Paeoniflorin alleviates neuroinflammatory response by suppressing HMGB1/RAGE pathway activation in MPTP induced mouse PD model

doi:10.21203/rs.3.rs-3863621/v1

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Paeoniflorin alleviates neuroinflammatory response by suppressing HMGB1/RAGE pathway activation in MPTP induced mouse PD model

https://doi.org/10.21203/rs.3.rs-3863621/v1

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Background

Numerous studies have highlighted that Paeoniflorin (PF) exerts various biological effects, including anti-inflammatory, antioxidative, and anti-apoptotic actions on Parkinson’s disease (PD). However, the underlying anti-inflammatory mechanism of PF on PD is still not fully understood.

Methods

In present study, we assumed that the neuroprotective effect and anti-inflammatory mechanism of PF occurs through modulating HMGB1/RAGE pathway. We verified this hypothesis on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)/1-Methyl-4-Phenylpyridinium Iodide (MPP⁺) induced PD model of mice and BV-2 cells. We performed behavioral tests (pole test, beam-cross test and traction test) to investigate the motor function, the protein level of tyrosine hydroxylase (TH) and the aggregation of microglia were evaluated by immunostaining. The pro-inflammatory cytokines and the expression of HMGB1, RAGE, and NF-κB were evaluated via ELISA and Western blot analyses respectively.

Results

The results showed that PF treatment could significantly improve motor function of PD mice in behavioral tests. Our data also revealed that PF could rescue dopamine neuron loss, inhibit the aggregation of microglial cells in the substantia nigra pars compacta in immunostaining, and lower the levels of pro-inflammatory cytokines in ELISA test. Both in vitro and in vivo experiments demonstrated that PF downregulates the expression of HMGB1, RAGE, and NF-κB in the PD models induced by MPTP/MPP⁺.

Conclusion

In this study, PF exerted neuroprotective effects on PD models by downregulating the activation of HMGB1/RAGE/NF-κB pathway to reduce neuroinflammatory response.

Parkinson’s disease

HMGB1

RAGE

Paeoniflorin

Neuroinflammation

Parkinson’s disease (PD) is the second most common neurodegenerative disease with pathological loss of dopaminergic neurons in substantia nigra pars compacta (SNpc)[1], which causes the typical motor dysfunction symptoms like bradykinesia, resting tremor, rigidity, and postural instability[2]. Although the mechanism of PD still remains unclear, previous studies have reached a consensus that aging, heredity, and environmental neurotoxins make a preponderant contribution to neuroinflammation, oxidative stress and mitochondria dysfunction, which play a key role in the progression of PD[3–5]. Besides the genetic predisposition, neuroinflammation has attracted great attention in basic studies. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), a typical neurotoxin for inducing PD model in mouse or rat, can evoke inflammatory response that promotes neurodegeneration of dopaminergic neurons specifically[6]. Τhe increasing activated microglia, which can be observed in MPTP-treated mice, is considered to contribute to the release of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6)[7], to promote the degeneration of dopaminergic neurons in SNpc[8].

High mobility group box 1 (HMGB1) has been reported to have a close relation to PD progression. Previous studies have presented that HMGB1 is highly expressed in peripheral blood of PD patients compared to the healthy group[9, 10]. Receptor for advanced glycation end products (RAGE) is known as one of the main receptors of HMGB1[11], and the interaction between HMGB1 and RAGE can lead to the activation of NF-κB, which plays a crucial role in producing proinflammatory cytokines such as TNF-α and IL-6[12]. Therefore, HMGB1/RAGE pathway and its downstream signaling factors may be closely associated with PD pathogenesis.

