Correction: Edaravone plays protective effects on LPS-induced microglia by switching M1/M2 phenotypes and regulating NLRP3 inflammasome activation
Year: 2026
Type: erratum
Abstract: Parkinson's disease (PD) is an age-related neurodegenerative disease around the world, whose pathogenesis is still unclear (Zou et al., 2015). Clinical signs include progressive dyskinesia with phenotypic severity, resting tremor, rigidity and bradykinesia, and may be accompanied with gastrointestinal dysfunction (Jankovic, 2008;Skjaerbaek, 2021). Classical pathological feature is the progressive loss of dopaminergic (DAergic) neurons within the nigrostriatal tract, related to decreased dopamine content in the striatum, which exacerbates motor function damage of patients. The prevalence of PD are unknown although the incidence is similar between Europeans and Han Chinese populations (Von Campenhausen et al., 2005;De Lau and Breteler, 2006). According to the clinical and etiological overlaps between PD and inflammation, recent preclinical researches have indicated that there are strong correlations between activated microglia and the progression of PD symptoms.Dramatically, postmortem studies in humans have shown the relationships between activated microglia and degeneration of dopaminergic neurons in nigra (Overmyer et al., 1999). However, once the stimuli occur in the brain, major innate immune cells in the brain will be activated and infiltrated locally to maintain homoeostasis of the lesion. Activated microglia are usually divided into two polarization states, termed "M1" (proinflammatory) phenotype and "M2" (anti-inflammatory) phenotype. In addition, in patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, the proportion of M1-polarized microglia increased. For simplicity, activated M1-type (classical) and activated M2-type (alternative) microglia models have has been widely used in vitro. Generally, M1-phenotype microglia are considered to have pro-inflammatory activity by increasing production of inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), leading to tissue damage.On the contrary, M2-phenotype microglia exert a neuroprotective effect by upregulating anti-inflammatory mediators and suppressing the pro-inflammatory response. Activated microglia can coexist as single or mixed phenotype, which reflects the complexity of microglia function and the dynamic changes of the environment in vivo (Sica and Mantovani, 2012).Increasing studies demonstrated that the release of neurotoxic factors of microglia might be associated with the NLRP3 Inflammasome (Gustin et al., 2015).The NOD-like receptor family, pyrin domain containing-3 protein (NLRP3) is a multi-protein complex that can regulate the maturation and secretion of the proinflammatory cytokines, including . Existing studies have demonstrated that inhibition of the NLRP3 Inflammasome activation could protect dopaminergic neurons (Gao et al., 2002;Gustin et al., 2015;Sarkar et al., 2017;Abas et al., 2018). Based on this theory, researchers are extensively seeking for new compounds to inhibit the activation of NLRP3 Inflammasome for therapeutic intervention against neurodegeneration in PD. Epidemiological studies have shown that chronic patients who use anti-inflammatory drugs have a low risk of developing idiopathic PD (Honglei Chen et al., 2003;Esposito et al., 2007). Since the neuroinflammation play an important role in the pathogenesis of PD, anti-inflammatory drugs are considered as candidates for the treatment of PD (Faust et al., 2009;Wang et al., 2017a).Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, EDA, Figure 1) is an ROS scavenger with a variety of biological activities such as anti-oxidative Suehiro et al. (2018), anti-inflammatory Zhang et al. (2019) and anti-tumor (Suehiro et al., 2018). Previous studies suggested that EDA could protect normal human bronchial epithelium from 6-OHDA-induced injury and improve motor function of rats through its anti-inflammation effect (Yuan et al., 2008). However, whether EDA can protect LPS-induced PD through mediating NLRP3 Inflammasome remains to be investigated. Based on the anti-inflammatory effect of EDA, we scrutinized whether EDA could inhibit