Application of mitochondrial glutamate transporter SLC25A22 in treatment of Parkinson's disease

Abstract

The invention discloses an application of mitochondrial glutamate transporter SLC25A22 in treatment of Parkinson's disease, and provides an application of SLC25A22 as a target in screening of drugs for treatment of Parkinson's disease and an application of a preparation for promoting expression of SLC25A22 in preparation of drugs for treatment of Parkinson's disease. Research finds that SLC25A22 promotes utilization of glutamic acid in MN9D cell mitochondria, synthesis of alpha-ketoglutaric acid and TCA circulation, and improves generation reduction of ATP in MN9D cells induced by MPP +, ROS accumulation and mitochondrial membrane potential decline, so that damage of MN9D cell activity is relieved, and finally loss of dopaminergic neurons is rescued.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of mitochondrial glutamate transporter SLC25A22 in the treatment of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease in the world after Alzheimer's disease, and it mostly occurs in the elderly population, imposing a heavy economic burden on patients, their families, and society. The main pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and the aggregation of α-synuclein (α-syn). The motor features of PD are mainly resting tremor, bradykinesia, and body rigidity. The phenotypes associated with the onset of PD are complex and diverse. Currently, studies have found that PD is related to neuroinflammation, oxidative stress, mitochondrial dysfunction, etc., but the specific pathogenesis is still unclear. At present, the treatment methods for PD include motor rehabilitation, surgical treatment, and drug treatment, but drug treatment is still the main treatment method. The therapeutic drugs include levodopa, carbidopa, etc. The mechanism of these drugs is to penetrate the blood-brain barrier as a dopamine (DA) precursor to supplement the lack of DA in the striatum of PD patients. However, these treatments can only partially improve the clinical symptoms of patients, and cannot effectively improve the loss of dopaminergic neurons. Moreover, the by-products are neurotoxic, and long-term drug exposure may bring irreversible consequences to PD patients. Therefore, finding an effective treatment method is the key research content of Parkinson's disease at present.

[0003] Butyric acid is one of the main short-chain fatty acids (SCFAs), which naturally exists in animals and is mainly produced by the metabolism of intestinal flora in the colon. It is also the main energy source of colon cells. Butyric acid is unstable in the body and often exists in the form of butyrate. Sodium butyrate (NaB) is a common butyrate, which has good anti-inflammatory effects and can repair the intestinal barrier in colitis. In addition, in diabetic nephropathy, it is believed that NaB can induce histone Kbu or H3K9bu to relieve renal damage caused by inflammation and fibrosis. In a stroke rat model, NaB activates PI41K / Akt through GPR3 / Gβγ, reducing neuronal apoptosis after stroke. Some studies have observed that in α-syn-induced PD rats, NaB reduces the levels of pro-inflammatory factors and restores dopamine levels, and the levels of autophagy-related genes and proteins are significantly improved, and the acetylation of histone H3 increases accordingly. Clinical reports have shown that the bacteria producing SCFAs in the intestines of PD patients decrease, and at the same time, a decrease in the content of SCFAs has also been detected in the fecal specimens of the patients. These short-chain fatty acids are the key to the physiological interaction between microorganisms and the host. In particular, the beneficial effects of sodium butyrate are known, and some studies have shown its role in regulating microglial activation and potentially inhibiting neuroinflammation in Parkinson's disease. It has been confirmed that gavage with NaB can relieve the motor disorders and the loss of dopaminergic neurons in the substantia nigra of PD mice, and can regulate the disorder of the intestinal flora in PD mice. However, the specific mechanism of the neuroprotective effect of NaB in the brains of PD mice is still unclear.

