
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00302-1
10.1016/j.redox.2024.103324
103324
Research Paper
Z-ligustilide provides a neuroprotective effect by regulating the phenotypic polarization of microglia via activating Nrf2-TrxR axis in the Parkinson's disease mouse model
Peng Shoujiao
Chen Yao
Wang Ran
Zhang Jiange jgzhang@shutcm.edu.cn
⁎
Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China
⁎ Corresponding author. jgzhang@shutcm.edu.cn
20 8 2024
10 2024
20 8 2024
76 1033244 7 2024
2 8 2024
18 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
The polarization phenotype of microglia is critical in the progression of Parkinson's disease (PD). Molecules that can polarize microglia toward the M2 phenotype represent a promising class of compounds for anti-PD medications. Z-ligustilide (ZLG) is a naturally occurring enol ester with diverse pharmacological properties, especially in neuroprotection. For the first time, we investigated the effect of ZLG on anti-PD and elucidated its underlying mechanism. The results primarily showed that ZLG attenuated motor deficits in mice and prevented the loss of dopaminergic neurons in the substantia nigra. Mechanistically, ZLG alleviates oxidative stress-induced apoptosis of microglia by triggering the endogenous antioxidant system. Besides, ZLG modulated phenotypic polarization of the microglia through the activation of the Nrf2-TrxR axis, leading to microglia polarization towards the M2 phenotype. Taken together, our research showed that ZLG is a prospective therapy candidate for PD by altering microglia polarization and restoring redox equilibrium through the Nrf2-TrxR axis.

Keywords

Nrf2
TrxR
Neuroprotection
Z-Ligustilide
Parkinson's disease
==== Body
pmc1 Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disease that affects elderly people on a global scale. Currently, PD impacts 0.3 % of the general population and 1–3% of individuals aged 65 and above, and its prevalence is projected to rise from 8.7 to 9.3 million by 2030 [1]. Typical pathology aspects of PD include the progressive death of dopaminergic neurons in the substantia nigra pars compacta and the formation of intraneuronal clumps called Lewy bodies [2]. Although the exact pathophysiology of PD remains incompletely known, several important intracellular processes have been implicated, including microglial polarization, mitochondrial dysfunction, oxidative stress, calcium accumulation, endoplasmic reticulum stress, and aggregation of misfolded proteins [3].

Microglia are immune cells that permanently stay in the brain. They perform an essential function in preserving the equilibrium of the central nervous system (CNS) [4]. There are two main types of microglial activation: classical activation, also known as the M1 phenotype, and alternative activation, also known as the M2 phenotype. M1 polarization is characterized by the secretion of proinflammatory cytokines, including TNF-, IL-6, IL-1, and IL-12. The polarization of M2 microglia is characterized by the presence of anti-inflammatory cytokines, namely IL-4 and IL-10, as well as particular markers such as Arg1, Ym1, Fizz1, and Klf4. Nevertheless, the transition from the M2-phenotype to the pro-inflammatory M1-phenotype is noticed in PD [5,6]. Generally, the transformation of physical characteristics exacerbates damage to neurons and suggests an unfavorable prognosis. Therefore, reversing the M1-to-M2 phenotypic shift in microglia is a highly promising therapeutic approach for PD [7].

Despite a limited understanding of microglial polarization mechanisms, significant evidence indicates that reactive oxygen species (ROS) play a crucial role in initiating M1 activation [8,9]. For instance, inhibiting NADPH oxidase, the primary enzyme accountable for the generation of ROS [10,11], decreases the proinflammatory M1 response. Rotenone, acting as a suppressor of the mitochondrial electron transport chain, enhances the activation of M1 by increasing ROS generation. In addition, because of enhanced anaerobic glycolysis, M1-activated microglia had higher intracellular ROS levels than M2-activated subtypes [12]. As a result, obliterating ROS is a potentially effective approach to redirect the phenotypic transformation of microglia toward the neuroprotective subtype.

Nrf2 is a crucial transcription factor in the body's natural antioxidant system, and thioredoxin reductase (TrxR) is a significant antioxidant gene regulated by Nrf2 [13,14]. TrxR is a selenoprotein that, along with its substrate Thioredoxin (Trx) and NADPH, forms the Trx system. NADPH supplies electrons to TrxR, which then catalyzes the reduction of oxidized Trx to its reduced form, known as reduced Trx [15]. Finally, reduced Trx interacts with downstream biological macromolecules to regulate and maintain redox equilibrium, cell proliferation, death, gene transcription, and other bodily activities. The Trx system is a crucial antioxidant system in the body, with TrxR serving as the primary functional protein within this system [16]. Studies have demonstrated that an excessive amount of TrxR has a protective impact on both cellular models of PD and mice models of PD. Therefore, TrxR is anticipated to emerge as a novel specific protein for the management of PD [17,18]. Collectively, the utilization of drug molecules that stimulate the Nrf2-TrxR Axis in microglia and consistently uphold redox equilibrium might enhance the M2 phenotype of microglia and ultimately provide the anti-PD effect.

Ligustilide (LG) is a primary bioactive compound derived from umbrella plants, specifically Ligusticum chuanxiong and Angelica sinensis. Its structure contains exocyclic double bonds, which distinguishes it into two isomers: (Z)-ligustilide (ZLG) and (E)-ligustilide (ELG) [19]. As a spatially prevalent conformation, the Z-type structure is considerably more stable and abundant in traditional Chinese medicine (TCM) than the E-type structure [20]. ZLG possesses a range of pharmacological activities, including antioxidant, anti-inflammatory, and neuroprotective properties [[21], [22], [23]]. Multiple studies have confirmed that ZLG could activate the Nrf2 pathway. Li et al. demonstrated that ZLG effectively inhibited the growth of acute myeloid leukaemia (AML) xenografts in mice, and Nrf2 knockout partially weakened its antitumor effect by inhibiting ferroptosis [24]. The application of related TCM (e.g., Ligusticum chuanxiong) is significant in relieving brain diseases, managing pain, and preventing cardiovascular and cerebrovascular illnesses. Additionally, it has therapeutic effects on Alzheimer's disease (AD), osteoporosis, and osteoarthritis [25,26]. Pharmacokinetic studies are crucial in the processes of discovering and developing drugs. Li et al. have reviewed the pharmacokinetic studies of ZLG, including its distribution in the brain. Results showed that after 1.2 h of administration to rats, the concentration distribution of ZLG in tissues was as follows: affected organs: cerebellum > brain > heart; clearing organs: spleen > kidney > liver. These data revealed that ZLG could pass the blood-brain barrier (BBB) and infiltrate the brain's extracellular fluid, particularly the cerebellum, implying that ZLG had a highly selective distribution in the brain [19]. Our study demonstrates, for the first time, that ZLG significantly relieved symptoms in mice with PD. Furthermore, ZLG exerted neuroprotective benefits by activating the Nrf2-TrxR axis and subsequently regulating microglial polarization.