microglia activation and alleviate dopaminergic neurodegeneration in PD models in vivo and in vitro.Previous study had showed that EDA could attenuate amyloid-β-induced proinflammatory response by inhibiting NLRP3 Inflammasome (Moehle and West, 2014). EDA has also been reported to have a therapeutic effect on retinal injury through regulating nuclear factor κB (NF-κB) signaling pathways in diabetic rats (Wan et al., 2019). Interestingly, EDA also could cross the blood-brain barrier (BBB) (Fang et al., 2014) and maintain integrity of BBB by activating the NFE2related factor 2 signaling pathway (Liu et al., 2019). However, there is currently no such evidence for the role of EDA between microglia and dopaminergic neurons in inflammation.The current study aims to identify the role of EDA in microglial polarization in PD, and to provide a basis for microglial-based diagnostic and therapeutic strategies. Specifically, these findings might give an insight into a potential therapeutic target for treating PD.Edaravone (≥ 99%) and lipopolysaccharide were purchased from Sigma-Aldrich (St.Louis, MO, United States). Primary antibodies against tyrosine hydroxylase, Iba-1, NLRP3, caspase-1, ASC, β-actin, Rabbit IgG and mouse IgG antibodies were bought from Proteintech Group (Chicago, IL, United States). Primary antibodies anti-Arg-1, anti-TNF-αand anti-CD11b/c were supplied by Abcam (Cambridge, MA, United States). MTT assay kit was obtained from Beijing Solarbio science and Technology Co., Ltd. (Beijing, China). Enzyme-linked Immunosorbant Assay (ELISA) for TNF-α, IL-1β and IL-18 were bought from Elabscience Biotechnology Co., Ltd. (Wuhan, China).Dulbecco's modified eagle medium (DMEM) and OPTI-MEM were provided by Gibco-BRL (Invitrogen Life Technologies, Carlsbad, United States). The small interfering RNA (siRNA) against NLRP3 was purchased from GenePharma (Shanghai, China).A total of 50 adult male Sprague-Dawley rats (200-230 g) were purchased from the Experimental Animal Center of the Nanjing Medical University (Nanjing, China; Specific pathogen-free Grade II; Certificate No. SCXK 2018-0011). All the animals were housed in a temperature (25 ± 2°C), light (12 h of daylight, 12 h of darkness) and humidity (60 ± 4%) environment. Adequate food and water were provided and available at any time. Rats were allowed to adapt to the environment for 1 week before experiments.All the animals were randomly allocated to five experimental groups (n = 10): Control (0.9% NaCl), EDA alone (10 mg/kg), LPS-model (LPS 5 μg), LPS + EDA (5 mg/kg) and LPS + EDA (10 mg/kg). DA neuronal damage was induced by receiving unilateral injection of LPS (5 μg) into the SN pars compacts, and the coordinate position as described in previous studies (Fang et al., 2014). After daily intragastric administration of EDA for 7 days, behaviors of rats were analyzed by rotarod test, pole test, and traction test. Then all animals devoted their lives and tissues were collected for subsequent experiments.BV-2 cells, a murine microglia cell line, were purchased from Wuhan University Cell Library (Wuhan, China). BV-2 cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a incubator with 5% CO2 at 37°C. BV-2 cells were pretreated with different doses of EDA, and 1 h later, LPS was added to the medium for 24 h. Then, the culture medium and protein were used for later experiments.BV-2 cells were seeded into 96-well plates at a density of 2 × 104/ ml for 24 h, then the supernatant was replaced by new medium containing 2% FBS, and the cells were exposed to LPS with or without EDA for another 24 h. After that, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was added for 4 h to verify cell viability. Using the microplate reader to measure the optical density values at 490 nm.According to the manufacturer's instructions, the levels of TNF-α, IL-1β and IL-18 were measured by ELISA (R&D Systems. United States). The microplate reader (Sunnyvale, CA, United States) was used to assay the absorbance at 450 nm, And the experiment was repeated at least three times.The total protein content was extracted from midbrain tissue and BV2 cells by using a lysis buffer containing protease inhibitors after washing with PBS three times. The protein concentration was measured with the BCA protein assay kit (Solarbio, pc0020).Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TBS (Solarbio) with Tween 20 (Sigma, P9416) and 5% skim milk for 2 h. Then, the PVDF was incubated with the following primary antibodies overnight at 4°C: TH (1:1,000), IBA-1 (1:800), NLRP3 (1:1,000), ASC (1:400), caspase-1 (1:800), Arg-1 (1:1,000), β-actin (1:2,000). After washing with TBST, the membrane was incubated with horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG (1: 2,000) for 1 h at room temperature. At last, the PVDF was treated with enhanced ECL reagent and protein bands were visualized on X-ray film utilizing enhance chemiluminescence system.Culture medium was removed and the slices were treated with 0.3% Triton X-100 for 30 min to fix. Then, the cells/tissues were orderly blocked with goat serum for 2 h, incubated with primary antibodies overnight at 4°C: Iba-1 (1:1,000), OX-42 (1:800), NLRP3 (1:800), and incubated with fluorescent-conjugated secondary antibody for 1 h.An Olympus microscope (Olympusr, Tokyo, Japan) was used to capture the Digital images of SN TH-positive neurons.Total RNA was extracted with trizol agent and purified with RNeasy kit. Iba-1, TNF-α, IL-1β, Arg-1, IL-10, and β-actin genes were amplified by using the forward and reverse primers. Real-time PCR was performed using a SYBR Green Supermix according to the instruction and then determined on a CFX96 real-time PCR detection system (Bio-Rad, CA, United States). The data analysis software provided with the system was used to normalize the target gene expression levels comparing with β-actin expression.BV-2 cells were chosen as an experimental model and transfected with siRNA oligos against NLRP3 (GenePharma, China). Cells were seeded in 24-well plates for 24 h. The mixture of RNA oligo and GP-siRNA-Mate Plus was used for siRNAs transfection.After 6 h of treatment, DMEM medium was instead of the transfection solution for an additional 24 h before subsequent test.Motor performance of rats was assessed by using a rotarod apparatus (RotaRod system).First, the rats were trained to adapt at a speed of 10 rpm/min before the experiment.After intragastric administration of EDA for 7 days, record the time of each rat spent on the rotary, with speed accelerating from 10 to 30 rpm in 5 min. Remove the rats which remain on the apparatus after 5 min and score their time as 5 min. Calculate rotation time for data analysis.To measure the bradykinesia for mice induced by LPS, a wooden pole (50 cm in length) with a ball of 1.25 cm radius placed on top was used in the pole test to determine climbing time for mice. Briefly, mice were placed on the top of a pole with a rough surface, and the different time taken by each mouse to get down completely was recorded. Results were repeated when mice stopped or climbed to the reverse direction.Each mouse was re-tested for three times one day and recorded as previously described.To evaluate muscle strength and equilibrium, A horizontal rope with a distance of 30 cm was used as described previously for determining the suspension time for mice. The time hanging on the wire was recorded and scored as the following criteria: 0-4s recorded as 0 point; 5-9 s recorded as 1 point; 10-14 s recorded as 2 point; 15-19 s recorded as 3 point; 20-24 s recorded as 4 point; 25-29 s recorded as 5 point, and above 30 s recorded as 6 point. The average of the score was calculated after performing daily in triplicate.Unless otherwise stated, all the data values represent means ± SEM. One-way analysis of variance was used to evaluate the significant differences between groups, and statistical analysis was performed by using the software Graphpad Prism (version 5.01).A value of p < 0.05 was viewed as statistically significant.In order to verify the degree of motor dysfunction in LPS-induced DA neuron damage, a significant clinical symptoms of PD patients, the behavior of rats was analyzed by rotarod test, pole test and traction test after intragastric administration of EDA for 7 days. As shown in Figures 2A-C, LPS-induced PD model rats showed an obvious increase in locomotor deficiency compared with