[0004] Mitochondria play an important role in the neurodegenerative process of Parkinson's disease. They play a key role in cellular energy production and cell signaling, using the bioenergetic state of the cell to determine whether the cell survives or degenerates. Synaptic damage and mitochondrial dysfunction are early events in the pathogenesis of Parkinson's disease, and alterations in mitochondrial structure and dynamics are associated with increased production of reactive oxygen species, abnormal intracellular calcium levels, and reduced mitochondrial ATP production. Neurons, especially dopaminergic neurons, have high energy demands and require high-quality mitochondrial bioenergetic function to maintain normal cell function and survival. Mitophagy is a process that selectively targets damaged or redundant mitochondria to lysosomes for elimination via the autophagy pathway and is essential for maintaining mitochondrial health. The two Parkinson's disease genes PINK1 and PRKN are involved in mitochondrial quality control in response to marked mitochondrial stress but are not required for all types of mitophagy. The LRRK2 Gly2019Ser mutation is the most common LRRK2 mutation in Parkinson's disease and has recently been shown to disrupt basal mitophagy in vivo, suggesting that other forms of mitophagy may play a role in neuronal survival. Recent literature suggests that other mitochondrial interactions are causative of Parkinson's disease. For example, aggregated α-synuclein has been reported to permeabilize the mitochondrial membrane and impair the electron transport chain to enhance oxidative stress-mediated apoptosis in neurons, and hypoxia may further exacerbate this process. Mitochondrial dysfunction and excessive production of reactive oxygen species promote the formation of soluble α-synuclein oligomers and insoluble fibrils. This suggests that studying mitochondrial dysfunction has important value for the treatment of Parkinson's disease.

[0005] Solute carrier family 25 member 22 (SLC25A22) is a mitochondrial glutamate transporter responsible for transporting glutamate from the cytosol into the mitochondrial matrix while importing protons, and is involved in glutamine metabolism and energy metabolism (such as the TCA cycle). Diseases associated with SLC25A22 include developmental and epileptic encephalopathies and early infantile epileptic encephalopathy. Downregulation of SLC25A22 expression in pancreatic ductal adenocarcinoma (PDAC) cells is associated with increased ferroptosis sensitivity. SLC25A22-dependent NAPDH synthesis blocks ferroptosis in PDAC cells by mediating the production of glutathione (GSH), and promotes the expression of stearoyl-CoA desaturase in PDAC cells in an AMP-activated protein kinase-dependent manner, resulting in the production of anti-ferroptotic monounsaturated fatty acids (MUFAs) and resistance to ROS accumulation. Studies have found that in radioresistant glioblastoma multiforme (GBM) cells, SLC25A22 is a mitochondrial bidirectional glutamate transporter, and upregulation of SL2C25A22 leads to the accumulation of cytoplasmic glutamate, and the accumulated cytoplasmic glutamate enhances the production of glutathione (GSH) and proline synthesis in radioresistant GBM cells. The increased GSH protects cells from the effects of reactive oxygen species (ROS) induced by ionizing radiation (IR).

[0006] Previous studies have shown that NaB can inhibit the loss of dopaminergic neurons in PD mice and can affect the rhythmic behavior of mice through gut genes. However, the mechanism by which NaB exerts its neurological effects in the brain remains unclear. Summary of the Invention

[0007] The object of the present invention is to provide an application of the mitochondrial glutamate transporter SLC25A22 in the treatment of Parkinson's disease in view of the above problems.

[0008] In order to achieve its object, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides an application of SLC25A22 as a target in screening drugs for the treatment of Parkinson's disease.

[0010] The drug promotes the expression of SLC25A22 or increases its protein activity.

[0011] Further preferably, the drug is a preparation overexpressing SLC25A22.

[0012] Further preferably, the drug is an SLC25A22 overexpression plasmid.

[0013] The second aspect of the present invention provides an application of a preparation for promoting the expression of SLC25A22 in the preparation of a drug for the treatment of Parkinson's disease.

[0014] The drug is an SLC25A22 overexpression plasmid.

[0015] The backbone vector of the overexpression plasmid is pcDNA3.1(+).

[0016] In any of the above-mentioned applications, SLC25A22 promotes the utilization of glutamate, the synthesis of α-ketoglutaric acid, and the TCA cycle in the mitochondria of MN9D cells, improves the reduction of ATP production, ROS accumulation, and mitochondrial membrane potential decline in MPP+-induced MN9D cells, thereby alleviating the impairment of MN9D cell activity and ultimately rescuing the loss of dopaminergic neurons.

[0017] In MN9D cells, the mitochondrial glutamate transporter SLC25A22 transports glutamate from the cytoplasm into the mitochondria, generates α-ketoglutaric acid to participate in the TCA cycle, restores cellular energy metabolism, and promotes GSH production to improve cellular mitochondrial dysfunction and alleviate the loss of dopaminergic neurons.

