Inhibition of NLRP3 inflammasome by glibenclamide attenuated dopaminergic neurodegeneration and motor deficits in paraquat and maneb-induced mouse Parkinson’s disease model

Xiaofei Qiua,d,1, Qinghui Wangb,1, Liyan Houc,1, Cuili Zhangd, Qingshan Wangc,*, Xiulan Zhaod,*

H I G H L I G H T S

Glibenclamide protected dopaminergic neurons in paraquat and maneb-intoxicated mice. Glibenclamide attenuated paraquat and maneb-induced NLRP3 inflammasome activation.
Glibenclamide suppressed paraquat and maneb-induced microglial activation and M1 polarization.
Glibenclamide suppressed paraquat and maneb-induced oxidative damage via iNOS and NOX2.

A B S T R A C T

Pesticides exposure can lead to damage of dopaminergic neurons, which are associated with increased risk of Parkinson’s disease (PD). However, the etiology of PD remains poorly understood and no therapeutic strategy is available. Previous studies suggested the involvement of NLRP3 inflammasome in the onset of PD. This study was designed to investigate whether glibenclamide, an inhibitor of NLRP3 inflammasome, could offer a reliable protective strategy for PD in a mouse PD model induced by paraquat and maneb. We found that glibenclamide exerted potent neuroprotection against paraquat and manebinduced upregulation of α-synuclein, dopaminergic neurodegeneration and motor impairment in brain of mice. Mechanistically, glibenclamide treatment blocked NLRP3 inflammasome activation evidenced by reduced expressions of NLRP3, activated caspase-1 and mature interleukin-1β in glibenclamide cotreated mice compared with those in paraquat and maneb group mice. Furthermore, glibenclamide treatment mitigated paraquat and maneb-induced microglial M1 proinflammatory response and nuclear factor-kB activation in mice. Finally, the increased superoxide production, lipid peroxidation, protein levels of NADPH oxidase 2 (NOX2) and inducible nitric oxide synthase (iNOS) induced by paraquat and maneb were all attenuated by glibenclamide. Overall, our findings demonstrated that glibenclamide protected dopaminergic neurons in a mouse PD model induced by combined exposures of paraquat and maneb through suppression of NLRP3 inflammasome activation, microglial M1 polarization and oxidative stress.

Keywords:
Glibenclamide
Parkinson’s disease Pesticide
NLRP3 inflammasome
Microglial activation