control rats in behavioral tests. At the same time, EDA significantly upregulated the time spent on the rod of LPS-induced rats. TH, a marker of TH+ dopaminergic neurons, acts as a rate-limiting enzyme in DA synthesis. Dopaminergic neurons survival in the SN were characterized by densitometric analysis. As shown in Figures 2D,E, there was no significant difference in the activity of DA neurons between the control group and EDA alone group, suggesting that EDA alone treatment had no effect on DA neurons. EDA ameliorated LPS-induced DA neuron loss in a dose-dependent manner, compared with LPS group.To further validate the neuroprotection effect of EDA on DA, the LPS-administrated protein expression of nigra TH was identified. As shown in Figure 2F, LPS reduced TH protein expression compared with control group and this reduction was weakened by EDA treatment.To observe the effect of EDA on polarization of LPS-induced microglia, immunofluorescence analysis was used to assay the expression of Argnise-1 (Arg-1).As shown in Figure 3A, the protein expression of Arg-1 was located in activated microglia and EDA could enhanced LPS-induced microglial activation. Notably, strong TNF-α and less Arg-1 mRNA expressed were detected in LPS-treated rats. After EDA treatment, the mRNA expression of M1 markers TNF-α and IL-1β decreased, while the expressions of M2 markers of anti-inflammatory cytokines Arg-1 and IL-10 were enhanced shown in Figures 3B-E.To further identify the regulatory mechanism of EDA on activation of microglia, BV-2 cell line was employed to investigate the improvement effect of EDA on LPS-induced microglial activation in vitro. BV-2 cell was treated with different dose of EDA or/and LPS. First, MTT assay was applied to confirm the optimal concentration of EDA and LPS for subsequent experiments (Figures 4A-C), and the results indicated that the concentration of EDA (50 mM) and LPS (100 ng/ml) had no significant effects on cytotoxicity and viability of normal BV-2 cells. Secondly, to discern the function of EDA on microglia morphology, protein Iba-1 (a specific microglial marker, including resting microglia and activated microglia) was evaluated. As indicated in Figures 4D,E, western blot analysis and RT-PCR assay revealed that EDA suppressed LPS-induced Iba-1 protein and Iba-1 mRNA expression.To explore the effect of EDA on LPS-stimulated phenotypic transformation of microglia, the expression of Arg-1 and TNF-α was detected by ELISA and RT-PCR (Figures 5A,B). The results showed that immunoreactivity of M1 marker (TNF-α) was in a high level and that of M2 marker (Arg-1) was low in LPS-treated cultures. However, TNF-α and Arg-1 expressions were significantly reversed in the EDA groups. This result suggested that EDA shifted the inflammatory M1 phenotype to the antiinflammatory M2 phenotype.The production of TNF-α, IL-10 and IL-1β in the cell supernatant were measured by ELISA, to assess the inhibitory actions of EDA on inflammatory cytokines expression and polarization of LPS-induced microglia. Then, the regulatory abilities of EDA on the production of M1 pro-inflammatory cytokines (TNF-α and IL-1β) and M2 anti-inflammatory cytokine were measured. As shown in Figures 5C-E, EDA reduced LPS-induced production of TNF-α, IL-1β in medium, while increased the production of IL-10 simultaneously.In order to investigate whether NLRP3 inflammasome was involved in the modulation of EDA in neuroinflammatory responses, the protein levels of NLRP3 were measured by immunofluorescence. As shown in Figure 6A, LPS significantly induced NLRP3 activation. Similarly, EDA inhibited LPS-induced increased expression of NLRP3, caspase-1, ASC proteins observe the between NLRP3 inflammasome and microglial NLRP3 siRNA siRNA a of NLRP3 gene was applied in subsequent The of NLRP3 expression with siRNA was by protein detection in BV2 cells and MTT assay indicated that NLRP3 siRNA cell viability In addition, NLRP3 siRNA significantly reduced the production of pro-inflammatory factors compared with LPS-treated (Figures the role of NLRP3 inflammasome on polarization of microglia was As indicated in Figures compared to groups, results that the of EDA on protein expression of Iba-1 