[0018] The beneficial effects of the present invention are as follows:

[0019] The present invention studies and discovers that NaB treatment can improve glutamate excitotoxicity in PD mice and the reduction of GSH in the substantia nigra. In MPP+-treated MN9D cells, SLC25A22 transports glutamate from the cytoplasm into the mitochondria, generates α-ketoglutaric acid to participate in the TCA cycle, restores cellular energy metabolism and GSH synthesis, and improves cellular mitochondrial dysfunction. The present invention discovers the molecular mechanism by which SLC25A22 participates in the neuroprotective effect of NaB on PD mice, providing new targets and new alternative drugs for the treatment of PD. Brief Description of the Drawings

[0020] Figure 1 Shows that NaB improves the motor dysfunction, α-syn accumulation, and DA neuron death in MPTP mice: (A) Schematic diagram of mouse treatment; (B) Movement trajectories of mice in the open field test; (C) Movement distance of mice in the central area of the open field test; (D) Average speed of mice in the open field test; (E) Movement time of mice in the pole climbing test; (F) Retention time of mice on the rotarod; (G) Protein immunoblot analysis of TH and α-syn protein content levels in the striatum of mice; (H-I) Quantitative analysis of the proteins in Figure G; (J) Immunofluorescence staining of dopaminergic neuron marker TH (green) and DAPI (blue) in the substantia nigra; (K) Quantitative analysis of TH+ dopaminergic neurons in the substantia nigra; n = 9 mice; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.001, ns indicates no statistical significance.

[0021] Figure 2Shows the differentially expressed genes in the midbrain of mice after NaB treatment: (A) Volcano plot of differentially expressed genes found by RNA sequencing in PDVS NC and PDVS NaB; (B) Venn diagram of the intersection of differential genes in NC VS PD and NaB VS PD; (C) Bubble plot of KEGG enrichment analysis, with multiple genes enriched in the tumor necrosis factor and glutathione metabolism signaling pathways; (D) TPM values of the six candidate genes in the NC group; (E) Relative expression levels of these six genes in the midbrain detected by qRT-PCR; (F) Representative immunoblot image of SLC25A22 in the midbrain; (G) Quantitative analysis chart of SLC25A22 immunoblot; n = 3 mice in each group; *p < 0.05, **p < 0.01, ***p < 0.001.

[0022] Figure 3 Shows that NaB feeding reduced the levels of inflammatory factors TNF-α and NF-κB in the striatum of MPTP mice: (A) Immunoblot images of NFκB, IL-6, and TNF-α in the midbrain tissue of mice; (B, C) Quantitative analysis charts of TNF-α and NF-κB immunoblots; n = 3 mice in each group; *p < 0.05, **p < 0.01, ***p < 0.001.

[0023] Figure 4 Shows that NaB feeding alleviated the excitatory neurotoxicity of glutamate in MPTP mice: (A) Immunoblot images of EAAT2 and GFAP in the striatum of mice; (B) (C) Quantitative analysis; (D) Glutamate content in the substantia nigra region of mice; (E) Image showing immunofluorescence staining of the activated astrocyte marker GFAP (green) and DAPI (blue) in the substantia nigra; (F) Quantitative analysis; n = 3 mice in each group; *p < 0.05, **p < 0.01, ***p < 0.001.

[0024] Figure 5 Shows that NaB treatment can improve the reduction of GSH in PD model mice: (A) Representative immunoblot images of SLC77A11 and GPX4 in the substantia nigra of mice; (B) (C) Quantitative analysis; n = 9 in each group; (D) Relative content of reduced glutathione in the substantia nigra of mice. n = 9 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no statistical significance.

[0025] Figure 6Decreased SLC25A22 expression was shown in DA neurons of MPTP mice and MPP+-treated MN9D cells: (A) Images showed immunofluorescence co-localization staining of SLC25A22 (red), dopamine neuron marker TH (green), and DAPI (blue) in the substantia nigra; (B) CCK8 assay was used to determine cell viability after treatment with different concentrations of MPP+; (C) CCK8 assay was used to determine cell viability after treatment with MPP+ and addition of different concentrations of NaB; (D) Representative immunoblot images of TH and SLC25A22 in MN9D cells; (E,F) Quantitative analysis of TH and SLC25A22 bands; (G) Immunofluorescence staining of SLC25A22 (green), Mitotracker (red), and DAPI (blue) in MN9D cells; n = 3 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ns means no statistical significance.