2 Reagents and methods

2.1 Reagents

Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Hoechst 33342, 2ʹ,7ʹ-dichlorofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), NADH, 5,5ʹ-Dithiobis-2-nitrobenzoic acid (DTNB), streptomycin, and penicillin were purchased from Sigma-Aldrich (St. Louis, MO, USA). NADPH was purchased from Roche (Mannheim, Germany). Fetal bovine serum (FBS) was from HyClone. Antibodies against TrxR1, Tyrosine Hydroxylase (TH), and actin were purchased from Sangon Biotech (Shanghai, China). Antibodies against Nrf2 and CD206 were provided by Cell Signaling Technology. Antibody against CD86 was purchased by Abcam. shRNA plasmids targeting the coding regions of the control non-targeting shRNA (shNT) and the rat Nrf2 gene (shNrf2) were purchased from Sangon Biotech (Shanghai, China). GeneTran III transfection reagent was purchased from Biomiga (CA, USA). Toluidine blue solution, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 1-methyl-4-phenylpyridinium (MPP+) were purchased from Beyotime (Nantong, China). Recombinant Escherichia coli TrxR and Trx were obtained from Cayman (MI, USA). Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore. ZLG was purchased by Yuanye Biotechnology (Shanghai, China), and its chemical configuration was confirmed by 1H NMR, 13C NMR, and HRMS (Figs. S1, S2, & S3). ZLG was dissolved in DMSO and kept at - 20 °C in a 100 mM stock solution. Analytical-grade reagents were utilized in all other cases.

2.2 Animal model

Male C57/B6J mice (12 weeks, 25–27 g) were kept at room temperature under a 12-h light and 12-h dark cycle. They were separated equally into groups (n = 10/group) and given saline, MPTP (30 mg/kg, Beyotime, ST1020), ZLG (20 mg/kg, Yuanye, B25452), and l-Dopa (20 mg/kg, Yuanye, B21710). ZLG or l-Dopa was given intraperitoneally (i.p.) 1 h before MPTP injection, and drug administration lasted 5 days. Following a week of behavior evaluations, subsequent analyses were conducted by sectioning the brain tissue. The Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine has approved this research (Ethics number PZSHUTCM2304210001).

2.3 Rotarod test, pole test, gait analysis, and forelimb grip strength test

As previously described, motor functioning in rodents was assessed using the rotarod test [27]. In brief, mice were instructed to rotate at rates of 10, 12, and 15 rp once daily for 3 days. The average duration of the mouse's presence on the rod was calculated for all 3 occasions.

The pole test was conducted to assess the extent of bradykinesia. Briefly, a mouse was positioned atop a 50 cm-long and 4 mm-diameter pole, and the average time it took for the mouse to descend to the ground was measured throughout three experiments.

Mouse gait analysis was conducted using the Gaitlab system (ViewPoint, France). The mice were trained to navigate an enclosed straight tunnel with a floor made of hard glass plate. At the same time, the mice walked on the glass plate without hesitation. The footprints, stride length, and swing speed of mice were analyzed using Gait behavior tracking software (Xinruan, China).

The forelimb muscle grip strength of mice was measured by an automated grip strength meter (Sansbio, China). Briefly, gently grasp the tail of the mouse and allow the mice to hold a horizontal bar with their forelimbs. Then, mice were gradually dragged backward until their grasp gave out. The maximal force was recorded during consecutive attempts (at least 5 times/mouse), and the average was set as the result.

2.4 Immunohistochemical (IHC)

Brain tissue sections were washed in PBS and treated in 3 % H2O2 for 10 min to inhibit endogenous peroxidase activity. After being washed in PBS, the sections were immersed in 10 % goat serum (CST, USA) and then treated with 0.1 % TritonX-100 (Sigma, USA) in PBS. Subsequently, they were incubated overnight at 4 °C with the anti-TH antibody. Immunoreactivity was detected using a biotinylated goat anti-mouse secondary antibody (Beyotime, China) and diaminobenzidine (Beyotime, China). Finally, the slides were observed under an optical microscope (Nikon, Japan).

2.5 HE staining and Nissl staining

The brain tissues were created by wax embedding. Initially, the sections underwent a dewaxing process using xylene, which was performed twice for 20 min each time. This was followed by a gradient ethanol treatment, where the sections were immersed in 100 % ethanol twice for 5 min each time and then in 75 % ethanol once for 5 min. After water washing, the wax slices were stained with hematoxylin and eosin. Ultimately, whole sections were analyzed utilizing an optical microscope (Nikon, Japan).

Neuronal injury and loss were assessed using Nissl staining. Briefly, the paraffin sections were stained for 5 min with a 1 % toluidine blue solution. After a 5-min rinse with 95 % ethyl alcohol, sections were washed twice in xylene for 5 min and sealed with neutral balsam. Finally, the slides were observed under an optical microscope (Nikon, Japan).

2.6 Cell cultures, generation of stable Nrf2 knockdown cells, and luciferase reporter gene assay

Microglia BV2 cells and neuron-like rat pheochromocytoma PC12 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were regularly cultivated in a medium of 1640/DMEM with 10 % FBS, 2 mM glutamine, 100 kU/L streptomycin, and 100 kU/L penicillin. They were kept in a humidified incubator with 5 % CO2 at 37 °C.

