Neuroprotective effect of green tea extract (-)-epigallocatechin-3-gallate in a preformed fibril-induced mouse model of Parkinson’s disease

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra (SN). The main bioactive component of green tea polyphenols (-)-epigallocatechin-3-gallate (EGCG) exerts protective effects against diseases such as neurodegenerative diseases and cancer. Therefore, this study investigated the effect of EGCG on the amelioration of neural damage in a chronic PD mouse model induced by α-synuclein preformed fibrils (α-syn-PFFs). A total of 20 C57BL/6J female mice were randomly divided into 3 groups: control group (saline, n = 6), model group (PFFs, n = 7), and prevention group (EGCG+PFFs, n = 7). A chronic PD mouse model was obtained by the administration of α-syn-PFFs by stereotaxic localization in the striatum. Behavioral tests were performed to evaluate PD-related anxiety-like behavior and motor impairments in the long-term PD progression. Tyrosine hydroxylase (TH) immuno-positive neurons and Ser129-phosphorylated α-syn (p-α-syn) were identified by immunohistochemistry. Pro-inflammatory and anti-inflammatory cytokines were measured by real-time quantitative PCR. EGCG pretreatment reduced anxiety-like behavior and motor impairments as revealed by the long-term behavioral test (2 weeks, 1 month, 3 months, and 6 months) on PD mice. EGCG also ameliorated PFF-induced degeneration of TH immuno-positive neurons and accumulation of p-α-syn in the SN and striatum at 6 months. Additionally, EGCG reduced the expression of pro-inflammatory cytokines while promoting the release of anti-inflammatory cytokines. EGCG exerts a neuroprotective effect on long-term progression of the PD model.

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease with chronic progression after Alzheimer’s disease [1]. The neuropathological characteristics of PD are the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta, the decrease in dopamine levels in the striatum, and the accumulation in the surviving neurons of Lewy bodies (LBs), in which α-synuclein (α-syn) is a significant component [2]. The clinical symptoms of PD are characterized by static tremor, rigidity, bradykinesia, postural instability, and other non-motor symptoms. Non-motor symptoms such as dementia, sleep disorders, constipation, and loss of smell can occur long before the onset of motor symptoms and the clinical diagnosis [3].

The native form of α-syn is primarily represented by unfolded soluble monomers or α-helically folded tetramers bound to the membrane, with a low tendency to aggregate [4]. Patients’ neurons in PD brains contain misfolded α-syn inclusion bodies that aggregate into oligomers and form insoluble amyloid structures rich in a beta-sheet structure, which is potentially neurotoxic. The misfolding and aberrant aggregation of α-syn and its phosphorylated variants in LBs and Lewy neurites (LNs) are involved in PD neuropathology [5,6]. Site-specific phosphorylation of α-syn fibrils at Ser129 and Y39 can exacerbate the neuropathology of endogenous α-syn aggregation [7]. The stereotactic injection of synthetic α-synuclein preformed fibrils (α-syn-PFFs) into the striatum has been used as a ‘seed’ to start the aggregation of soluble endogenous α-syn and occurrence of LB/LNs pathology in vivo to construct the chronic PD mouse model [8].

Much research in recent decades has focused on the well-documented health benefits of the polyphenols in green tea for the prevention of several diseases including cancer (colorectal, skin, prostate, breast, lung, and liver cancer), diabetes, cardiovascular diseases, and neurological diseases [9–14]. (-)-epigallocatechin-3-gallate (EGCG) is the main molecule in green tea extracts with extensive biological activities [15]. Recent studies from computational simulations revealed that EGCG attenuates the α-syn protofibril-membrane interactions by forming interactions with the model membrane, thus restoring the membrane integrity, and that oxidized EGCG remodels the toxic α-syn fibrils into non-toxic aggregates by non-covalent interactions [16,17]. These results suggest that EGCG exerts neuroprotective effects, being potentially effective in delaying PD development.