[0026] Figure 7 Overexpression of SLC25A22 was shown to alleviate the decrease in cell viability and the reduction in TH protein expression after MPP+ treatment: (A) CCK8 assay was used to determine cell viability; (B) Representative immunoblot images of SLC25A22 and TH in MN9D cells; (C)(D) Quantitative analysis of TH and SLC25A22; n = 3 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ns means no statistical significance.

[0027] Figure 8 Overexpression of SLC25A22 was shown to alleviate the decrease in GSH and the increase in ROS induced by MPP+: (A) Representative immunoblot images of GPX4 and SLC7A11 in MN9D cells; (B)(C) Quantitative analysis; (D)(E) Contents of reduced GSH and Glu in MN9D cells; (F) RT-qPCR was used to detect the relative mRNA expression levels of GCLM, GCLC, and GS; (G) Detection of intracellular NADP+ and NADPH contents; (H) ROS probe was used to label the reactive oxygen species in MN9D cells; (I) ROS fluorescence quantitative analysis; n = 3 in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ns means no statistical significance.

[0028] Figure 9Overexpression of SLC25A22 alleviates mitochondrial dysfunction in MN9D cells by promoting Glu synthesis into α-AKG: (A) Detection of intracellular glutamate dehydrogenase activity; (B) Detection of α-ketoglutarate dehydrogenase activity; (C) Representative immunoblotting of α-ketoglutarate dehydrogenase subunit OGDHL and glutamate dehydrogenase subunit GLUD1 in MN9D cells; (D) Quantitative analysis of OGDHL; (E) Quantitative analysis of GLUD1; (F) Detection of intracellular α-ketoglutarate content; (G) Detection of ATP content in MN9D cells; (H) Labeling of mitochondrial membrane potential in MN9D cells with JC-1 probe; (I) Quantitative analysis of JC-1; n = 3 for each group; *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no statistical significance. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereby.

[0030] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0031] Research method of the present invention: 27 male C57BL / 6J mice at 7 weeks old were used to establish a PD mouse model. These mice were randomly and evenly divided into a normal control group (Normal control, NC), a model group (MPTP), and a NaB treatment group (NaB), with 9 mice in each group. In addition to injecting an equal volume of normal saline into the mice in the NC group, the mice in the MPTP group and the NaB group were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP, 30 mg / (kg*d)] for 5 consecutive days to establish a subacute PD model. After the modeling was completed, the mice in the NaB group were continuously gavaged with NaB [NaB, 300 mg / (kg*d)] for 14 days, and the mice in the MPTP group and the NC group were gavaged with an equal volume of normal saline. After the treatment, the motor function of the mice was detected by behavioral experiments, and 3 mice were randomly selected from each group for RNA sequencing. Immunoblotting was used to detect the expression of TH, α-syn, NF-κB, TNF-α, GFAP, and EAAT2 in the striatum of the mice. The number of dopaminergic neurons and activated astrocytes in the substantia nigra of the mice was detected by immunofluorescence. The content of glutamate and glutathione in the striatum was detected. Through RNA sequencing analysis, it was found that SLC25A22 was significantly highly expressed in the middle brain tissue of mice and was significantly differentially expressed in MPTP mice. Therefore, we selected SLC25A22 as the research object of this topic. In order to explore the specific mechanism of SLC25A22 in protecting dopaminergic neurons, we constructed an overexpression plasmid of SLC25A22, overexpressed SLC25A22 in MN9D cells, and stimulated them with MPP+. CCK-8 was used to detect cell viability. The contents of glutamate, glutathione, and α-ketoglutaric acid in MN9D cells were detected. And the activities of glutamate dehydrogenase and α-ketoglutaric acid dehydrogenase were detected. RT-qPCR was used to detect GCLM, GCLC, and GS. Immunoblotting was used to detect the expression levels of TH, SLC7A11, GPX4, OGDHL, and GLUD1 proteins in the cells. We also detected the ATP content, NADP+ / NADPH, ROS, mitochondrial membrane potential, and mitochondrial morphology.