BV2 cells (2 × 105 cells/well) were seeded in a six-well plate. Following a day of recovery, the cells were transfected with control non-targeting shRNA (shNT) and shRNA plasmids that targeted the coding regions of the mouse Nrf2 gene (shNrf2). The cells were picked after two days by adding G418 (0.5 mg/mL), and they were then cultured in full DMEM in a humidified incubator at 37 °C with 5 % CO2. Western blotting was used to assess the effectiveness of transfection.

BV2-ARE-Luc cells were cultured following our previous research protocol [28]. The cells were cultivated in a 96-well plate and subjected to ZLG or tert-butylhydroquinone (t-BHQ, a positive control) for 24 h. Subsequently, the previous culture media was discarded, and D-fluorescein sodium salt was introduced. The luciferase activity was measured using a microplate reader (Thermo, USA). The data were represented as a percentage relative to the control.

2.7 MTT assay and lactate dehydrogenase (LDH) release assay

In the MPP + model, BV2 cells (1 × 104 cells/well) were grown in 96-well plates for a day and pretreated with ZLG for 12 h. MPP+ (800 μM) was added to the cells and incubated for another 12 h. The MTT assay was used to assess cell viability, as previously mentioned in our study [29]. In a 12-well plate, BV2 cells were seeded at a density of 1 × 105 cells/well and incubated for 1 day. After 12 h of ZLG treatment, 800 μM MPP+ was added and incubated for another 12 h. The supernatant was then collected to evaluate LDH activity, as described in our earlier study [30].

2.8 Hoechst 33342 staining and estimation of caspase-3 activity

Cells at a density of 2 × 105 cells/well were cultured in a 12-well plate for 1 day, then pretreated with ZLG for 12 h. After adding 800 μM MPP+, the cells were incubated for 5 h. The samples were stained with Hoechst 33342 (5 μg/mL) for 15 min. After washing with PBS, the cells were viewed and photographed using a fluorescence microscope (Nikon, Japan).

Cells at a density of 4 × 105 cells/well were plated in 60 mm dishes for 1 day and treated with ZLG for 12 h. After 12 h of treatment with 800 μM MPP+, cells were collected and lysed with RIPA buffer. As per our published work, the lysate was taken to evaluate caspase-3 activity [31].

2.9 Annexin V/propidium iodide (PI) staining

Cells were collected and rinsed with ice-cold PBS following treatment. Then, a 500 μL solution containing PI and Annexin V/FITC (Zoman Biotech, Beijing, China) was used to resuspend the cells. The cells labeled with two markers were seen using a flow cytometer (CytoFLEX, Backman Coulter). The results were condensed using CellQuest software (CytoFLEX, Backman Coulter).

2.10 Estimation of intracellular ROS levels

Cells at a density of 1 × 105 cells/well were plated in 12-well plates for 1 day, then pretreated with ZLG for 12 h. After 5 h of incubation with 800 μM MPP+, the cells were treated with a fresh FBS-free media containing DCFH-DA (10 μM) or DHE (10 μM) for 30 min. Then, the cells were viewed and photographed with a fluorescence microscope (Nikon, Japan).

2.11 Estimation of intracellular thiols (SH) and malondialdehyde (MDA)

DTNB titration was utilized to monitor intracellular thiols [32]. In brief, following a 12-h pretreatment of the cells with ZLG followed by a 12-h treatment with 800 μM MPP+, the proteins were isolated from the cells and subsequently quantified utilizing the Bradford procedure. The measurement of total intracellular thiols was done as previously mentioned [31]. MDA level was detected based on a commercial reagent kit (Beyotime, China).

2.12 Quantitative real-time PCR

The BV2 cells, with a density of 4 × 105 cells per well, were cultured in 60 mm dishes for 24 h. Subsequently, they were exposed to 40 μM ZLG for the specified duration. The total RNA from the cells was isolated using RNAiso Plus (TaKaRa, Dalian, China), and reverse transcription was performed using the Primescript™ RT reagent kit (TaKaRa). RT-PCR analysis was conducted utilizing a Power SYBR Green PCR Master Mix and an Mx3005PRT-PCR System (Agilent Technologies). The gene primers were acquired from Sangon (China). The housekeeping gene used in this study was glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the relative mRNA levels were calculated using the 2−ΔΔCT method.

2.13 Immunofluorescence (IF)

Following the treatment of the cells with a 4 % paraformaldehyde solution, the sections were subjected to a blocking step using a 10 % goat serum for a duration of 1 h. Subsequently, the sections were subjected to overnight incubation at 4 °C with primary antibodies, followed by a 2-h incubation at room temperature with secondary antibodies. Subsequently, after being rinsed with PBS, the sections were sealed using an anti-fluorescence quenching agent containing 4,6-diamidino-2-phenylindole (DAPI). The photographs were obtained utilizing a confocal scanning microscope (Nikon, Japan).

2.14 Western blot analysis

The cells were collected and lysed after the indicated time of treatment. As stated in our published work, we produced the protein from entire cells [33]. Denatured samples containing 10 μg of protein per lane were loaded and separated using sodium dodecyl sulfate-polyacrylamide gels. Next, the proteins were deposited onto PVDF membranes and subsequently examined using particular primary and secondary antibodies. The signals were observed using a chemiluminescence kit and measured using IPP software.