1. Introduction

Parkinson’s disease (PD) usually progresses several decades with gradual loss of nigral dopaminergic neurons (Hou et al., 2018a) and formation of abnormal inclusions (Wang et al., 2015). clinical and pathological features of PD have been extensively used to explore the key molecular events that provoke neurodegeneration and evaluate the therapeutic effect of PD. Generally, some neurotoxins-induced rodents PD models are commonly used to mimic human sporadic PD, and stereotactic injection of human αsynuclein expressing viral vectors into substantia nigra (SN) of adult rats or transgenic mice are developed for gene-based PD models (Dauer and Przedborski, 2003).1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), rotenone, and 6-hydroxydopamine (6-OHDA) are the most commonly used neurotoxins to induce dopaminergic neurodegeneration. MPTP is highly lipophilic and readily across the blood-brain barrier (BBB). In brain, MPTP is + converted to toxic 1-methyl-4-phenylpyridinium ion (MPP ), which potently inhibits complex I of the mitochondrial electron transport chain, rapidly leads to decreases ATP content, and selectively kill the monoaminergic neurons. Rotenone is also lipophilic and similar in ability to inhibit complex I, but its neurotoxicity was proved to be more widespread rather than only dopaminergic neurons (Höglinger et al., 2003). 6-OHDA may play a role as an endogenous neurotoxin. Iron is abundant in the substantia nigra pars compacta (SNpc) and can react in a Fenton-type reaction with dopamine and hydrogen peroxide to produce 6-OHDA (Pezzella et al., 1997). However, the SNpc dopaminergic neuronal death induced by 6-OHDA was achieved by local stereotaxic injection, due to unable to cross BBB. Recently, epidemiological evidences support that geographical overlap in agricultural use of paraquat and maneb are related to PD (Costello et al., 2009; Wang et al., 2011), and raise the possibility of multiplehit models of PD (Thiruchelvam et al., 2000). Previous study demonstrated that paraquat preferentially acts on nigrostriatal dopaminergic neurons in the brain due to similar structure with + MPP (Shimizu et al., 2001). Increased neurotoxicity of paraquat is observed when co-administrated with maneb (Choi and Xia, 2014; Zhou et al., 2004). Moreover, rodents intoxicated with combined paraquat and maneb displayed several important features of PD, including nigral dopaminergic neurodegeneration, α-synuclein aggregation and motor deficits, which represent early events in the pathogenesis of PD in humans and may be used for the etiological and therapeutic studies of PD (Dixit et al., 2013; Norris et al., 2007; Uversky, 2004).
Although the exact molecular mechanisms of PD remain unclear, chronic neuroinflammation has long been identified as a key contributor to the progressive neurodegeneration in neurological disorders (Hou et al., 2020a; Wang et al., 2020). Microglial activation is one of the hallmarks of neuroinflammation (Barnum and Tansey, 2010). Lots of activated microglia are detected in the SN of PD patients and human with selfadministered MPTP as well as experimental rodent PD models (Hou et al., 2020a; Imamura et al., 2003). Furthermore, high levels of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) are reported in the plasma of PD patients compared with individuals without PD (Menza et al., 2010). Recently, we and others have demonstrated that in addition to causing dopaminergic neurodegeneration, microglial activation can also be signaled by damaged neurons, resulting in reactive microgliosis and additional neuron death (Hou et al., 2019a). This vicious cycle formed between dysregulated microglia and injured neurons may underlay the progressive nature of neurodegeneration in PD (Gao and Hong, 2008; Wang et al., 2014). Interrupting the neurotoxic vicious cycle through inactivation of microglia might represent an effective strategy for the treatment of PD.
The NLRP3 inflammasome, an intracellular multiprotein complex, is composed of NLRP3 scaffold, apoptosis-associated speck-like protein (ASC) adaptor, and procaspase-1 (Abderrazak et al., 2015). It has been reported that NLRP3 inflammasome can be activated by pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), leading to caspase-1 activation and subsequent production of IL-1β and other cytokines (Hong et al., 2019; Swanson et al., 2019; He et al., 2016). Recent studies revealed an important role of NLRP3 inflammasome in neuroinflammation and related dopaminergic neurotoxicity in PD. Codolo et al. reported that α-synuclein, the main component of PD characteristic inclusions, was able to induce activation of NLRP3 inflammasome to produce IL-1β in human monocytes (Codolo et al., 2013). Furthermore, elevated levels of IL-1β and IL-18 were detected in the serum and cerebrospinal fluid (CSF) of PD patients (Dursun et al., 2015; Mogi et al., 1996; Zhang et al., 2016). Additionally, MPTP, 6-OH-DA and rotenone, three PD-related toxins, had all been shown to be able to activate NLRP3 inflammasome and amplify IL1β production in both microglial cells and experimental animals (Mao et al., 2017; Zhou et al., 2016; Liu et al., 2017). Moreover, genetic deletion of caspase-1 alleviated microglial activation, IL-1β production and dopaminergic neurodegeneration (Qiao et al., 2016). These findings suggested that NLRP3 inflammasome might be an upstream target for suppressing microglial activation and related neurodegeneration in PD.
As a proof of concept, we tested the neuroprotective efficacy of glibenclamide, an inhibitor of NLRP3 inflammasome in a mouse PD model induced by combined paraquat and maneb (P + M). We found that glibenclamide significantly reduced the α-synuclein upregulation, dopaminergic neurodegeneration in the nigrostriatal system and motor deficits in P + M-intoxicated mice. Mechanistically, suppression of chronic microglial activation, M1 polarization and oxidative stress through inactivation of NLRP3 inflammasome contributed to the neuroprotective effects of glibenclamide.