Advances in the treatment of PD patients have been made in recent decades. However, it is important to emphasize that both pharmacological medications and surgical interventions have been unsatisfactory due to their unwanted adverse effects and their inability to effectively slow down or inhibit the death of dopaminergic neurons [18]. Hence, PD prevention and early intervention are of the utmost importance. In this respect, EGCG holds great potential as a natural product. A recent study reported an independent negative correlation between daily tea consumption and PD risk in a large, well-established Chinese cohort [19]. Until now, most studies focused on acute or subacute PD mouse models, which do not accurately mimic the PD chronic neurodegenerative process, and no report demonstrated the neuroprotective effect of EGCG in the PFF-induced PD mouse model. Therefore, this study aimed to assess the potential neuroprotective effect of EGCG on a PD chronic mouse model induced by PFFs. The investigation involved the use of behavioral tests, pathological analysis, and the detection of inflammatory cytokines. The findings of this study offer experimental evidence supporting the use of EGCG as a natural dietary intervention for the prevention of PD in the future.

Methods

Western blot and preformed fibrils preparation

Protein concentration was measured by the BCA protein quantification kit (Solarbio, China). The protocol to obtain pure α-syn protein samples and the specific procedure to perform western blot were described in our previous studies [20,21]. The primary antibody was mouse-derived anti-α-syn (Cell Signaling Technology, 2642S, Massachusetts, USA). The thawed α-syn was centrifuged, and the supernatant was collected, diluted and shaken for 7 days at 37 °C. The PFF-containing supernatant possessed a turbid and cloudy appearance. Following ultrasonication at 4 °C, the diluted PFFs were placed onto a carbon-coated copper mesh (ZXBR, China). Uranium acetate 2% was added dropwise to the copper mesh for 30 s to 2 min and dried in ambient air. The samples were observed by transmission electron microscopy (HITACHI, Japan).

Animals and treatment

Six-week-old female C57BL/6 N mice weighing between 25 and 30 g were purchased from the Henan Provincial Laboratory Animal Center and acclimatized in SPF-class animal rooms for 1 week. Subsequently, they were randomly divided into groups that remained housed in SPF-class animal rooms: control group (saline, n = 6), model group (PFFs, n = 7), and prevention group (EGCG+PFFs, n = 7). An additional animal was included in each of the last two groups due to the potential depletion of animals during the experiment because of the neurotoxic nature of PFFs. The experimental protocol is shown in Fig. 1c. The sham group and PFFs group both received intraperitoneal injections of 0.9% saline at the same time every day for the first 7 days. Additionally, EGCG (Sigma-Aldrich; Merck KGaA, Germany) at a dose of 10 mg/kg dissolved in saline was intraperitoneally administered to the EGCG+PFFs group. PFFs (1 μg/μl, 3 μl) were injected after 7 days from the construction of the model by stereotaxic localization in the left-brain striatal region in the PFFs group and PFFs+EGCG group, while, the saline group was injected with 0.9% saline (3 μl). An EGCG-only group was not set up for this experiment because previous work showed no significant difference in striatal dopamine concentration between this group and the control group [22]. The management of laboratory animals and experiments was in accordance with the regulations on Laboratory Animal Management of School of Basic Medical Sciences and the guidelines of the Ethics Committee of Zhengzhou University. One mouse from each of the PFFs and EGCG+PFFs groups was sacrificed after 3 months for brain tissue validation based on Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates and experienced laboratory operators [23]. Three animals from each group were used after 6 months to collect and section the brain and the other three were used for tissue homogenization.

Fig. 1.

Preparation and identification of α-syn-PFFs. (a) Western blotting of the purified α-syn (14 kDa). (b) α-syn-PFFs by transmission electron microscope. Scale bar: 100 nm. (c) Experimental schedule.

The location in the mouse brain for the stereotaxic injection was also based on Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates with anterior fontanelle as the zero point (AP: -0.98 mm; ML: +2.5 mm; DV: -3.5 mm), and then, 3 μl of prepared PFFs (1 μg/μl) were injected with a microinjector at a rate of 0.2 μl/min [23].