2.15 Estimation of Trx and TrxR activities

To evaluate enzyme activity, the treated cells were lysed in RIPA buffer. The Bradford technique was used to determine the total protein in the cells. The cell lysate was subsequently produced to quantify the Trx and TrxR activities using the methodology outlined in our prior study [34]. Briefly, a cell extract with 20 μg of total protein was mixed with 100 mM Tris-HCl (pH 7.4), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and either 60 nM recombinant rat TrxR1 (for measuring Trx activity) or 4 μM E. coli Trx (for measuring TrxR activity). The mixture was then incubated at 37 °C for 30 min. Add 200 μL of 1 mM DTNB in 6 M guanidine hydrochloride (pH 8.0) to terminate the reaction. The same treatment was applied to a blank sample that contained all components except Trx (for TrxR assay) or TrxR (for Trx assay). The blank value was subtracted from the corresponding absorbance value of the sample, and the absorbance at 412 nm was measured. Equal quantities of DMSO were added to both the control experiments. The activity was then measured and represented as a percentage relative to the control.

2.16 Elisa kit test

The levels of IL-1β, IL-6, TNF-α, IL-4, and IL-10 were quantified using commercially available kits (Boster, China). Following the treatment, the liquid portion of the sample (or serum) was analyzed using ELISA. The ELISA assays were performed following the manufacturer's procedure. The absorbance was determined using standard curves.

2.17 Establishment of neuron-microglia co-cultured system

A transwell dish-based neuron-microglia co-culture model was used to elucidate the role of ZLG in neuroprotection. The membrane filter has a pore size of 0.4 μm, which enables intercellular communication through diffusible components without direct contact. An apical chamber was seeded with BV2 cells (5 × 103/well) that had been pre-treated with ZLG (10, 20, and 40 μM) for a duration of 12 h, while PC12 cells (1 × 104/well) were seeded in the basal chamber. After co-culturing for 12 h, the transwell dish holding both PC12 and BV2 cells was treated with MPP + for 12 h. PC12 cell viabilities were conducted with MTT assay.

2.18 Statistical analyses

The statistical significance between groups was assessed using the Student's t-test. Multiple comparisons were made using a one-way analysis of variance. Statistical significance was set at P < 0.05.

3 Results

3.1 ZLG alleviates dopamine reduction and motor deficits in the MPTP-induced PD model

The study first investigated the neuroprotective effects of ZLG on the MPTP-induced PD model in C57/B6J mice. The ZLG structure and workflow for animal experiments were illustrated in Fig. 1A & B, respectively. Before subcutaneous injection of MPTP (30 mg/kg), mice in the treatment group were injected with ZLG at a 20 mg/kg dose for 5 days. The positive control group was injected with l-dopa at a dose of 20 mg/kg, which underwent cerebral conversion into dopamine. The TH enzyme is involved in the synthesis of dopamine. IHC was performed on substantia nigra and striatum sections to analyze the impact of ZLG on the expression of TH and dopaminergic neurons. MPTP treatment was observed to reduce the quantity of TH-positive neurons in the substantia nigra and striatum. However, ZLG or l-dopa treatment reversed this phenomenon (Fig. 1C & D). In order to detect the protection of ZLG on MPTP-induced damage, HE staining was used to observe the integrity and orderliness of the substantia nigra in mice. The results indicated that MPTP promoted nuclear contraction and cell count reduction, but ZLG treatment inhibited pathological damage and restored normal cell arrangement (Fig. 1E). Nissl staining was used to assess neuronal injury and loss in the substantia nigra. As shown in Fig. 1G, MPTP triggered damage to the neurons in the substantia nigra indicated by the Nissl bodies. Meanwhile, ZLG treatment increased the number of Nissl-positive neurons and provided neuronal protection.Fig. 1 ZLG attenuated dopamine reduction and motor deficits in the MPTP-induced PD model. (A) Chemical structure of Z-ligustilide (ZLG). (B) Experimental workflow of animal treatments. (C) TH (tyrosine hydroxylase) expression in the substantia nigra and striatum was assessed by IHC. Scale bars: 500 µM. (D) Quantitative analysis of TH-positive neurons in the substantia nigra and striatum by IPP software. (E) Representative images of HE staining in the substantia nigra. (F) The rotarod test was used to assess motor function. Scale bars: 20 µM. (G) Neuronal injury and loss in the substantia nigra were assessed by Nissl staining. Scale bars: 20 µM. (H) The pole test was used to assess motor function. (I) Walk chart of mice (left forelimb: LF; left hindlimb: LH; right forelimb: RF; right hindlimb: RH). (J) Step regularity index of mice. (K) Measurement of forelimb muscle grip strength. Data represent the means ± SD of five independent experiments. **p < 0.01 vs. the sham group; ^p < 0.05, and ^^p < 0.01 vs. the MPTP-treated group.

Fig. 1

To assess motor behavior, the rotarod and pole assessments were implemented. Mice in the MPTP group spent less time on the rod than the control group, while treatment with ZLG and l-dopa increased their duration on the rod (Fig. 1F). Likewise, ZLG and l-dopa reduced the duration of the pole test for rodents treated with MPTP (Fig. 1H). Besides, gait analysis and a grip strength meter were employed to further assess mice's motor function and muscle strength. Gait analysis indicated that ZLG treatment significantly improved the accuracy of the step-walk chart and the step regularity of mice compared to the mice in the MPTP group (Fig. 1I & J). Interestingly, the results showed that the forelimb grip of the MPTP-treated mice was significantly weaker than that of the sham group, but ZLG treatment could enhance the grip strength of the mice (Fig. 1K). These findings suggest that ZLG treatment reduces MPTP-induced motor impairments by inhibiting the loss of TH-positive neurons in the substantia nigra and striatum.