2. Materials and methods

2.1. Animal dosing

12 weeks old Male C57BL/6 mice were provided by Jinan Pengyue Experimental Animal Breeding Co. LTD (Jinan, China). After acclimation for 5 days, mice were randomly divided into control, P + M and P + M+Gli groups (n = 15 mice/group). Mice were intraperitoneally injected with paraquat (10 mg/kg.bw, SigmaAldrich, St. Louis, MO, USA) and maneb (30 mg/kg.bw, SigmaAldrich, St. Louis, MO, USA) for consecutive 8 weeks (twice per week). Glibenclamide (1 mg/kg.bw, Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally administrated 30 min. prior to P + M. The dosages of paraquat, maneb and glibenclamide were chosen based on previous reports (Hou et al., 2017; Zhang et al., 2014; Hou et al., 2020b). The procedures of the study were approved by Ethics Committee for Animal Experiments of School of Public Health, Shandong University.

2.2. Gait analysis

Gait performance was measured based on previous protocol (Jayabal et al., 2015). Stride length (the distance between subsequent left or right limbs) and stride distance (the distance between forelimbs or hindlimbs) were measured and analyzed.

2.3. Rotarod test

The rotarod test was performed based on previous protocol (Jurado-Arjona et al., 2019). The start speed of the rotarod system was 4 rpm, which accelerated to 40 rpm in 100 s. The maximum test time of each animal was set to 240 s. The latency to fall was measured. Three consecutive trials were performed for each mouse with 30 min. interval. The mean latency for the three trails was used for the analysis.

2.4. Immunohistochemistry

Whole brains of mice were removed, fixed in 4% paraformaldehyde, and serially frozen sections (30 mm) were prepared. Immunohistochemistry was performed in brain sections in each group as described previously (Wang et al., 2015, 2014; Hou et al., 2017). Sections were incubated with 1% H2O2, followed by blocking solution (0.4 % Triton X-100, 1% BSA, 4% horse or goat serum) for 10 min. And then, sections were washed and were placed into primary antibody solution overnight at 4℃. The primary antibodies include TH (1:4,000; EMD Millipore, Temecula, CA, USA), Iba-1 (1:5,000; Wako Chemicals, Richmond, VA, USA) and ITGAM (CD11b; 1:200; AbD Serotec, Raleigh, NC, USA). The next day, brain sections were incubated with appropriate secondary antibody and the immunostaining was visualized by 3,3′-diaminobenzidine (DAB).

2.5. Cell counts

The TH-immunoreactive (THir) cells in the SNpc region was visually quantified under a microscope. According to the anatomical atlas, the boundary of SN was outlined under the magnification of 10 objective lens. Eight sections selected every forth section from a total 24 sections/brain were used for the quantification of THir cells based on previous reports (Wang et al., 2014; Zhang et al., 2004). The amount of THir cells were counted individually by two pathologists and any disagreement was resolved by consensus after joint review.

2.6. Western blot analysis

Sample tissues were homogenized in extraction buffer (pH 7.4, 10 mM 2-hydroxyethyl, 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.05 % NP-40, 5 mM sodium fluoride, 1 mM sodium vanadate, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride and 1% protease inhibitor) and then were centrifuged at 12,000 g at 4℃ for 10 min. The supernatants were collected and the protein concentrations were quantified using the BCA assay kit (Pierce Biotechnology, USA). Equal amount of protein sample was heated at 100℃ for 10 min. and then was loaded to the SDS-PAGE gel, followed by transfer to the nitrocellulose membrane. The membranes were incubated with anti-NLRP3, anti-4HNE, anti-caspase-1, anti-IL-1β, anti-NF-kB, anti-p-NF-kB (Cell Signal Technology, Beverly, MA, USA), anti-α-synuclein (phosphor S129) (Abcam Cambridge, USA), anti-p47phox, or anti-gp91phox antibodies (BD Transduction Laboratories, San Jose, CA, USA) at 4 C overnight and then followed by HRP-linked secondary antibody for 2 h. Antibodies against GAPDH and β-actin (Sigma-Aldrich, St. Louis, MO, USA) were included as loading controls.