and was by NLRP3 study showed that EDA could the microglia phenotype from proinflammatory M1 to anti-inflammatory M2 inhibit the neuroinflammatory response. In addition, the effect of EDA on polarization of microglia on the of NLRP3 EDA can inhibit the M1-type polarization of microglia by NLRP3 and further inhibit the inflammatory response involved by microglia. In this study suggested that EDA the polarization of microglia, and an role through inhibiting the activity of NLRP3 inflammasome the of Parkinson's disease is to be related to chronic neuroinflammation and factors et al., et al., 2019). the remain that neuroinflammation is associated with pathogenesis of has been reported that in neurodegenerative and receptor pyrin domain three activated inflammatory and microglia as as inflammatory cytokines such as IL-1β play important et al., Inflammasome is an important of the immune which has three receptor protein and caspase-1 et al., 2018). NLRP3 is an inflammatory which in microglia, and its may to diseases et al., 2021). In the of activation of microglia will to the production of IL-1β and IL-18 by NLRP3, are pro-inflammatory factors et al., Activated NLRP3 of caspase-1, then whose can IL-1β into an et al., 2019). have that mice without NLRP3 or caspase-1 are to DA neuron loss in nigra by and et al., et al., have shown that activated microglia and have increased significantly in the nigra of PD patients et al., 2019). microglia release proinflammatory and The of factors can damage to neurons. The neurons the secondary activation of microglia, which further damage the neurons. studies also suggested such as and have effects in by regulating and activation et al., et al., 2018). inhibiting the activation of microglia and inflammasome NLRP3 is a therapeutic EDA is a of have shown that EDA can inhibit the production of and microglia induced by et al., This study that EDA can inhibit the neuroinflammatory response by microglia activation. results were with previous studies that EDA by inhibiting neuroinflammation by have high and can two of proinflammatory M1 phenotype and anti-inflammatory M2 phenotype different microglia are by and will into M1 phenotype and release TNF-α, and which TNF-α is one of the markers of M1 phenotype microglia. This result in the of activated microglia the activation of M1 phenotype microglia is a response of the activation can damage neurons. In the M2 phenotype is to by IL-10 the microglia are into the M2 phenotype, and which is the marker of the M2 phenotype microglia. M2 cells anti-inflammatory factors that can M1 activation and the (Moehle and West, 2014). Based on the two of has been whether the the cytokines the from to et al., 2014). In Parkinson's disease, the M1 phenotype of microglia is and the inflammatory response is inhibiting M1 activation alone provide et al., 2007). the polarization of microglia to the M2 phenotype and the of be of for the treatment of Parkinson's to the of used anti-inflammatory drugs are in the treatment of of compounds can improve by inhibiting M1 and a compounds can the polarization of microglia to the M2 phenotype. This study showed that EDA can inhibit the expression of microglia M1 phenotype markers and the M2 polarization of microglia, the anti-inflammatory phenotype, which may to the EDA can cross the which for EDA to and system this study showed that EDA inhibited the activation of NLRP3 reduced the M1 phenotype of microglia and the cells to to the M2 all these result in an improvement in neuroinflammatory response. The current results the potential of EDA in the treatment of study that EDA microglial polarization to M2 phenotype through of NLRP3 findings provide a new evidence that EDA might have value as a agent for neuroinflammatory diseases The mRNA expression of M2 markers Arg-1 and IL-10 were were expressed as a of the control group and were the ± from rats. < compared with control < compared with LPS
Source: Frontiers in Pharmacology
Authors: Jiping Li, Xinping Dai, Liuyi Zhou, Xinxiu Li, Dongxiao Pan
Language: English
Subfield: Neurology
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
OpenAlex ID: W7155181216
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