3.2 ZLG alleviates MPP + -induced BV2 cell damage and apoptosis

Given the significant involvement of microglia in PD progression, our subsequent investigation aimed to determine if ZLG had a protective effect against MPP + -induced cell death in BV2 cells. As shown in Fig. 2A, BV2 cells treated with MPP + for 12 h exhibited approximately 50 % cell mortality compared to the control group. On the contrary, when cells were pretreated for 12 h with ZLG at nontoxic concentrations (10, 20, and 40 μM) and subsequently subjected to MPP + injury, the proportion of deceased cells was reduced to approximately 20 %. To validate the cytoprotective effects of ZLG, the amount of LDH leakage after the MPP + insult was quantified. As illustrated in Fig. 2B, introducing MPP + resulted in a fourfold augmentation in LDH release. The pretreatment of BV2 cells with ZLG significantly reduced the leakage of LDH. To conduct a more comprehensive analysis of the bioactivity of ZLG, we systematically observed its impact on the apoptosis of BV2 cells. As seen in Fig. 2C & D, the double staining results indicated that ZLG could considerably lower MPP + -induced apoptotic cells. MPP + induced apoptosis in BV2 cells, as shown by the formation of highly fluorescent, condensed masses representing apoptotic nuclei; in contrast, almost no apoptotic nuclei were seen in control cells (Fig. 2E). ZLG pretreatment significantly reduced the number of apoptotic nuclei in BV2 cells. Caspase-3 activation is a universal metabolic feature of all apoptotic cells. Therefore, we conducted additional measurements to quantify the extent of caspase-3 activation. As shown in Fig. 2F, The cellular caspase-3 was activated by MPP+, and ZLG effectively reduced the level of caspase-3 activation in a concentration-dependent manner. Altogether, ZLG at safe dosages prevented apoptosis in MPP + -damaged BV2 cells while simultaneously offering cytoprotection.Fig. 2 ZLG reduced MPP + -induced BV2 cell damage and apoptosis. (A) Protection from MPP + -induced BV2 cell injury. BV2 cells were grown in a 96-well plate for one day, then treated with ZLG (10, 20, 40 μM) for 12 h. After 12 h in a fresh medium with 800 μM MPP+, cell viability was evaluated using the MTT assay. (B) Detection of the concentration of LDH in the medium. The cells were treated with ZLG (10, 20, 40 μM) for 12 h, followed by 800 μM MPP + for another 12 h. The LDH activity was determined. (C) Annexin V/PI double-staining assay for apoptosis analysis. (D) Quantification of living, apoptotic, and necrotic cells. (E) Visualization of nuclear structure using Hoechst 33342 dye under a Nikon inverted fluorescence microscope. Scale bars: 20 μM. (F) The activation of cellular caspase-3 was assessed using a colorimetric test. Data represent the means ± SD of three independent experiments. **p < 0.01 vs. the control group; ^p < 0.05, and ^^p < 0.01 vs. the MPP + -treated group.

Fig. 2

3.3 ZLG reduces MPP + -induced oxidative damage in BV2 cells

To determine if ZLG reduced oxidative stress in BV2 cells, ROS generation was evaluated using DCFH-DA, a sensitive fluorescent sensor for ROS detection. Our findings demonstrated that pretreatment of the cells with ZLG significantly and dose-dependently decreased the accumulation of ROS (Fig. 3A & B). Superoxide anions are generally harmless. However, the chemicals generated by interacting with hydroxyl groups can cause damage to cellular DNA [35]. Here, we utilized DHE staining to estimate the presence of superoxide anions. As shown in Fig. 3D & E, the findings demonstrated that ZLG effectively eradicated endogenous superoxide anions in BV2 cells. The concentration of total thiols in BV2 cells was measured by DTNB titration. Fig. 3C showed that pre-treating BV2 cells with ZLG effectively inhibited the depletion of cellular thiols caused by MPP+ in a dose-dependent manner. High levels of malondialdehyde (MDA) in live organisms can lead to the formation of cross-linked polymers in important biological components, including proteins and nucleic acids, which can cause cell damage and toxicity [36]. The findings in Fig. 3F demonstrate that ZLG effectively decreases the synthesis of MDA caused by MPP+ in BV2 cells.Fig. 3 ZLG restored redox balance in MPP + -damaged BV2 cells. (A) Pretreatment with ZLG significantly alleviated ROS accumulation induced by MPP+, as determined using DCFH-DA staining. Scale bars: 20 μm. (B) Quantification of ROS fluorescence intensity by ImageJ. (C) Effects of ZLG on the total cellular thiols. (D) Pretreatment with ZLG significantly alleviated superoxide anion accumulation induced by MPP+, as determined using DHE staining. Scale bars: 20 μm. (E) Quantification of the fluorescence intensity in Fig. 3D by ImageJ. (F) Effects of ZLG on the MDA. Data are presented as means ± SD of three independent experiments. **p < 0.01 vs. contro, and ^^p < 0.01 vs. MPP + -treated cells.

Fig. 3

3.4 ZLG mitigates nerve damage by regulating the Nrf2-TrxR axis in BV2 cell

The findings above demonstrated that ZLG effectively maintained redox equilibrium in BV2 cells. The analysis of the chemical structure of ZLG revealed the presence of a single Michael acceptor moiety, which serves as a fundamental structure in numerous Nrf2-ARE inducers [37]. Thus, we postulated that ZLG might stimulate the cellular ARE activation. Here, we developed BV2-Luc-ARE cells to assess the impact of ZLG on Nrf2 transcription (Fig. 4A). Fig. 4B demonstrates that applying ZLG (40 μM) to BV2-Luc-ARE cells notably increased the transcriptional activity of Nrf2, which was similar to the impact observed with the positive control t-BHQ. In addition, the immunofluorescence results demonstrated that ZLG (40 μM) facilitated the movement of Nrf2 into the nucleus (Fig. 4C & D), with the highest activity observed after 1 h of ZLG treatment. As an antioxidant gene controlled by Nrf2, TrxR is a crucial element of the Trx system and a significant protein for preserving redox equilibrium [14]. In Fig. 4E, ZLG (40 μM) treatment of BV2 cells dramatically increased TrxR gene expression, with the highest induction observed at 6 h. Following this, we determined the effect of ZLG on TrxR expression in MPP + -damaged BV2 cells by western blotting. The findings demonstrated that ZLG counteracted the inhibitory effect of MPP + on TrxR expression in BV2 cells (Fig. 4F & G). The functions of TrxR and its substrate Trx are essential for maintaining redox balance. Consequently, we assessed the enzyme activities using the enzyme cycling method. Notably, our results demonstrated that ZLG restored the activities of TrxR and Trx in BV2 cells, which had been depleted by MPP+ (Fig. 4H & I). These findings suggested that ZLG might regulate cellular redox balance by activating the Nrf2-TrxR axis.Fig. 4 ZLG modulated the Nrf2-TrxR axis in MPP + -damaged BV2 cells. (A) Construction of BV2-ARE-Luc cells. (B) ZLG promotes ARE transcriptional activation. BV2-ARE-Luc cells were treated with ZLG or tBHG (positive control) for 24 h, and the luciferase reporter assay was performed. (C) The translocation of Nrf2 into the nucleus by IF. Scale bars: 20 μm. (D) Quantification of relative fluorescence intensity in individual cells by Image J. (E) Effects of ZLG on the TrxR mRNA expression by qRT-PCR in BV2 cells. (F) Effects of ZLG on the TrxR protein expression by western blotting in MPP + -treated BV2 cells. (G) IPP software quantified the relative blot intensity of each band. (H & I) Up-regulation of TrxR and Trx activities by ZLG in MPP + -treated BV2 cells. Data are presented as means ± SD of three independent experiments. *p < 0.05, and **p < 0.01 vs. control, and ^p < 0.05 and ^^p < 0.01 vs. MPP + -treated cells.