2.7. Real-time PCR analysis

Total RNA was isolated from the midbrain tissues using Ambion1 TRIzol Reagent (Invitrogen, USA). The concentrationTM and purity of the extracted RNA were measured by NanoDrop 2000c spectrophotometer (Thermo Scientific Inc., USA) and the 260/280 ratio was between 1.8–2.0 to ensure the samples were not contaminated by proteins or organics. Complementary DNA (cDNA) was synthesized from RNA samples using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc, Japan). Real-time PCR was conducted using the SYBR1 Premix EX TaqTM (Tli RNaseH Plus) (Takara Bio Inc, Japan) on Roche LightCycler480 Ⅱ Real-Time PCR system with the cycling conditions of initial denaturation at 95℃ for 30 s, PCR of 40 cycles at 95℃ for 5 s and 60℃ for 30 s, followed by melting step at 95℃ for 5 s, 60℃ for 1 min and continuous 95℃ for 1 cycle, respectively. Each specific primer pairs are listed in Table 1. The mRNA levels of all genes were normalized to GAPDH (Wang et al., 2014; Hou et al., 2017).

2.8. ROS measurement

DCFH-DA was used to detect the content of ROS in mouse brain. The tissue homogenates were prepared in a 1:10 (w/v) homogenization system on ice. A final concentration of 5 mM of DCFH-DA was used in the reaction system, including 25 ml tissue homogenate, 1.45 mL PBS and 30 ml of fluorescent dye. The reaction system was incubated at 37 C for 15 min. and then was centrifuged at 12,500 g for 10 min. Five milliliter PBS was used to re-suspend the pellet and then was placed at 37 C for 1 h. The fluorescence intensity was detected at 488 nm excitation wavelength and an absorption wavelength of 525 nm. The standard curve was used to calculate the ROS content of each sample.

2.9. Statistical analysis

Data were presented as mean SEM and were analyzed by SPSS 20.0 software. Differences among means of rotarod test were analyzed using repeated measures analysis of variance. Others were analyzed by one-way ANOVA with treatment as the independent factors. When ANOVA showed significant differences, Tukey’s post hoc testing was used for multiple comparisons. A p value less than 0.05 was believed statistically significant. 3. Results

3.1. Glibenclamide ameliorated P + M-induced degeneration of dopaminergic neurons

To explore whether glibenclamide was able to protect dopaminergic neurons in PD, P + M-intoxicated mice were administrated with glibenclamide and dopaminergic neurons in the SN and striatum were stained with antibody against TH. Consistent with previous report (Che et al., 2018; Hou et al., 2018b), P + M exposure resulted in dopaminergic neurodegeneration in the SNpc as shown by 31.4 % decrease of THir cells compared with control. P + M-induced loss of dopaminergic neuron was significantly ameliorated by glibenclamide. Consistently, P + M-induced degeneration of striatal fibers of dopaminergic neuron was also mitigated by glibenclamide as shown by elevated expressions of TH in the striatum of mice treated with combined glibenclamide compared to P + M alone (Fig. 1A and B). There is no significant difference of nigral THir cell number and striatal TH expression between glibenclamide alone and control groups (Fig. 1A–C).
The phosphorylation of α-synuclein at Ser129 site is essential for its aggregation and neurotoxicity (Chen and Feany, 2005). We therefore determined the effects of glibenclamide on P + Minduced Ser129-phosphorylation of α-synuclein in mice. Results revealed significant elevation in α-synuclein (phospho S129) expressions in P + M-treated mice. Glibenclamide significantly mitigated P + M-induced α-synuclein (phospho S129) upregulation (Fig. 1D).

3.2. Glibenclamide improved motor function in mice intoxicated with P + M

To determine whether glibenclamide-elicited protection on dopaminergic neurons was associated with functional improvement, the rotarod activity and gait disturbance of mice were determined. We found that glibenclamide markedly attenuated the reduction of rotarod activity induced by paraquat and maneb (Fig. 2A). In agreement with rotarod activity test, glibenclamide also alleviated gait abnormality in P + M-intoxicated mice. Quantification of footprint revealed a longer stride length of hindlimb in glibenclamide co-treated mice than in P + M alone group (Fig. 2B and C). The enlarged stride distance in P + M-intoxicated mice was also attenuated by glibenclamide (Fig. 2D).