Paeoniflorin (PF), a monoterpene glycosidic extract from the root of Paeonia albiflora Pall, has been widely reported for its pharmacological effects on the neuronal system. Some studies have reported that PF was widely used in the treatment of epilepsy[13], cerebral ischemia[14] and subarachnoid hemorrhage[15]. In a previous study, PF was found to attenuate neuroinflammation by activating the adenosine A1 receptor in MPTP-induced PD mouse models[16]. Another research also suggested that PF treatment could improve the impaired motor performance and decrease the loss of dopaminergic neurons in MPTP-treated mice via lowering monoamine oxidase-B (MAO-B) and caspase-3/9 activities, and increasing Akt activity[17]. Moreover, the anti-oxidation and anti-apoptosis effects of PF were demonstrated in PC12 cell PD model[18, 19]. However, the potential mechanism by which PF alleviates PD-associated neuroinflammation is still not fully understood.

In this study, the protective effect of PF was investigated in PD mice induced by MPTP. Furthermore, we explored whether PF exerted its neuroprotective effect against the neurotoxicity of MPTP/1-Methyl-4-Phenylpyridinium Iodide (MPP⁺) by modulating the HMGB1/RAGE pathway.

Chemicals and antibodies

The chemicals and antibodies used in this study are as follows. For the PD model, MPTP (M0896) and MPP⁺ (D048) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). PF (purity ≥ 98%) was purchased from Nanjing jingzhu bio-technology Co., Ltd (Nanjing, China)

The ELISA kits for mouse TNF-α, IL-1β and IL-6 were purchased from Wuhan Servicebio Technology Co., Ltd (Wuhan, China).

The antibodies for western blotting are as follows: anti-HMGB1 antibody (ab18256), anti-RAGE antibody (ab216329), anti-NF-κB antibody (ab16502), anti-β-actin antibody (ab8227) and anti-GAPDH antibody (ab8245) were purchased from Abcam (Cambridge, UK).

Antibodies for immunostaining are as follows: anti-tyrosine hydroxylase (TH) antibody (ab137869), anti-Iba-1 antibody (ab178847) were purchased from Abcam (Cambridge, UK), horseradish peroxidase-conjugated secondary immunoglobulin G was obtained from Beyotime (Shanghai, China).

Animals and treatments

A total of fifty male C57BL/6 mice (8 weeks old, weighing 20 ~ 24g) were obtained from the Animal Laboratory of Peking University People's Hospital and were maintained in a clean animal room with ambient temperature 22°C ~ 25°C, relative humidity 50%~60% and a 12h/12h dark-light cycle. All mice had free access to food and water.

The mice were randomly divided into 5 groups: (1) control, (2) MPTP, (3) MPTP + PF (10mg/kg), (4) MPTP + PF (20mg/kg), (5) MPTP + PF (40mg/kg) (n = 10 in each group). The mice of low, middle and high dose PF groups were weighed and constantly given PF for 14 days intragastrically. Mice in the MPTP treated group and each PF treated group were given an intraperitoneal injection of 30mg/kg MPTP for 7 days after a 2-day pretreatment with saline and PF. The control group was also given the same volume of saline intraperitoneally throughout the study.

Cell culture and treatment

The microglia cell line of mouse BV-2 was purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The BV-2 cells were seeded into 6-well plates and cultured at 37 ℃ in MEM medium supplemented with 10% fetal bovine serum for 24 hours. Then, 0.1 mM MPP⁺ was added into each well and incubated for 6 hours. Finally, 0.1 µM. 1 µM or 10 µM of PF was added separately. The cells were collected for further analysis 24 hours later.

Behavioral test

To measure bradykinesia symptoms of parkinsonism, the pole test was performed according to a protocol reported by a previous study with slight adjustments[20]. Briefly, a 60cm high wooden pole in 8mm diameter with a rough surface was placed vertically. Then, the mouse was positioned head downward on the top of the pole, and the time it took for the mouse to climb down the pole was recorded. The test was repeated three times and the average time was calculated for statistical analysis.

To test for postural instability, the beam-cross test was performed after two days of training. The mouse was placed at one end of an elevated beam (100cm long, 2cm wide). The time it took to cross the beam was recorded and the test would be repeated three times.

