 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 the subsequent pro-inflammatory cytokines excretion. In addition, EA-mediated anti-neuroinflammation and further DA neuroprotection from LPS-induced neurotoxicity was not shown upon microglial NLRP3 siRNA treatment. Conclusions: This study demonstrated EA has a profound effect on protecting DA neurons against LPS-induced neurotoxicity via the suppression of microglial NLRP3 inflammasome signaling activation. western blot analysis with an anti-Iba-1antibody (B). Scale bar = 100 μm. The effects of EA on NLRP3 inflammasome signaling activation in BV-2 cells were detected via western blotting (B). The ratio 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 pro-inflammatory factors, such as TNF-αIL-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.


Introduction
Parkinson's disease (PD) is a chronic progressive neurodegenerative disease characterized by a deep selective loss of dopamine (DA) neurons in the substantia nigra (SN) [1].
Clinical manifestations include static tremor, slow movement, postural instability, stiffness and other motor disorders [2]. So far, only a few drugs have been available for PD treatment and most of them just relieve symptoms, not prevent the death of DA neurons [3].
It was recognized that age-related excessive oxidative stress led to DA auto-oxidation, αsynuclein accumulation, and glial cells activation [4], which are the main causes of neuroinflammation [5]. Furtherly, neuroinflammation, characterized by microglia activation and infiltrating T cells at sites of neuronal injury, was considered to be a prominent contributor to the pathogenesis of progressive PD [6]. Microglia are natural immune barriers in the immune system. While stimulated by external conditions, such as brain damage, inflammation and pathogens, microglia could be activated and secrete a range of pro-inflammatory factors [7]. These factors led to the damage of adjacent DA neurons, which in turn elicited more microglia to reactivate in response, forming a selfpropelling cycle [8]. The inflammasome involving NLRP3 (NLR family, pyrin domain containing 3)-CASP1 (caspase 1) can regulate the maturation of IL-1β and IL-18 [9,10].
Additionally, nod-like receptor protein 3 (NLRP3) inflammasome was highly located in microglia and involved in the process of neuroinflammation [11]. Activation of NLRP3 inflammasomes had been discerned to play an important role in the progression of neurodegenerative diseases [12]. Therefore, inhibition of NLRP3 inflammasomes activation could be an important breakthrough point in PD drug therapy.
Ellagic acid (2, 3, 7, 8-tetrahydroxybenzopyrano [5, 4, 3-cde] benzopyran-5-10-dione, EA) is a natural polyphenol that has been widely found in soft fruits, nuts and other plant tissues. EA exhibits a number of pharmacological activities, such as anti-oxidant and antiinflammatory effects [13]. Recent studies confirmed EA exerted neuroprotection against aging, ischemic stroke and neurodegenerative disorders. For example, EA generated neuroprotection and improved cognitive dysfunctions in sporadic Alzheimer's disease animal model [14]. In addition, EA was indicated to confer neuroprotection against ischemic stroke [15]. However, the underlying mechanisms remain unclear. In this study, rat substantia nigral stereotaxic single injection of lipopolysaccharide (LPS)-elicited DA neuronal loss was performed to investigate EA-exerted neuroprotection and the underlying mechanisms as well. These findings would provide evidence for the future application of EA on PD treatment.

Animal and Treatment
Male Wistar rats (200-250 g, 8-10 weeks old) were bought from the Experimental Animal Center in the Third Military Medical University. All experimental procedures were carried out in accordance with Chinese Guidelines of Animal Care and Welfare and this study received an approval from the Animal Care and Use Committee of Zunyi Medical University (Zunyi, China). Rats were acclimated to their environment for 1 week before the experiments. All the animals were randomly allocated to five experimental groups with six rats in each group: control, EA alone (50 mg/kg), LPS, LPS+EA (10 mg/kg) and LPS+EA (50 mg/kg). With anesthetized by 7% chloral hydrate (0.5 ml/100 g, v/w), rats received a single LPS (10 µg in 5 µl PBS) unilateral injection into the SN pars compacts followed by the coordinates 5.2 mm posterior to bregma, 1.9 mm lateral to the midline, and 8.0 mm ventral to the surface of the skull [16]. Daily intragastric injection of EA (10 and 50 mg/kg) was administrated for 7 consecutive days after LPS injection. Control animals were injected with a single PBS into SN pars compacts. Afterwards, animals were sacrificed and the biochemical analysis was performed.