3.3. Glibenclamide inhibited the activation of NLRP3 inflammasome induced by P + M

To explore whether inactivation of NLRP3 inflammasome contributed to the neuroprotective effects of glibenclamide, we compared the status of NLRP3 inflammasome activation between P + M alone and glibenclamide co-treated mice. Western blot analysis of midbrain homogenates revealed elevated expressions of NLRP3, cleaved caspase-1 and mature IL-1β in mice intoxicated with P + M compared with vehicle, indicating activation of NLRP3 inflammasome (Fig. 3A–C). Interestingly, glibenclamide treatment mitigated P + M-induced activation of NLRP3 inflammasome by showing reduced levels of NLRP3, cleaved caspase-1 and mature IL-1β in glibenclamide co-treated mice compared with P + M alone group (Fig. 3A–C).

3.4. Glibenclamide mitigated P + M-induced proinflammatory microglial activation

To investigate whether the neuroprotective effects of glibenclamide were related to suppression of microglial activation, we determined the effects of glibenclamide on P + M-induced activation of microglia. Microglia in the SN were stained with two microglial markers, Iba-1 and CD11b. As illustrated in Fig. 4A, microglia in P + M-intoxicated mice exhibited hypertrophied morphology and increased density of Iba-1 and CD11b staining, indicating microglial activation. Morphological observation and quantitative analysis revealed that glibenclamide suppressed P + M-induced microglial activation by showing ramified microglial morphology and reduced Iba-1 and CD11b expression in glibenclamide co-treated mice compared with P + M alone group (Fig. 4A and B).
Activated microglia can be classified into two subgroups, i.e., detrimental M1 and beneficial M2 phenotype (Hou et al., 2019a, b). To further demonstrate the effects of glibenclamide on microglial phenotype, the gene transcripts of M1 and M2 markers were determined. As seen in Fig. 4C, P + M intoxication significantly elevated the mRNA levels of iNOS, TNFα and IL-1β, three microglial M1 markers, in midbrains of mice. A similar increase of microglial M2 marker, elevated mRNA level of Arg-1 was also observed in P + M-intoxicated mice (Fig. 4C), although no significant difference of CD206 and Ym-1, the other two markers for M2 microglia, was detected between P + M and vehicle groups. However, P + Minduced elevation of iNOS and TNFα mRNA levels was markedly attenuated by glibenclamide (Fig. 4C), indicating that glibenclamide significantly abrogated microglial M1 activation in P + Mtreated mice.

3.5. Glibenclamide abrogated P + M-induced NF-kB activation

Due to the important role NF-kB pathway in microglial activation and M1 polarization (Yao and Zu, 2020), we explored the effects of glibenclamide on the activation of NF-kB pathway. As shown in Fig. 5, compared with vehicle, P + M intoxication led to elevation of the levels of both phosphorylated NF-kB and total NF-kB in midbrain of mice. Interestingly, the elevation of phosphorylated NF-kB but not total NF-kB in P + M-intoxicated mice was abrogated by glibenclamide, indicating suppression of NF-kB pathway.

3.6. Glibenclamide suppressed P + M-induced oxidative damage

Oxidative stress is usually associated with neuroinflammation, collaterally resulting in neurodegeneration (Wang et al., 2015; Hou et al., 2020a; Bermudez et al., 2016). We therefore determined the production of ROS in mice treated with P + M with or without glibenclamide by using DCFH-DA, a widely used probe for ROS detection. As shown in Fig. 6A, exposure to P + M resulted in increase of ROS production, which was significantly inhibited by glibenclamide. In agreement with inhibition of ROS production, the elevated levels of 4-hydroxynonenal (4-HNE), an unsaturated aldehydes derived from lipid hydroperoxidase (Uchida, 2003), in P + M-intoxicated mice were also blocked by glibenclamide (Fig. 6B).
Previous studies demonstrated that NOX2 and iNOS are two critical enzymes for both neuroinflammation and oxidative damage (Mander and Brown, 2005; Gao et al., 2011). The indicating that blocking NOX2 and iNOS might, at least partly, contribute to anti-inflammatory and anti-oxidative capacity of glibenclamide.