Traction test was performed on a horizontal wire in 1.5mm diameter (30cm high from flat surface). The forepaws of the mouse would be hung on the wire and its behavioral performance would be scored according to the following criteria:

1 = no hind paw can catch the wire.

2 = one hind paw can catch the wire.

3 = both hind paws can catch the wire.

All mice were pre-trained 2 days before receiving agent treatments, and every behavioral test was repeated three times.

Immunohistochemistry and immunofluorescence

After behavioral assessment, mice of each group underwent cardiac perfusion with 4°C saline under anesthesia induced by 2% isoflurane inhalation. The whole brains were removed and midbrain tissues were isolated on ice and then fixed in 4% paraformaldehyde. After fixation, brains were embedded in paraffin according to standard procedures and serially sectioned at 6 µm (Leica, Germany) for staining. In the immunohistochemistry assay, brain sections were deparaffinized with xylene and rehydrated through a series of ethanol solutions (from 100 to 75%). Antigen retrieval was performed by soaking the sections in boiling citrate buffer (pH 6.0) in a microwave for 15 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H₂O₂) for 30 min. Then sections were incubated with 5% normal goat serum (Thermo Fisher, USA) for 1 h at room temperature. Next, the brain sections were probed with anti-Tyrosine hydroxylase (TH) primary antibody (1:200) solution at 4°C overnight. Then, the sections were incubated with secondary antibody at room temperature for 1 h. After being incubated with 3,3′-diaminobenzidine (DAB) (Thermo Fisher, USA) for 10 min, the sections were counterstained with hematoxylin. Images were taken using a light microscope (Leica DM1000, Wetzlar, Germany).

For immunofluorescence staining, the brain sections were incubated with anti-Iba-1 antibody (1:200) at 4°C overnight. Then, after the washing stage, the sections were incubated in Cy3-labeled goat anti-rabbit IgG and FITC-labeled goat anti-mouse IgG (Beyotime, China) for 2h at room temperature. Then, the sections were washed using PBS and co-stained with DAPI. Images were taken using fluorescence microscope (Leica DM1000, Wetzlar, Germany).

Enzyme-linked immunosorbent assay

Midbrain tissue samples were collected from mice to determinate the protein levels of inflammatory factors. The levels of TNF-α, IL-1β and IL-6 were measured by enzyme-linked immunosorbent assay kits (TNF-α, IL-1β and IL-6) according to the manufacturer’s protocol. A Microplate reader (BioTek, China) was used to test the absorbance of all inflammatory cytokines.

Western Blotting analyses

Midbrain or cells specimens were homogenized 1:10 (w/v) in homogenization buffer (containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate, 1 mM sodium vanadate, 1% Triton X-100, and 1 mg/mL leupeptin; pH 7.5) supplemented with protease inhibitor cocktail. After centrifugation at 12 000 g for 10 minutes, protein lysates were quantified by bicinchoninic acid method (Bio-Rad). Then equal amounts of protein samples (each 40 µg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking in 5% skim milk in Tris-buffered saline (TBS, 150 mM NaCl, 20 mM Tris–HCl, pH 7.4), the membranes were incubated with primary antibodies (HMGB1, 1:1000; RAGE, 1:1000; NF-κB, 1:500; GAPDH, 1:1000 and β-actin, 1:2000) at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies for 45 min and visualized with a chemiluminescent HRP substrate (Millipore) in a ChemiDoc™MP Imaging System (Bio-Rad). The optical density of each strip was assessed using Image J software.

Statistical analysis

Data are presented as the mean ± standard error of mean (SEM). Prism software version 9 (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis, one-way ANOVA followed by Tukey’s post hoc test was performed for multiple comparisons of each group. The results were considered statistically significant when the p-value < 0.05.