Fig. 4

3.5 ZLG promotes M2 microglial polarization in MPP + -induced BV2 cell

Significant evidence indicates that ROS has a crucial role in initiating M1 activation. Therefore, we conducted a study to examine whether ZLG has the potential to reduce ROS levels by regulating the Nrf2-TrxR axis, leading to polarization of microglia towards the M2 phenotype. CD86 and CD206 serve as indicators of M1 and M2 polarization, respectively. Upon MPP + stimulation, the relative fluorescence intensity (RFI) of CD86 (red) significantly increased, whereas CD206 (green) exhibited minimal changes (Fig. 5A–C). The treatment of ZLG at concentrations of 10, 20, and 40 μM resulted in a significant reduction in RFI of CD86. Conversely, the incubation with ZLG at concentrations of 10, 20, and 40 μM significantly enhanced the RFI of CD206. Our findings demonstrated that ZLG could promote M2 microglia polarization while inhibiting M1 polarization in MPP + -induced BV2 cells.Fig. 5 The effect of ZLG on microglia polarization and activation in MPP + -damaged BV2 cells. (A) The representative images of co-incubation of CD206 and CD86 in MPP + -induced BV2 cells. Nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. (B & C) Quantification of CD206 (green, B) and CD86 (red, C) fluorescence intensity by ImageJ. (D–H) The productions of IL-1β (D), IL-6 (E), TNF-α (F), IL-4 (G), and IL-10 (H) in MPP + -induced BV2 cells were measured using ELISA assay. (I) ZLG-treated BV2 salvage co-cultured PC12 cells after MPP + damage. Data are presented as means ± SD of three independent experiments. *p < 0.05, and **p < 0.01 vs. control, and ^p < 0.05 and ^^p < 0.01 vs. MPP + -treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

To further investigate the influence of ZLG on MPP + -induced microglia polarization, the concentration of M1/M2 phenotypic indicators was measured using ELISA. Fig. 5D–F showed that MPP + treatment of BV2 cells significantly elevated IL-1β, IL-6, and TNF-α levels. Pre-incubation with ZLG effectively lowered the concentrations of these pro-inflammatory cytokines. In contrast, the levels of M2 phenotypic cytokines (IL-4 and IL-10) were significantly higher in the ZLG groups compared to the MPP + group (Fig. 5G & H). These findings demonstrated that ZLG might shift the balance of microglial activation from pro-inflammatory M1 to anti-inflammatory M2-phenotypes via activating the Nrf2-TrxR axis.

Subsequently, we assessed whether BV2 cells treated with ZLG exhibited neuroprotective properties on PC12 cells co-cultured with them following the MPP + stimulation. BV2 cells and PC12 cells were cultured in the upper and lower compartments of the Transwell dish, respectively. The presence of MPP + caused a decrease in the viability of PC12 cells. Furthermore, when co-cultured with BV2 cells, the loss in PC12 cell viability induced by MPP+ was even more severe. Remarkably, the ZLG pre-treated BV2 cells significantly increased the vitality of PC12 cells by up to 70 % (Fig. 5I). The transition of ZLG-treated microglia from the deleterious M1-phenotype to the protective M2-phenotype might account for their neuroprotective effect.

3.6 ZLG regulates polarization of microglia in MPTP-induced PD mice

Next, we further investigated the regulatory effect of ZLG on microglial polarization in MPTP-induced PD mice. Following MPTP-induced damage, there was a considerable rise in the RFI of CD86+Iba1+ cells. However, treatment with ZLG at a dose of 20 mg/kg effectively reduced the RFI of CD86+Iba1+ cells (Fig. 6A & B). Although MPTP induction slightly reduced the RFI of CD206+Iba1+ cells, the treatment with ZLG considerably increased the RFI of CD206 (Fig. 6C & D). The results of our study showed that ZLG had the ability to enhance the polarization of M2 microglia and suppress the polarization of M1 microglia in MPTP-induced PD mice. Subsequently, the levels of M1/M2 phenotypic markers in the serum were quantified using ELISA. Fig. 6E showed that MPTP significantly elevated the level of IL-1β, IL-6, and TNF-α in mouse serum. However, pre-incubation with ZLG effectively lowered the concentrations of these pro-inflammatory cytokines. Moreover, in comparison to the MPTP group, the ZLG treatment group exhibited considerably elevated levels of M2 phenotypic cytokines (IL-4 and IL-10) (Fig. 6F). These results showed that in MPTP-induced PD mice, ZLG could activate the Nrf2-TrxR axis, which might tip the microglial activation balance from pro-inflammatory M1 to anti-inflammatory M2-phenotypes.Fig. 6 The effect of ZLG on microglia polarization and activation in MPTP-induced PD mice. (A) The representative images of co-incubation (CD86 and Iba-1). Nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. (B) The fluorescence intensity of CD86+ Iba1+ cells was quantified by ImageJ. (C) The representative images of co-incubation (CD206 and Iba-1). Nuclei are counterstained with DAPI (blue). Scale bar: 20 μm. (D) The fluorescence intensity of CD206+ Iba1+ cells was quantified by ImageJ. (E) The productions of IL-1β, IL-6, andTNF-α in mouse serum were measured via ELISA assay. (F) The productions of IL-4 and IL-6 in mouse serum were measured via ELISA assay. Data are presented as means ± SD of three independent experiments. **p < 0.01 vs. sham, and ^p < 0.05 and ^^p < 0.01 vs. MPTP-treated mice. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