Behavioral tests

Four behavioral experiments were performed at 2 weeks, 1 month, 3 months, and 6 months after stereotaxic injection into the brain. The open field apparatus and elevated plus maze apparatus were obtained from Xinruan Information Technology Co., Ltd. (Shanghai, China). The rotating bar fatigue instrument was obtained from Beijing Zhongshidichuang Co., Ltd. (Beijing, China). The instrument was cleaned with 50% ethanol to remove the animal odor before and after each test.

The open field test and elevated plus maze test were performed by placing the mice in the chamber for 1 min for adaptation and then, their trajectory under dim light for 5 min was recorded using the Xinruan software. The open field test was performed by initially placing the animals in the center of the test chamber (45 cm × 45 cm × 45 cm). The elevated plus maze was composed of open arms (5 cm × 50 cm) and closed arms (5 cm × 50 cm × 10 cm) intersecting with a central junction area of 5 cm × 5 cm. The mice were initially placed at the outermost end of the open arm, facing the central area. One day before the rotating rod test, the mice were trained by being placed for 20 min on the rod rotating at 10 rpm/min. The next day the mice were placed on the stationary rotating rod device and the speed was increased from 5 to 40 rpm/min within 300 s. The time from start to drop was recorded and repeated three times for each mouse. The suspension experimental apparatus was constructed by fixing a smooth wire with a diameter of 1.5 mm and a length of 30 cm at a height of 50 cm above the ground on two parallel pieces of cardboard using a glue gun [24]. The two front mouse paws were placed in the mid-point of the 30 cm horizontal wire. The time to falling was recorded within 300 s and repeated three times to determine the average value for each mouse.

Immunohistochemistry

Animals were anesthetized by an intraperitoneal injection of pentobarbital sodium solution (80 mg/kg), and perfused with pre-cooling saline via the left ventricle until the lungs and liver appeared white. The whole brains were fixed with 4% paraformaldehyde, dehydrated with 15%, 25%, and 30% sucrose and stored at 4 °C. Then the dehydrated brain tissues were cut into 25 µm-thick sections according to the instructions of the frozen sectioning machine (Leica, Germany) and stored in cryoprotective solution at −20 °C for further use.

Striatal and SN sections were rinsed with PBST for 15 min and treated with 3% H₂O₂ for 10 min. Then the sections were treated with PBST and blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with mouse-derived anti-Ser 129-phosphorylated α-synuclein (p-α-syn) (Wako, 015-25191, Japan) and rabbit-derived anti-tyrosine hydroxylase (TH) (Millipore, AB152, USA) for 12 h at 4 °C, and rinsed with PBST for 15 min. The SABC (Mouse/Rabbit IgG)-POD Kit (Solarbio, SA0041, China) and the SABC (Rabbit IgG)-POD Kit (Solarbio, SA0021) were utilized for staining. Next, the slides were counterstained with hematoxylin. Finally, the sections were processed by ethanol gradient dehydration, permeabilized with dimethyl benzene, sealed with neutral balsam, and further scanned by the Motic EasyScan (Motic, China). The appropriate images were selected according to Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates. The quantitative evaluation of immunohistochemistry results was analyzed using ImageJ (Fiji) based on the grayscale values in the selected staining areas [25]. The relative density of p-α-syn fibrils compared to the sham group was measured by counting p-a-syn immuno-positive fibrils colored in brown.