Rotarod Test
Rotarod test was performed to study the muscular coordination. It contained cylindrical arrangement of the thin steel rods with two parts by compartmentalization to permit the detection of two rats at the same time. Prior to the start of the test, rats were allowed to remain stationary at 0 rpm. Then, the rotational speed was steadily increased to 10 rpm in 30 s interval till rats fell off the rungs. The duration time each rat stayed on the rod was recorded and calculated for analyzing the behavior changes of rats [17].

Immunohistochemical Analysis and Cell Counting in the SN
Rat brains were cut with a horizontal sliding microtome into 35 μm transverse free-floating sections and immunostained with the corresponding antibodies. Briefly, brain slices were incubated with 0.3% Triton X-100 and blocked with goat serum. Subsequently, brain slices were incubated with anti-TH (1:500), OX-42 (1:800), and NLRP3 (1:800) antibodies at 4 °C overnight, respectively [18]. inflammasome were obtained and the fluorescence intensity was calculated using ipwin32 software.

Cell Culture and Treatment
Mouse microglial BV-2 cell lines were obtained from China Center for Type Culture Collection (Wuhan, China). Cultures were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in the humidified atmosphere of 5% CO 2 and 95% air [19]. DA neuron MN9D cell lines were purchased from the Cell Culture Center in the Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). MN9D cells were cultured in DMEM medium with 10% FBS and 1% penicillinstreptomycin on an atmosphere with 5% CO 2 at 37 °C in the humidified atmosphere of 5% CO 2 and 95% air [20].

MTT Assay
Cell viability was evaluated by MTT assay. BV-2 and MN9D cells were cultured in 1×10 5 /well in 96-well plates for 24 h. Afterwards, cells were treated with different 7 concentrations of EA for 30 min followed by LPS (100 ng/ml) or 6-OHDA (100 μM) treatment for another 24 h and then incubated with MTT solution (5 g/l) for 4 h. Formazan crystals in the cells were solubilized using 200 μl dimethyl sulfoxide (DMSO) and the absorbance was detected by an automated microplate reader within 490 nm wavelength [21].

Western Blot Analysis
Total protein content was extracted from rat midbrain tissue and BV-2 cells using a lysis buffer containing protease inhibitors. Protein levels were quantified by BCA assay [22].
Protein (10 μg) from each sample was subjected to SDS-PAGE gel under the reduced conditions. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes.
The membranes were blocked with 5% nonfat milk for 2 h at room temperature and then

ELISA
The levels of TNF-α, IL-1β, and IL-18 were measured by ELISA according to the manufacturer's instructions. The microplate reader was used to measure the absorbance at 450 nm [23].

Immunofluorescence Staining
Activated microglia were identified with an anti-OX-42 antibody. Cells were fixed with paraformaldehyde (4%) for 30 min. Later, cells were permeabilized using triton X-100 (0.3%) for 15 min. Then, cells were blocked using goat serum blocking solution for 60 min at 37 °C. Thereafter, cells were incubated with 1:800 dilution of anti-OX-42 antibody 8 overnight at 4 °C. Following overnight incubation, cells were incubated in dark for 30 min with goat anti-rabbit secondary antibody (1:1000) or goat anti-mouse secondary antibody (1:1000). Cells were also counterstained with DAPI for 5 min [24]. After rinsing cells with PBS, representative fluorescence images were obtained using EVOS® Floid® Cell imaging station. The fluorescence intensity was calculated by using ipwin32 software.