3.7 The effect of inhibiting Nrf2-TrxR axis on MPP + -damaged BV2 cells

To ascertain if ZLG provided protection through the Nrf2-TrxR axis, we performed Nrf2 knockdown test in BV2 cells. The effectiveness of shRNAs (shNrf2-814, shNrf2-1713, shNrf2-294, and shNT) in reducing protein expression was confirmed using Western blot analysis (Fig. 7A), and the bands were quantified using IPP software (Fig. 7B). The expression of cellular Nrf2 was significantly reduced in cells transfected with shNrf2s. Among these shNrf2s, we selected shNrf2-294 for further research. In order to further explore the regulatory influence of Nrf2 on TrxR, we analyzed the mRNA and protein expression of TrxR following Nrf2 knockdown. As shown in Figs. S5 and S6, the mRNA and protein expression of TrxR in BV2-shNT cells was significantly higher than that in BV2-shNrf2 cells, indicating that Nrf2 positively regulates TrxR. As expected, the activities of TrxR and Trx did not undergo significant changes in MPP + -induced BV2-shNrf2 cells as compared with those in MPP + -induced BV2-shNT cells (Fig. 7C & D), indicating that the Nrf2-TrxR axis had been suppressed. Subsequently, we assessed the defensive impact of ZLG against oxidative damage on the transfected cells. ZLG exhibited a comparable defensive pattern in BV2-shNT cells, identical to what was observed in regular BV2 cells. However, the level of protection significantly decreased in BV2-shNrf2 cells, as the protective effect of ZLG was nearly eliminated after MPP + -induced cell death (Fig. 7E). Additionally, the capacity to scavenge ROS in BV2-shNrf2 cells was detected using a DCFH-DA staining experiment. BV2-shNrf2 cells pretreated with ZLG were unable to eradicate the ROS brought by MPP+ (Fig. 7F). In summary, the findings suggest that the capacity of ZLG to preserve redox homeostasis and exert cytoprotection was completely nullified when the Nrf2-TrxR axis was blocked.Fig. 7 The effect of inhibiting Nrf2-TrxR axis on MPP + -damaged BV2 cells. (A) Evaluation of Nrf2 knockdown efficiency by western blotting. PC12 cells were transfected with shRNAs specifically targeting the mouse Nrf2 gene (shNrf2s) or nontargeting shRNA (shNT), and (B) the quantification of the relative blot intensity of each band was performed by IPP software. (C) The activity of TrxR in MPP + -damaged BV2-shNT cells and BV2-shNrf2 cells. (D) The activity of Trx in MPP + -damaged BV2-shNT cells and BV2-shNrf2 cells. (E) Impairment of cytoprotection of ZLG in MPP + -damaged BV2 cells with Nrf2-TrxR axis inhibition. (F) The impact of ROS level in MPP + -damaged BV2 cells with Nrf2-TrxR axis inhibition. Scale bar: 20 μm. Data are presented as means ± SD of three independent experiments. **p < 0.01 vs. control, and ^^p < 0.01 vs. MPP + -treated cells.

Fig. 7

3.8 The impact of suppressing Nrf2-TrxR axis on microglia polarization and activation

Subsequently, our focus was on studying the role of the Nrf2-TrxR axis in regulating microglia polarization and activation using the BV2-shNrf2 cells. The images in Fig. 8A demonstrated that ZLG could not reverse the transformation of microglia from the M1 phenotype (CD86, red) to the M2 phenotype (CD206, green). The RFI of CD86 and CD206 was measured using ImageJ software (Fig. 8B & C). These data confirmed that the Nrf2-TrxR axis was involved in ZLG-modulated M1/M2 polarization in MPP + -induced BV2 cells. In addition, the ZLG treatment successfully decreased the levels of IL-1β, IL-6, and TNF-α, while increasing the levels of IL-4 and IL-10 in BV2-shNT cells induced by MPP+. In contrast, when Nrf2 transfection was performed, the inhibitory effects of ZLG on IL-1β, IL-6, and TNF-α, as well as the promotion effects of ZLG on IL-4 and IL-10, were effectively blocked (Fig. 8D–H). Remarkably, following the MPP + stimulation, the neuroprotective effects of ZLG-treated BV2-shNrf2 cells were no longer present on the co-cultured PC12 cells (Fig. 8I). These findings indicate that the Nrf2-TrxR axis is essential for the biological actions of ZLG, such as controlling the polarization of microglia, releasing inflammatory substances, and providing neuroprotective benefits.Fig. 8 The effect of inhibiting Nrf2-TrxR axis on microglia polarization and activation in MPP + -damaged BV2 cells. (A) The representative images of co-incubation of CD206 and CD86 in MPP + -induced BV2-shNrf2 cells. Nuclei were counterstained with DAPI (blue). Scale bar: 10 μm. (B & C) Quantification of CD206 (green, B) and CD86 (red, C) fluorescence intensity by ImageJ. (D–H) The productions of IL-1β (D), IL-6 (E), TNF-α (F), IL-4 (G), and IL-10 (H) in MPP + -induced BV2-shNT cells and BV2-shNrf2 cells were measured via ELISA assay. (I) ZLG-treated BV2-shNrf2 cells failed to salvage co-cultured PC12 cells after MPP + damage. Data are presented as means ± SD of three independent experiments. *p < 0.05, and **p < 0.01 vs. control, and ^p < 0.05 vs. MPP + -treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