PF improved the motor function in PD mouse model induced by MPTP

To confirm the neuroprotective effect of PF, the pole test, beam-cross test and traction test were performed respectively to estimate the motor function of MPTP-induced model in this study. In the pole test, the results showed that MPTP-treated mice spent a longer time descending the pole compared with the control group (p < 0.001, vs control group), while PF (20mg/kg, 40mg/kg) treatment decreased the descending time significantly (p < 0.001, vs MPTP group, Fig. 1A). Similarly, MPTP-treated mice spent a longer time crossing the beam in beam-cross test, while PF treatment at 20mg/kg and 40mg/kg significantly decreased the crossing time (p < 0.001, vs MPTP group, Fig. 1B). In addition, MPTP injection markedly reduced movement score (p < 0.001, vs control group) in traction test while 20mg/kg (p < 0.05, vs MPTP group) and 40mg/kg (p < 0.001, vs MPTP group) PF treatments increased movement score significantly (Fig. 1C).

PF prevented the dopaminergic neuronal loss from the neurotoxin of MPTP

Dopaminergic neuronal damage was detected via immunohistochemical staining of dopaminergic neuronal marker TH (Fig. 2A). Dopaminergic neurons in SNpc of control group were evidently observed, whereas a significant loss of dopaminergic neurons was observed in the MPTP-treated group (p < 0.001, vs control group, Fig. 2B). Besides, PF administration (10mg/kg, 20mg/kg, 40mg/kg) could rescue dopaminergic neuronal death induced by MPTP in a dose-dependent manner compared with the MPTP-treated group (p < 0.001, vs MPTP group, Fig. 2B). The results of TH immunostaining revealed that PF could provide neuroprotective effect in MPTP-induced PD mice.

PF attenuated inflammatory responses by inhibiting microglia aggregation in SNpc of MPTP-treated mice

An obvious overexpression of TNF-α, IL-1β and IL-6 was detected in midbrains of the MPTP-treated group (p < 0.001, vs control group, Fig. 3A, B, C), and the treatments of PF (10mg/kg, 20mg/kg and 40mg/kg) suppressed the expression of the cytokines mentioned above ((p < 0.05 and p < 0.001, vs MPTP group, Fig. 3A, B, C). Additionally, the immunofluorescence staining showed that microglia (Iba-1 positive) aggregated in the SNpc of MPTP-treated mice, while the PF administration (40mg/kg) could markedly inhibit the aggregation of microglia (Fig. 3D). These results confirmed that PF exhibited its anti-inflammation effect by preventing aggregation of microglia to reduce the expression of proinflammatory cytokines.

PF suppressed HMGB1/RAGE/ NF-κB pathway activation in vivo

To investigate whether PF could modulate the activation of the HMGB1/RAGE/NF-κB pathway to exert its anti-inflammatory effect, we conducted Western Blot analysis both in MPTP-treated mice and all the other groups. As shown in Fig. 4A, we detected the protein expression levels of HMGB1, RAGE and NF-κB in each mouse group. Obviously, we observed that HMGB1, RAGE and NF-κB showed a significant overexpression in the MPTP-treated group (p < 0.001, vs control group, Fig. 4B, C, D), while treatment with PF (20mg/kg, 40mg/kg) dose-dependently ameliorated the increased expressions of HMGB1, RAGE and NF-κB induced by MPTP (p < 0.01 and p < 0.001, vs MPTP group, Fig. 4B, C, D).

PF inhibited HMGB1/RAGE/ NF-κB pathway activation in MPP⁺ treated cells

The anti-inflammatory effect of PF was also investigated in BV-2 cells treated with MPP⁺. As shown in Fig. 5A, MPP⁺ administration could significantly activate the HMGB1/RAGE/NF-κB pathway, as indicated by the upregulation of the expressions of HMGB1, RAGE and NF-κB (p < 0.001, vs control group, Fig. 5B, C, D). In accordance with the in vivo experiment, PF treatment (mainly 1 µM and 10 µM) could markedly downregulate the expression levels of HMGB1, RAGE and NF-κB (p < 0.01 and p < 0.001, vs MPP⁺ group, Fig. 5B, C, D).