4. Discussion

This study demonstrated that glibenclamide, an NLRP3 inhibitor, exhibited potent dopaminergic neuroprotection and improved motor performance of mice against P + M-induced toxicity. Glibenclamide significantly inhibited M1 polarization of microglia and the production of pro-inflammatory cytokines. Mitigated oxidative damage was also found in glibenclamide and P + M co-treated mice. Furthermore, NF-kB, NOX2 and iNOS were identified to be critical for the inhibitory effects of glibenclamide against neuroinflammation and oxidative stress.
A wealth of evidence supports that the activation of NLRP3 inflammasome is detrimental in neurological diseases (Welberg, 2014). In brains of Alzheimer’s disease (AD) patients, the expression of active caspase-1 was significantly increased (Guan and Han, 2020). Genetic deletion of NLRP3 or caspase-1 in APP/ PS1 mice significantly decreased deposition of amyloid-β and improved the cognitive function of mice (Heneka et al., 2013; Ju et al., 2020). Likewise, NLRP3 inflammasome activation was also detected in the serum of PD patients and the midbrains of PD model mice (Zhou et al., 2016; Bu et al., 2015). Therefore, NLRP3 inflammasome inactivation should be beneficial for combating neurodegenerative diseases including PD. NLRP3 inflammasome activation was observed in midbrain of mice treated with P + M. Furthermore, we found inactivation of NLRP3 inflammasome by glibenclamide exhibited potent inhibitory effects on P + Minduced dopaminergic neurotoxicity. In agreement with our findings, Mao et al. demonstrated that casepase-1 inhibitor, AcYVAD-CMK, suppressed the expression of NLRP3 inflammasome components in LPS- and 6-OHDA-intoxicated rats, which was accompanied by reduced number of rotations and dopaminergic neurodegeneration in the SN (Mao et al., 2017). Recently, neuroprotection via NLRP3 inflammasome inactivation were also verified in experimental animals suffering cerebral ischemia/ reperfusion injury (He et al., 2017), intracerebral hemorrhage (Cheng et al., 2017) and n-hexane exposure (Hou et al., 2020c).
The present study indicated that the neuroprotection elicited by inhibition of NLRP3 inflammasome might be ascribed to suppression of neurotoxic M1 microglial activation and oxidative stress. This is in line with previous findings from other neurological disease. Huang et al. reported that Salvianolic acid B abolished depression through suppression of oxidative stress and neuroinflammation via inhibiting NLRP3 inflammasome activation in chronic mild stress-treated rats (Huang et al., 2019). In an ischemic stroke model, the activation of NLRP3 inflammasome exaggerated microglial M1 activation and inactivation of NLRP3 inflammasome by NOSH-NBP switches microglial phenotype from M1 to M2 (Ji et al., 2017). Furthermore, the regulation of NLRP3 inflammasome activation on microglial M1 polarization was observed in APP/PS1 mouse model of AD since genetic ablation of NLRP3 or caspase-1 gene mitigated M1 microglial inflammatory responses (Heneka et al., 2013). In this study, P + M exposure in mice resulted in elevation of both M1 and M2 activated microglia. Glibenclamide treatment significantly mitigated M1 activated microglia in P + Mtreated mice, suggesting that NLRP3 inflammasome inactivation was beneficial to the balance of M1 and M2 microglial states. However, present results showed that the Arg-1 mRNA level of P + M-treated mice was higher than the value of glibenclamide cotreated mice. It is accepted that M2 activated microglia are further classified into M2a, M2b and M2c subtypes. M2a is a healing phase with Arg-1, CD206 and Ym-1 as markers; M2b phase exhibits high levels of immune response characterized by the production of the pro-inflammatory cytokines TNFα and IL-6, while M2c acts as a clamp on pro-inflammatory immune response. Microglial activation is dynamic and mixed processes, in which phenotypes and states switches over time by various pro- and anti-inflammatory drivers (Bell-Temin et al., 2015). In addition, large numbers of peripheral monocytes can be recruited and infiltrated into the brain during neuroinflammation, M2 macrophage is associated with the elevation of Arg-1 expression (Dey and Hankey Giblin, 2018). Thus, the conflicting data might be ascribed to the difference in targets of glibenclamide and diversity of microglia activation.