4 Discussion

Currently, numerous studies are being conducted on ZLG in neuroprotection. However, these studies primarily concentrate on investigating its effects on cerebral ischemia-reperfusion injury and AD. ZLG improves neuronal damage in the hippocampus caused by cerebral ischemia-reperfusion through the activation of the PINK1/Parkin pathway [38]. In addition, in a mouse model of AD, liposomes loaded with ZLG decrease mitochondrial abnormalities and improve cognitive performance by activating the PKA/AKAP1 signaling pathway [39]. There is a shortage of comprehensive investigation on the pharmacological impacts of ZLG on PD, particularly regarding the effectiveness and probable mechanism of ZLG in treating PD. The current study was conducted to clarify the anti-PD impact of ZLG and its underlying mechanism. The results of our investigation demonstrated that ZLG positively impacted PD in mice, namely in terms of reducing dopamine levels and improving motor function. Additionally, our work provided insights into the potential mechanism of action of ZLG on PD, suggesting that it may operate through the Nrf2-TrxR axis.

Neuroinflammation and ROS are universally believed to be involved in CNS diseases. To obtain therapeutic success with central disease, the prevention of neuroinflammatory responses and oxidative stress is implemented [40]. Mounting evidence has confirmed that activated microglia play a crucial role in inflammatory processes, carrying out specialized immunological activities to preserve physiological homeostasis. Upon activation, microglia can undergo phenotypic polarization, resulting in either a classical phenotype (M1, pro-inflammatory) or an alternate phenotype (M2, anti-inflammatory) [41]. Hence, the concurrent suppression of the M1 phenotype and stimulation of the M2 microglia hold great potential as a therapeutic approach for PD [42]. Numerous studies have focused on investigating the regulatory mechanisms of microglial polarization. One particular area of interest for researchers is the influence of ROS levels on microglial polarization. Cao et al. indicated that ROS is pivotal in macrophage-mediated acute inflammation. Their further study demonstrated that reactive oxygen species (ROS) act as signaling molecules that control the polarization of M1 macrophages through the involvement of ataxia-telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (Chk2), which are essential kinases in the signaling pathway responsible for DNA damage response [43]. Elevated levels of ROS in microglia result in the activation of the pro-inflammatory M1 phenotype, whereas moderate levels of ROS lead to the activation of the anti-inflammatory M2 phenotype. Hence, our initial inquiry revolved around the potential of ZLG to modulate the polarization phenotype of microglia by diminishing levels of ROS.

Nrf2 is generally regarded as a regulator of the endogenous antioxidant system. After being activated, Nrf2 moves into the nucleus and attaches to specific DNA sequences to control downstream molecules' transcription [44]. TrxR is a crucial antioxidant gene located in the downstream region of Nrf2. Trx and TrxR are integral constituents of the Trx system, which regulates numerous cellular signaling pathways and is the most significant component of the intracellular redox milieu [17]. According to our experimental findings, ZLG can prevent ROS in microglia by triggering the Nrf2 pathway and preserving the intracellular redox balance. Additional research further indicates that ZLG stimulates the gene expression of TrxR. TrxR typically decreases the cysteine residue of oxidized Trx, and the reduced Trx functions as an antioxidant by facilitating the reduction of other proteins through the interchange of cysteine thiol-disulfide bonds. Using the enzyme cycling approach, we assessed the impact of ZLG on TrxR and Trx activity. The findings indicate that ZLG substantially enhances the activity of TrxR and Trx in microglia. Based on these findings, ZLG activates the Nrf2-TrxR axis to reduce oxidative damage in microglia.

ZLG effectively reduces ROS in microglia through the Nrf2-TrxR pathway, perhaps influencing microglia polarization. Significantly, our empirical findings corroborate this notion. ZLG suppressed the microglial M1 polarization caused by MPP+. On the other hand, the alternative M2 activation state spans a wide range of responses in comparison to the M1 activation state. Typically, the M2 activation state is linked to mending and scavenging, which contrasts with the pro-killing condition of the M1 phenotype. Another significant discovery in this study is that ZLG partially reversed the inhibition of M2 microglia caused by MPP+, hence enhancing the production of anti-inflammatory proteins. Microglia are specialized macrophages that are found exclusively in the nervous system. They have a crucial function in forming neuronal circuits and regulating tissue balance [45,46]. Their alterations are intricately connected to the onset and progression of PD [47]. We demonstrated through BV2-PC12 co-culture experiments that ZLG exerts prominently neuroprotective effects. ZLG's regulatory function in maintaining redox homeostasis and microglia polarization is eliminated by Nrf2 knockdown and Trx system inactivation.

5 Conclusion

Overall, the current study showed that the ZLG treatment reduced the severity of the MPTP-induced PD mice model. Research on mechanism suggests that ZLG has the potential to restore the balance of redox homeostasis in microglia, thus preserving their normal functioning and safeguarding them against oxidative damage. Besides, ZLG treatment could also shift the balance of microglial activation from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype by protecting microglia and polarizing microglia towards the M2 phenotype. A deeper mechanism suggests that ZLG exerts a protective effect and regulates polarization by regulating the Nrf2-TrxR axis in microglia. We schematically presented the molecular mechanism of ZLG in Fig. 9. The Nrf2 shRNA transfection and downregulated TrxR activity confirm the role of the Nrf2-TrxR axis in the ZLG-modulated microglia polarization. Transgenic mice could be used in further investigation.Fig. 9 Mechanism diagram of ZLG exerting neuroprotective effects.

Fig. 9

CRediT authorship contribution statement

Shoujiao Peng: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization. Yao Chen: Methodology, Investigation. Ran Wang: Project administration, Methodology, Investigation. Jiange Zhang: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgments

This work was supported by funding from the Shanghai Science and Technology Development Fund from the central leading local government (grant number: YDZX20223100001004 ), the Shanghai Pujiang Program (grant number: 23PJ1411200 ), and the Science and Technology Development Fund of SUTCM (grant number: 23KFL003 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103324.
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