Real-time quantitative PCR

Animals were anesthetized by an intraperitoneal injection of pentobarbital sodium solution (80 mg/kg), and perfused with pre-cooling saline via the left ventricle until the lungs and liver appeared white.The samples of striatum and SN of the mouse brains were separated on ice by experienced laboratory operators based on Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates, and then frozen at - 80 °C [23]. The total RNA was extracted from the striatum and SN of brain tissue using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions, and RNA concentration and purity were measured using NanoDrop^TM One (Thermo Fisher Scientific) at OD260/OD280. In a 1.5-ml RNase-free EP tube, 3 μl of total RNA and 1 μl of Oligo (dT) (Takara, Japan) primer were added. RNase-free water was then supplemented to 14 μl, and the tube was placed in a water bath at 70 °C for 5 min and in an ice bath for 5 min. The solution was reverse transcribed, placed in a water bath at 37 °C for 90 min, and then in a water bath at 70 °C for 5 min. The concentration and purity of single-strand cDNA were measured and then, the cDNA was stored at −80 °C. The PCR reaction system was performed on ice using the synthesized cDNA as template, each sample was performed in duplicate and gene expression was detected by fluorescence quantitative PCR (Thermo Fisher Scientific). Mouse primers for each gene are listed in Table 1 with GAPDH as the internal reference. Amplification was run at 95 °C for 180 s followed by 40 cycles of 95 °C for 10 s, 58 °C for 15 s, and 72 °C for 13s, then 95 °C for 15 s as a final elongation step. The relative quantification of mRNA expression was calculated by 2^-∆∆Ct algorithms. All three groups followed exactly the same protocols.

Table 1.

Primer sequences for qPCR analysis

Gene (mouse)	Sequence
TNF-α	Forward:5’-CTGAACTTCGGGGTGATCGG-3’
	Reverse:5’-GGCTTGTCACTCGAATTTTGAGA-3’
IL-1β	Forward:5’- AGTGTGGATCCCAAGCAATACCCA-3’
	Reverse:5’- TGTCCTGACCACTGTTGTTTCCCA-3’
IL-6	Forward:5’- ACTTCACAAGTCCGGAGAGG-3’
	Reverse:5’- TGCAAGTGCATCATCGTTGT-3’
TGF-β	Forward:5’- ACCGCAACAACGCCATCTAT-3’
	Reverse:5’- TGCCGTACAACTCCAGTGAC-3’
IL-4	Forward:5’- GGTCTCAACCCCCAGCTAGT-3’
	Reverse:5’- GCCGATGATCTCTCTCAAGTGAT-3’
IL-10	Forward:5’- CTTACTGACTGGCATGAGGATCA-3’
	Reverse:5’- GCAGCTCTAGGAGCATGTGG-3’
GAPDH	Forward:5’- TGCCCCCATGTTTGTGATG-3’
	Reverse:5’- TGTGGTCATGAGCCCTTCC-3’

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com) and SPSS (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0; IBM Corp., Armonk, New York, USA). Shapiro-Wilk Normality test was performed to show that all the data was normally distributed. The results of the behavioral tests were compared by one-way analysis of variance (ANOVA) followed by the least significant difference test. The data for immunohistochemistry and quantitative PCR (qPCR) were compared using one-way ANOVA followed by Dunnett’s multiple comparison test. Results were expressed as mean ± SEM and a value of P < 0.05 was considered statistically significant.

Results

α-synuclein purification and preformed fibrils preparation

The α-syn proteins were prepared and purified as previously described [20], and the successful preparation was confirmed by western blot (Fig. 1a). The PFFs had an average length of 50 nm (Fig. 1b).

Effect of EGCG pretreatment on behavioral tests in a preformed fibril-induced Parkinson’s disease mouse model

The timeline of the behavioral tests is shown in Fig. 1c. The open field test (Fig. 2a and b) and the elevated plus maze test (Fig. 2c and d) were performed to detect the anxiety level of mice [26]. The open field test revealed that the injection of PFFs significantly enhanced the anxiety-like behavior at 6 months compared with the sham group (P < 0.01). The elevated plus maze test revealed that the exploration distance in the open arm of the group injected with PFFs were significantly reduced at 3 months and 6 months compared to those of the control group (P < 0.05 and P < 0.05). The results of the two behavioral tests demonstrated the successful construction of a PFF-induced chronic PD mouse model. The rotating rod test (Fig. 2e) and the suspension test (Fig. 2f) were conducted to detect muscle strength and motor ability related to PD symptoms. The fall latency of the PFFs group was significantly shorter than that of the sham group in both the rotating rod test and the suspension test at 6 months (P < 0.05 and P < 0.05). Notably, the result of the mean drop latency from the rotating rod test in the EGCG+PFFs group at 1 month suggested that EGCG treatment could ameliorate the motor impairment symptoms induced by the injection of PFFs (P < 0.05) (Fig. 2e).