[0032] Results: The results of behavioral experiments showed that NaB treatment could improve the locomotor speed and limb strength of MPTP mice. Western blot results showed that NaB treatment up-regulated TH and down-regulated the expression of α-syn in the striatum of MPTP mice. Immunofluorescence results showed that NaB treatment up-regulated the decrease of dopaminergic neurons in the substantia nigra of MPTP-induced mice. The results of midbrain RNA sequencing analysis showed that genes were enriched in the TNF signaling pathway and glutathione metabolism signaling pathway. The differential gene SLC25A22 was down-regulated in dopaminergic neurons of MPTP mice. Western blot results showed that NaB treatment down-regulated the expression of NF-κB and TNF-α in the striatum. Western blot results showed that NaB improved the down-regulation of EAAT2, up-regulation of GFAP, down-regulation of SLC7A11 and GPX4 in the striatum of MPTP mice, and up-regulated the decrease of Glu and GSH in the MPTP group. Overexpression of SLC25A22 in MN9D cells increased cell viability, protein expression of TH, SLC7A11, GPX4, and mRNA expression of GCLM, GCLC, and GS after MPP+ treatment. Overexpression of SLC25A22 reduced Glu in cells, increased GSH in cells, and alleviated the accumulation of intracellular ROS, the decrease of NADP+ / NADPH ratio, the decrease of mitochondrial membrane potential, the decrease of ATP production, and the damage of mitochondrial morphology compared with MPP+ treatment. The activities of glutamate dehydrogenase and α-ketoglutarate dehydrogenase in cells increased, and the protein expression of OGDHL and GLUD1 was up-regulated.

[0033] The specific research process is as follows:

[0034] Example 1

[0035] Experimental Materials and Methods

[0036] 1.1 Breeding of Experimental Animals and Establishment of Models

[0037] Male C57BL / 6J mice at 7 weeks of age with a body weight of about 20-22 g were selected for this experimental study and purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were raised under normal conditions at a temperature of about 25 °C, a humidity of about 55%, and a light-dark cycle of 12 hours of light and 12 hours of darkness. The mice had free access to water and food. After one week of adaptive feeding, the mice were randomly and evenly divided into the NC group, PD group (Parkinson's disease group), and NaB group. All animal studies in this experiment were approved by the Ethics Committee of Chongqing Medical University.

[0038] Model establishment: Mice in the PD group and NaB group were intraperitoneally injected with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) solution at a concentration of 3 mg / mL for 5 consecutive days. The dosage was 0.1 ml per 10 g of mouse body weight. The NC group was intraperitoneally injected with the corresponding volume of normal saline. After 5 days of model establishment, mice in the NaB group were intragastrically administered with NaB (sodium butyrate) solution at a concentration of 30 g / ml at a dosage of 0.1 ml per 10 g of mouse body weight, while mice in the NC group and PD group were intragastrically administered with the corresponding volume of normal saline for 14 days.

[0039] 1.2 Main reagents and instruments

[0040] Antibodies against TH, GAPDH, Bmal1, and occludin were purchased from Proteintech Group, Inc. (USA); the NFκB antibody was purchased from Cell Signaling Technology; MPTP, sodium butyrate, and MPP + were purchased from Sigma-Aldrich (USA); Trizol was purchased from Takara; the reverse transcription kit and SYBR GREEN were purchased from ABclonal; the RIPA lysis buffer and BCA protein quantification kit were purchased from Beyotime Institute of Biotechnology; the ECL chemiluminescence solution was purchased from Millipore; the PCR instrument, fluorescence quantitative PCR instrument, electrophoresis apparatus, and gel imaging system were purchased from Bio-Rad; the open field test chamber was purchased from Shenzhen Rewod Life Science Co., Ltd.

[0041] 1.3 Behavioral experiments

[0042] (1) Open field test

[0043] The open field test was used to measure the spontaneous activity and exploratory behavior of mice. The open field test chamber was a box with dimensions of 50 cm × 50 cm × 40 cm (length × width × height). Before the experiment, the test chamber was ensured to be clean and odor-free. Then, the experimental mice were quickly placed in the central area of the test chamber and the experimenter immediately left. The entire experiment lasted for 5 minutes. The SMART 3.0 software was used to measure and record the movement trajectory images of the mice, the total movement distance, the movement distance in the central area, and the number of times crossing the central area. After each mouse completed the experiment, the entire test chamber was wiped with 75% alcohol to remove the feces, urine, and odor left by the previous mouse.