In the present study, we investigated the inflammatory response in PD models induced by MPTP and focused on exploring the anti-inflammatory and neuroprotective effects of PF which could be a potential agent for PD therapy by suppressing the aggregation of microglia and the activation of the HMGB1/RAGE//NF-κB pathway.

Neurotoxic agents such as MPTP have been demonstrated to induce Parkinsonian manifestations in various experimental animals. In rodents, C57BL/6 mice exhibit the highest sensitivity to MPTP[21]. MPTP can accumulate in SNpc of mice and hinder mitochondrial energy metabolism, generating reactive oxygen species. This results in oxidative stress, activating neuroinflammatory responses and leading to extensive dopamine neuronal death[22]. Although the pathogenic mechanisms of Parkinson's disease remain unclear, neuroinflammation is widely acknowledged to play a significant role in its progression[6]. Furthermore, numerous studies have found that MPTP-treated mice exhibit substantial glial cell accumulation in SNpc, accompanied by elevated levels of cytokines such as TNF-α, IL-1β, and IL-6[6, 8]. These phenomena are closely associated with the occurrence of neuroinflammation, making the MPTP-induced PD model an ideal choice for investigating drugs that exhibit anti-inflammatory effects to mitigate dopaminergic neuron loss. In this study, MPTP-treated mice exhibited significant motor dysfunction in behavioral tests, and we observed a considerable loss of dopaminergic neurons and aggregation of microglial cells in the SNpc of MPTP-treated mice. Additionally, the pro-inflammatory molecules TNF-α, IL-1β, and IL-6 were significantly elevated in the SNpc of MPTP-treated group, demonstrating that MPTP can induce neuroinflammation and damage dopaminergic neurons in mice, aligning with previous research findings.

Compared to cell replacement therapy and traditional dopamine supplementation-based treatments for Parkinson's disease, traditional Chinese medicine extracts, such as PF, offer a novel approach for pharmacological intervention[23]. PF, a bioactive ingredient extracted from the traditional Chinese medicine Paeonia albiflora Pall, has been studied extensively, showing neuroprotective effects through various mechanisms, including anti-inflammatory, anti-oxidation, and anti-apoptotic actions[16, 18, 24–27]. In this study, our data indicated that PF exerts dose-dependent anti-inflammatory effect, primarily evidenced by the inhibition of microglial cell aggregation in the SNpc of MPTP-induced PD mice. Therefore, PF treatment could significantly reduce the expression levels of corresponding cytokines, while the loss of dopaminergic neurons notably decreased in the SNpc, thereby improving the motor function of MPTP-treated mice.

To further investigate the potential anti-inflammatory mechanism of PF in the MPTP-induced PD model, we assessed the expression levels of molecules in the HMGB1/RAGE/NF-κB pathway. Currently, HMGB1 has been confirmed to be associated with various neurodegenerative diseases. Clinical studies conducted by Yang et al. and Baran et al. have shown elevated levels of HMGB1 in the peripheral blood of PD patients[9, 10]. Besides, HMGB1 acts as a secreted pro-inflammatory cytokine, inducing inflammation through interactions with receptors like RAGE and Toll-like receptors (TLRs)[28, 29]. Sasaki et al. demonstrated that inhibiting HMGB1 using monoclonal antibodies protects rat models from the neurotoxic effects of 6-OHDA[30]. Similarly, Santoro et al. indicated that neutralizing HMGB1 in the MPTP mouse model leads to decreased RAGE and TNF-α levels, inhibits activation of microglia and mitigates dopaminergic neuron death[31]. This suggests that downstream signaling cascades of RAGE, such as NF-κB are involved in HMGB1-mediated pro-inflammatory effects. Consistent with these findings, the Western Blot results of our study revealed that PF dose-dependently reduced the levels of various signaling molecules in the HMGB1/RAGE/NF-κB pathway in MPTP-treated mice. In vitro experiments also demonstrated that PF similarly downregulated the expression levels of HMGB1, RAGE and NF-κB in BV-2 cells treated with MPP⁺. In short, PF exerted its anti-inflammatory effects in the brain of PD mouse model by downregulating the activation of HMGB1/RAGE/NF-κB pathway.