Fig. 2.

Behavioral tests. (a) Automated tracking of mice in the open field for 5 min. (b) Time spent in the center zone in the open field test. (c) Automated tracking of mice in the elevated plus maze for 5 min. (d) Ratio of distance in the open zone in the elevated plus maze test. (e) Mean latency to fall in the rotating rod test. (f) Mean latency to fall in the suspension test. Within-group: *P < 0.05; between-group: #P < 0.05, ##P < 0.01; n = 6, 7, 7/group (Sham, PFFs, EGCG+PFFs) (2 weeks, 1 month); n = 6, 6, 6/group (3 months, 6 months). CA, closed arm; OA, open arm.

Histochemical evaluation of EGCG pretreatment in a preformed fibril-induced Parkinson’s disease mouse model

The level of TH immuno-positive neurons was detected by immunohistochemical staining in the SN and striatum of PD mice at 6 months to verify the construction of the PD mouse model and explore the neuroprotective effect of EGCG (Fig. 3). Immunohistochemical staining in the striatum showed that TH immuno-positive fibers were relatively abundant in the sham group (0.15 ± 0.001, P < 0.001) and EGCG+PFFs group (0.12 ± 0.011, P < 0.05) compared to the PFFs group (0.09 ± 0.002) at 6 months (Fig. 3a and b). Similarly, the relative number of TH immuno-positive neurons in the SN of the PFFs group (565.37 ± 18.55) was also lower than that in the sham (1346.66 ± 11.64, P < 0.001) and EGCG+PFFs group (855.80 ± 4.43, P < 0.001) groups at 6 months (Fig. 3c and d).

Fig. 3.

Immunostaining of tyrosine hydroxylase (TH) immuno-positive fibers and cells in the striatum and substantia nigra (SN). (a and b) TH immuno-positive fibers in the striatum. Scale bar: 100 μm. (c and d) TH immuno-positive neurons in the SN. Scale bar: 400 μm. *P < 0.05, ***P < 0.001, n = 3/group.

The accumulation of p-α-syn fibrils in Lewy bodies and Lewy neurites is the pathological hallmark of PD. Therefore, the p-α-syn in the striatum of the mouse model was further detected at 6 months to evaluate the pathological process of PD (Fig. 4). The results showed that the relative density of p-α-syn fibrils at 6 months was significantly increased in the PFFs group compared to the sham group (PFFs group: 0.16 ± 0.006; sham group: 0.10 ± 0.007, P < 0.001), and that the increase tended to be suppressed by the pretreatment with EGCG (0.13 ± 0.006, P < 0.05, Fig. 4b).

Fig. 4.

Immunostaining of p-α-syn fibrils in the striatum. Scale bar: 100 μm. *P < 0.05, ***P < 0.001, n = 3/group.

Effect of EGCG on preformed fibril-induced neuroinflammation

The pro-inflammatory and anti-inflammatory cytokines associated with the neuroinflammatory response of the striatum and SN regions were further measured by qPCR at the transcription level to investigate the mechanism of action of EGCG in the process (Table 2).

Table 2.

The expression of inflammatory cytokines was measured using qPCR

Relative mRNA level of SN	2-∆∆Ct		Group comparisons	Significance
	PFFs	EGCG + PFFs
TNF-α	1.15 ± 0.32	1.06 ± 0.04	PFFs vs. sham	NS
			PFFs vs. EGCG + PFFs	NS
IL-6	1.55 ± 0.22	1.14 ± 0.17	PFFs vs. sham	**
			PFFs vs. EGCG + PFFs	*
IL-1	1.53 ± 0.26	1.18 ± 0.23	PFFs vs. sham	*
			PFFs vs. EGCG + PFFs	NS
TGF-β	3.04 ± 0.28	3.91 ± 0.19	PFFs vs. sham	***
			PFFs vs. EGCG + PFFs	**
IL-10	1.32 ± 0.11	1.70 ± 0.12	PFFs vs. sham	**
			PFFs vs. EGCG + PFFs	***
IL-4	1.21 ± 0.11	1.69 ± 0.01	PFFs vs. sham	**
			PFFs vs. EGCG + PFFs	**