RNA Transfection
BV2 cells were cultured and seeded in a 6-well plate at a density of 1 × 10 5 cells/ml. The

Statistical Analysis
Results were indicated as mean ± standard error of the mean (SEM). Statistical significance was analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Upon ANOVA demonstrating the significant differences, pairwise comparison between means was evaluated by Bonferroni's post hoc test with correction. A value of p<0.05 was considered statistically significant.

EA attenuated LPS-induced DA neuronal damage in the SN in vivo
Neuroprotective effects of EA on LPS-induced DA neuronal damage were investigated in rats. As shown in Figure 1A, LPS reduced the time rat stayed on the rod, compared with control group. However, EA attenuated LPS-caused decrease in the time rat remained on the rod. To further confirm EA-mediated DA neuroprotection, TH-positive neuronal number and TH protein expression were determined. As shown in Figure 1B, EA ameliorated LPSinduced decrease in TH protein expression. Consistent with TH protein detection results, TH-positive neuronal counting showed EA protected against LPS-induced DA neuronal loss ( Figure 1C).

EA ameliorated LPS-elicited activation of microglia and NLRP3 inflammasome signaling in vivo
Next, the effects of EA on microglia and NLRP3 inflammasome activation were investigated. To verify the connection between NLRP3 inflammasome and microglia, the immunofluorescence double calibration site was conducted. As shown in Figure 2A, the NLRP3 inflammasome was activated and located in activated microglia. EA attenuated LPSinduced activation of NLRP3 inflammasome in microglia. Also, EA inhibited Iba-1 protein expression induced by LPS (Figure 2B). In addition, EA suppressed LPS-induced activation of NLRP3 inflammasome signaling (figure 2C) and pro-inflammatory cytokines (1L-1β, TNF-α and 1L-18) protein expressions (figure 2D).

EA had no direct neuroprotective effects on DA neurons
To further confirm whether EA produced direct neuroprotective actions on DA neurons, the effects of EA on 6-OHDA -induced DA neuronal damage in vitro were determined. First, as shown in Figure 3A, 6-OHDA induced neurotoxicity was not attenuated by EA treatment in MN9D cell-enriched cultures. Next, in TH-positive neurons counting analysis ( Figure 3B and C), 6-OHDA caused TH-positive neuronal loss and EA didn't protect from 6-OHDAinduced neuronal damage. Similar results were indicated in TH protein detection shown in Figure 3D. These results demonstrated that EA didn't generate direct neuroprotection on 10 DA neurons.

EA inhibited microglial NLRP3 inflammasome activation in vitro
The effects of EA on microglia and NLRP3 inflammasome signaling activation were further confirmed in vitro. First, BV-2 cells were employed to examine the effects of EA on LPSinduced microglial activation. As shown in Figure 4A, immunofluorescence staining assay indicated that EA reduced LPS-induced microglia activation. Also, EA decreased LPSinduced higher protein expression of Iba-1 ( Figure 4B). Then, EA inhibited the activation of microglial NLRP3 inflammasome signaling induced by LPS (Figure 4B). In addition, EA eliminated LPS-induced production of pro-inflammatory factors, such as TNF-α, IL-1β and IL-18 in the culture medium ( Figure 4C).

NLRP3 inflammasome signaling inactivation was involved in EA-mediated antiinflammatory properties
To investigate the role of NLRP3 inflammasome signaling in EA-mediated antineuroinflammation, NLRP3 siRNA was performed in BV-2 cell cultures. First, as shown in Figure 5A, NLRP3 siRNA was transfected into microglia cells and the successful transfection with NLRP3 siRNA was evaluated by NLRP3 protein level in BV-2 cells.
Furthermore, NLRP3 siRNA and EA inhibited LPS-induced NLRP3 inflammasome signaling activation, respectively. However, no significant difference of NLRP3, caspase-1 and procaspase-1 protein expressions between LPS+EA and LPS+EA+NLRP3 siRNA groups was discerned ( Figure 5B). In addition, the effects of EA on pro-inflammatory factors excretion with NLRP3 siRNA administration were measured. In parallel with NLRP3 inflammasome signaling analysis, EA didn't reduce LPS-induced release of proinflammatory factors again after NLRP3 siRNA treatment (Figure 5C). These observations indicated EA attenuated microglia-induced neuroinflammation via the inhibition of NLRP3 inflammasome signaling activation.