In conclusion, our study reveals that PF protects mice and BV-2 cells from the neurotoxicity of MPTP/MPP⁺, and exhibits its anti-inflammatory effects by suppressing the aggregation of microglial cells and reducing pro-inflammatory cytokine levels. It also demonstrates that the anti-inflammatory effects of PF are achieved through modulating the HMGB1/RAGE/NF-κB pathway. Thus, we believe that PF may become a novel choice for PD therapy and HMGB1 is one of the crucial therapeutic targets for the treatment of PD.

PD Parkinson’s disease

SNpc substantia nigra pars compacta

MPTP 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine

MPP⁺ 1-Methyl-4-Phenylpyridinium Iodide

TNF-α tumor necrosis factor-alpha

IL-1β interleukin 1 beta

IL-6 interleukin 6

HMGB1 high mobility group box 1

RAGE receptor for advanced glycation end products

PF paeoniflorin

MAO-B monoamine oxidase-B

TH tyrosine hydroxylase

TLRs toll-like receptors

Funding This work was supported by funding from the National Key R&D Plan of the Ministry of Science and Technology (No. 2019YFE0117100).

Author contributions YL conceived and designed the experiments, YZ and SP performed the experiments, YL, SP, LW and collected and analyzed data, YL, SP, LW and RL drafted and revised the manuscript. All authors read and approved the final manuscript.

Conflict of interest The authors have no competing interests to declare that are relevant to the content of this article.

Data availability The data during the current study are available from the corresponding author on reasonable request.

Ethics approval All experimental procedures were approved by the Ethic Committee of Peking University People’s Hospital (approval No. 2021PHE042), and were performed according to the guidelines for the Care and Use of Laboratory Animals

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No competing interests reported.

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Paeoniflorin alleviates neuroinflammatory response by suppressing HMGB1/RAGE pathway activation in MPTP induced mouse PD model

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Chemicals and antibodies

Animals and treatments

Cell culture and treatment

Behavioral test

Immunohistochemistry and immunofluorescence

Enzyme-linked immunosorbent assay

Western Blotting analyses

Statistical analysis

Results

PF improved the motor function in PD mouse model induced by MPTP

PF prevented the dopaminergic neuronal loss from the neurotoxin of MPTP

PF attenuated inflammatory responses by inhibiting microglia aggregation in SNpc of MPTP-treated mice

PF suppressed HMGB1/RAGE/ NF-κB pathway activation in vivo

PF inhibited HMGB1/RAGE/ NF-κB pathway activation in MPP⁺ treated cells

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

Paeoniflorin alleviates neuroinflammatory response by suppressing HMGB1/RAGE pathway activation in MPTP induced mouse PD model

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

Chemicals and antibodies

Animals and treatments

Cell culture and treatment

Behavioral test

Immunohistochemistry and immunofluorescence

Enzyme-linked immunosorbent assay

Western Blotting analyses

Statistical analysis

Results

PF improved the motor function in PD mouse model induced by MPTP

PF prevented the dopaminergic neuronal loss from the neurotoxin of MPTP

PF attenuated inflammatory responses by inhibiting microglia aggregation in SNpc of MPTP-treated mice

PF suppressed HMGB1/RAGE/ NF-κB pathway activation in vivo

PF inhibited HMGB1/RAGE/ NF-κB pathway activation in MPP+ treated cells

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

PF inhibited HMGB1/RAGE/ NF-κB pathway activation in MPP⁺ treated cells