Relative mRNA level of striatum	2-∆∆Ct		Group comparisons	Significance
	PFFs	EGCG + PFFs
TNF-α	1.51 ± 0.08	1.07 ± 0.08	PFFs vs. sham	***
			PFFs vs. EGCG + PFFs	***
IL-6	1.43 ± 0.15	1.41 ± 0.22	PFFs vs. sham	*
			PFFs vs. EGCG + PFFs	NS
IL-1	1.45 ± 0.15	1.21 ± 0.04	PFFs vs. sham	**
			PFFs vs. EGCG + PFFs	*
TGF-β	13.82 ± 9.43	26.58 ± 18.89	PFFs vs. sham	NS
			PFFs vs. EGCG + PFFs	NS
IL-10	1.38 ± 0.14	1.66 ± 0.09	PFFs vs. sham	NS
			PFFs vs. EGCG + PFFs	*
IL-4	1.34 ± 0.20	1.77 ± 0.20	PFFs vs. sham	**
			PFFs vs. EGCG + PFFs	*

*P < 0.05, **P < 0.01, ***P < 0.001, n = 3/group.

The treatment with PFFs significantly increased the nigral levels of interleukin-6 (IL-6) (P < 0.01), IL-1 (P < 0.05), and striatal levels of IL-6 (P < 0.05), IL-1 (P < 0.01) and tumor necrosis factor-α (TNF-α) (P < 0.001) compared with the levels in the control group, indicating the increased neuroinflammation in the related brain regions. This increased pro-inflammatory cytokine expression in the PFFs group was significantly reduced by EGCG treatment (nigral levels of IL-6: P < 0.05; striatal levels of IL-1: P < 0.05, TNF-α: P < 0.001). Furthermore, EGCG treatment significantly increased the expression of the anti-inflammatory factors transforming growth factor-β (TGF-β) (P < 0.01), IL-10 (P < 0.001), and IL-4 (P < 0.01) in the SN, and anti-inflammatory factors IL-10 (P < 0.05) and IL-4 (P < 0.05) in the striatum. In general, these results suggest that EGCG pretreatment may alleviate PFF-induced neuroinflammation in the nigrostriatal pathway, thus exerting a neuroprotective role in a PD mouse model.

Discussion

The prevalence of PD is increased significantly associated with global population aging. PD not only seriously affects the health of the elderly but also increases the burden on families and society [27]. The accumulation of LBs in the SN increases with time, and the gradual degeneration of dopaminergic neurons contributes to the worsening of PD [28]. However, there is a lack of effective therapeutic options for PD patients. Traditional therapies, such as levodopa replacement therapy, only provide symptomatic relief [29]. Hence, early diagnosis and early intervention of PD are currently under active investigation [21,30].

PD exhibits a strong correlation with nutritional factors, prompting numerous studies to investigate the association between food and PD risk, especially tea [31,32]. A Mendelian randomization study has found that green tea consumption reduces the risk of PD and indicates that a larger intake of green tea is linked to a slower rate of PD progression [33]. Ingestion of 3 or more cups of tea daily were discovered to have association with a reduction of PD risk [34]. Previous in vivo studies on mice revealed that EGCG prevents the loss of striatal dopaminergic neurons caused by MPTP [35,36]. The natural origin of EGCG and its ability to be absorbed through the digestive tract and cross the blood-brain barrier have also attracted wide attention as a potential clinical therapeutic agent for PD in recent years [37].