EA targeted microglial NLRP3 inflammasome to produce DA neuroprotection
Since microglia were the target of EA-generated DA neuroprotection, whether this neuroprotection resulted from inhibiting microglial NLRP3 inflammasome activation was then explored. As shown in Figure 6 Neuroinflammation is considered to be a most common feature of the aging brain and neurodegenerative disorders including PD. It is primarily mediated by activated glial cells and accompanied by the secretion of pro-inflammatory mediators [26]. As the first defense of immune surveillance, microglia readily become activated and predominately participate in inflammatory response. In addition, astroglia could amplify microgliamediated neuroinflammation and result in the feedback loop of neuroinflammatory reactions [27]. In this regard, understanding the molecular mechanisms of PD can be found by investigating the microglial neuroinflammation [28]. Therefore, to target microglia-derived pro-inflammatory cytokines might offer promising therapeutic approaches for PD management. Inflammasomes are multi-protein complexes responsible for intracellular sensors of environmental and cellular stress [29]. Inflammasomes consist of NLRP family, the signaling adapter apoptosis associated speck-like protein containing a caspase recruitment domain (ASC) and pro-inflammatory precursor pro-caspase-1 [30]. In addition, NLRP3 inflammasome is particularly located in microglia and involved in the response of neuroinflammation [11]. Upon cellular stress, assembly of NLRP3 inflammasome triggers caspase-1 activation and further caspase-1-mediated production of IL-1β and IL-18, thereby initiating neuroinflammation [31]. In PD patient brains, the NLRP3 inflammasome is potentially activated by insoluble a-synuclein aggregates and oxidative stress [32]. However, deficiency of NLRP3 inflammasome attenuates motor dysfunction and DA neurodegeneration in PD mouse model [33]. Thus, inhibition of NLRP3 inflammasome activation might be beneficial for PD intervention. In the present study, EAmediated DA neuroprotection was discerned in neuron-microglia co-cultures but not in neuron-enriched cultures, implying that microglia were at least essential for EA-generated neuroprotection. Moreover, we found EA reduced pro-inflammatory factors production induced by LPS. Furtherly, EA-inhibited microglial activation and subsequent proinflammatory factors release was attributed to the inhibition of microglial NLRP3 inflammasome activation. This finding was revealed by the following observations: 1) EA inhibited microglial NLRP3 and pro-caspase-1 activation and IL-1β production. 2) EA could not further suppress LPS-induced pro-inflammatory factors release and produce DA 13 neuroprotection after neuron-microglia co-cultures treated by NLRP3 siRNA.
To date, current PD therapy is focused on the symptoms control and fails to delay the progressive neurodegenerative process. Actually, various side-effects of the available drugs present huge challenges for long-term application. Therefore, more potential candidates from animal studies to clinical trials was indicated. Therefore, an urgent approach for novel anti-inflammatory alternatives design was prompted [29]. Recent studies have confirmed EA could cross blood-brain barrier [34]. Here, the present study demonstrated that EA protected from LPS-induced DA neurotoxicity and modulation of microglial NLRP3 inflammasome signaling activation was revealed to participate in this neuroprotection. However, the current study on EA only stays in the animal experiments, and the follow-up hopes to be used as a clinical drug application for the next research.

Conclusions
This study demonstrated EA has a profound effect on protecting DA neurons against LPSinduced neurotoxicity via the suppression of microglial NLRP3 inflammasome signaling activation. These findings suggest EA might be a potential benefit for PD treatment.