Mechanistic studies revealed the involvement of multiple molecules including NF-kB, NOX2 and iNOS in the antiinflammatory and anti-oxidative effects of glibenclamide. NFkB, an important transcription factor, is critical for gene transcriptions of a variety of proinflammatory genes. M1 microglial classic stimulators, such as LPS and IFN-g, are shown to stimulate activation of NF-kB and then increase expressions of M1 microglial markers, including iNOS, CD86 and MHC-II (Orihuela et al., 2016). INOS and NOX2 are major free radical– generating enzymes in activated microglia and play important roles in controlling microglial activation and oxidative stress. Genetic deletion or pharmacological inhibition of iNOS suppressed neuroinflammation and oxidative damage in multiple models of PD (Gao et al., 2011; Liberatore et al., 1999). NOX2 might also play an essential role in neuroinflammation mediated by microglia, as inactivation of NOX2 attenuated M1 microglial response in mice treated with LPS, MPTP, paraquat or maneb (Wang et al., 2015; Hou et al., 2020a; Wang et al., 2014; Hou et al., 2019b). The regulatory effects of NOX2 on neurotoxic microglial M1 activation might be related to the activation of NF-kB via increasing intracellular ROS level. Taetzsch et al. found that H2O2 derived from NOX2 was able to amplify microglial M1 activation by acting on p50 NF-kB (Taetzsch et al., 2015). In the present study, inactivation of NLRP3 inflammasome by glibenclamide mitigated NF-kB activation and expressions of NOX2 and iNOS in P + M-treated mice. Currently, it is not clear how NLRP3 inflammasome regulates activation of NF-kB, NOX2 and iNOS and therefore affects microglial M1 polarization. Further studies should be warranted to investigate the mechanisms of how NLRP3 inflammasome regulates activation of NF-kB, NOX2 and iNOS.
Glibenclamide is a hypoglycemic medication used to treat type 2 Diabetes Mellitus primarily by acting on the ATP-sensitive potassium channels (KATP) (Shepherd et al., 2017). KATP channels are abundantly distributed in SN of brain, and are considered to play an important role in maintaining the membrane potential and mitochondrial matrix volume during ATP decline. Thus, the effects of KATP channels in the development of PD induced by mitochondrial-directed neurotoxicants such MPTP and rotenone had been widely investigated, but the results were quite inconsistent. For example, Kou et al. demonstrated that daily intraperitoneal injection (i.p.) of 30 mg/kg glibenclamide to C57BL/6 mice for 2 weeks significantly potentiated the reduction of striatal dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins induced by a single dose of 20 mg/kg of MPTP treatment (Kou et al., 2006). Contrarily, Abdelkader et al. demonstrated that 3 mg/kg glibenclamide (i.p.) for 3 consecutive weeks exhibited evidently neuroprotective effects in rotenoneinduced PD mouse model, possibly by anti-inflammatory and antiapoptotic effects (Abdelkader et al., 2020). The varied effects of glibenclamide on different neurotoxins might be ascribed to the different doses employed in different studies. The hypoglycemic activity induced by 30 mg/kg glibenclamide might potentiate the neurotoxicity in MPTP-intoxicated mice model, while glibenclamide of nonhypoglycemic doses might primarily exhibit the antiinflammatory and anti-oxidation effects. Indeed, recent study showed that 5 mg/kg glibenclamide treatment for 8 weeks, significantly suppressed neuroinflammation in 5XFAD transgenic mice by regulating microglial activity (Ju et al., 2020). In another study, mice treated orally with 5 mg/kg glibenclamide for consecutive 21 days effectively prevented the emotional and cognitive defects induced by chronic stress, without any effects on serum glucose levels and neurobehavioral performance in glibenclamide alone treated mice (Rosado et al., 2021).
Taken together, our study demonstrated that the inactivation NLRP3 inflammasome by glibenclamide conferred neuroprotective effects in a mouse PD model induced by paraquat and maneb. Inhibition of microglial M1 polarization and oxidative stress, at least partly, contributed to the neuroprotective effect of glibenclamide. These results suggest the important roles of NLRP3 inflammasome in dopaminergic neurodegeneration in PD and the therapeutic potential of glibenclamide on PD treatment.

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