In this study, the neuroprotective properties of EGCG on the chronic progression of PD were assessed by constructing, for the first time, a chronic mouse model generated by PFFs subjected to EGCG pretreatment. It is worth emphasizing that the EGCG was administered before the onset of PD (intraperitoneal injection, 1 time/day for 7 days), which differs from the order of other similar studies [22,38]. Given that EGCG currently is unable to entirely halt the course of PD, we prefer to use it as a component to prevent or delay the onset of PD. The concentration of EGCG in mice can reach a relatively constant level after 7 consecutive days of EGCG administration, considering the metabolic half-life of EGCG. At this time, the stable concentration of EGCG played its neuroprotective role after PFFs injection, thus inhibiting the accumulation of α-syn during the rapid recruitment of PFFs in the early stage [39]. However, the results of the open field test suggested that the function of EGCG lasted only 3 to 6 months when EGCG was administered before the injection of PFFs. In future, it would therefore be preferable to continue EGCG intake throughout the whole stage of the follow-up experiment.

The exogenous addition of PFFs can cause endogenous α-syn misfolding, thus constructing the chronic animal PD model [40]. This chronic modeling method can fully simulate the disease process, which is helpful in studying the changes in PD-related behaviors and pathology over time. Consequently, a stereotaxic intra-striatal injection of PFFs into the mouse brain was performed to construct a PD model. Abnormal aggregation of endogenous α-syn leads to the loss of homeostasis of other proteins and membrane structures, such as mitochondria and lysosomes in neurons, all involved in the formation of LBs [41]. Moreover, abnormal α-syn can be transferred among cells in the brain, and neurons gradually lose their function in this process [42].

The gradual decline in neuronal function significantly impairs the behavioral performance. Long-term and multiple behavioral experiments revealed that EGCG ameliorates the anxiety of PD mice, enhances the spontaneous motor ability of mice, and improves muscle strength and the function of motor coordination. Furthermore, immunohistochemistry results showed that EGCG pretreatment significantly reduced the loss of dopaminergic neurons in the SN region, increased the density of TH immuno-positive fibers in the striatum, and reduced the accumulation of p-α-syn at 6 months. Clearly, more sensitive detection of dopamine metabolites by HPLC-ECD and other techniques as well as the determination of neuronal death and apoptosis will be necessary in the future to further confirm this point. Nevertheless, EGCG pretreatment significantly reduced the reduction in immuno-identifiable dopaminergic neurons, a finding that appeared to be correlated with the outcome of behavioral tests.

Recent studies have indicated that the neuroprotective effect of EGCG is also closely linked to its anti-inflammatory property [43,44], and that IL-1β, IL-2, IL-6, IL-10, and TNF-α may be used as biomarkers to assess the neuroinflammation in PD [45]. Consequently, the related transcriptional expression of inflammatory factors in different brain areas was investigated in our study. Since microglia are important executors of the neuroinflammatory response, polarization of microglia may be involved in the process of EGCG’s anti-inflammatory effect [46]. Research has indicated that EGCG may have a potential preventive effect on PD by reducing the first uptake of toxic α-syn short fibrils by microglia [47]. The results of our study indicated that EGCG exerted anti-inflammatory properties by inhibiting the release of pro-inflammatory cytokines and promoting the release of anti-inflammatory cytokines at the transcriptional level. However, further investigation is required to examine the intricate mechanism involved.

Our study still has some limitations due to the limited sample size. It would be better to use quantitative analysis by western blot for the detection of TH and p-α-syn. Furthermore, it is necessary to increase the sample size in future follow-up research. In conclusion, the successful construction of a chronic model of PD induced by PFFs was obtained in mice according to various behavioral and histochemical perspectives, and confirmed the potential preventive ability of EGCG in PD mice to protect dopaminergic neurons, revealing the abilities of EGCG in delaying PD development in a mouse model, suggesting the potential value of the green tea extract EGCG as a clinical PD preventive intervention component.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82101506), the Scientific project of Beijing Life Science Academy (No. 2023000CC0140), China Postdoctoral Science Foundation (No. 2023M733887), and National College Students Innovation and Entrepreneurship Training Program of Zhengzhou University (No. 2021cxcy363).

Conflicts of interest

There are no conflicts of interest.