[0044] (2) Pole climbing test

[0045] The device for the pole climbing exercise is a smooth wooden stick with a diameter of 1 cm and a length of 50 cm, and a small wooden ball with a diameter of 2 cm fixed at the upper end of the stick. To prevent slipping, the stick and the small wooden ball are wrapped with gauze. The mouse is placed on the wooden ball with its head down and tail up, and the time it takes for the mouse to climb down the entire stick is recorded. A 3-day training was conducted before the formal experiment. The experiment was repeated 3 times, with each experiment interval of 15 min, and the average value was taken.

[0046] (3) Rotarod test

[0047] The mouse is placed on the rotating rod and rotated at a speed of 4 rpm. The rotating rod accelerates uniformly at 40 rpm within 5 minutes; and the falling time of the mouse is recorded. Three rotating tests are conducted on the animal, with an interval of 1 hour between each test. The average value is taken.

[0048] 1.4 Sampling

[0049] After the mouse is anesthetized, it is fixed on a foam box, and its chest cavity and heart are exposed. A scalp needle is inserted into the heart apex along the left ventricle, and at the same time, the right auricle is cut open. After seeing dark red blood gushing out, pre-cooled normal saline is quickly injected into the heart. When the color of the liver becomes lighter, the lungs are not significantly enlarged, and there is no obvious fluid leakage from the mouth and nose, it indicates successful perfusion. For some mice, the brain is directly dissected and the brain tissue is stored at -80 °C; for the other part of the mice, 4% paraformaldehyde solution is perfused immediately after the perfusion of normal saline. When it is observed that the limbs and tail of the mouse tremble and spasm and the body becomes stiff, it indicates successful perfusion with paraformaldehyde. The mouse brain is taken and soaked in 4% paraformaldehyde solution for standby.

[0050] 1.5 Tissue sequencing

[0051] Three mice are randomly selected from each group and sent fresh midbrain tissue to Shanghai Sangon Biotech Co., Ltd. for transcriptome sequencing.

[0052] 1.6 Western blot

[0053] After mincing tissues such as the substantia nigra and striatum of mice as finely as possible, transfer them into RIPA lysis buffer containing PMSF in advance for homogenization, and perform the operation on ice. Then centrifuge at 14000 r / min for 5 min, and the supernatant is the extracted protein solution. After measuring the protein concentration by the BCA method, add 5x protein loading buffer to the protein solution, boil at 100 °C for 10 min, and then take the same mass of protein for SDS-PAGE gel electrophoresis. Transfer the membrane at a constant current of 250 mA according to 1 KD = 1 min, block with 10% skim milk blocking solution at room temperature for 2 h, incubate with TH (diluted 1:1000, Sanko), SLC25A22 (diluted 1:1000, Sanko), NFκB (diluted 1:500, CST), TNF-α (diluted 1:1000, Sanko), GAPDH (diluted 1:5000, Sanko), GFAP (diluted 1:5000, Sanko), EAAT2 (diluted 1:5000, Sanko), GPX4 (diluted 1:500, Sanko), SLC7A11 (diluted 1:500, Sanko), OGDHL, GLUD1, primary antibodies on a shaker in a 4 °C refrigerator for 12 - 16 h, wash the membrane 3 times with TBST, 10 min each time, incubate with secondary antibody (diluted 1:5000, Boster) on a shaker at room temperature for 1 - 1.5 h, wash the membrane 3 times with TBST, 10 min each time. Detect with an ECL chemiluminescence kit and image with a gel imaging system.

[0054] 1.7 Immunofluorescence

[0055] Use a cryostat to cut mouse brain tissue into 5-μm-thick sections (CM1950, Leica, Wetzlar, Germany). Collect the sections containing the substantia nigra-striatum part. Select a total of 10 representative sections containing the main part of the substantia nigra from 2.92 mm to 3.52 mm anterior to the bregma from the sections collected from each mouse for TH staining. Briefly, after repairing the slides with sodium citrate buffer (pH value, 6.0), incubate the brain sections in PBS containing 0.2% v / v Triton X 100 for 10 min, and then incubate in 5% v / v goat serum at 37 °C for 1 h. Incubate the samples with rabbit anti-TH (1:200, EP1532Y, Abcam, Cambridge, UK) primary antibody overnight at 4 °C. After rewarming for 0.5 h, detect the primary antibody with a FITC-labeled appropriate goat anti-rabbit antibody (1:500, A-11008, Thermo Fisher, Waltham, MA, USA). Detect the cell nuclei with DAPI solution (Solarbio, Beijing, China). Record representative images with the aid of a fluorescence microscope (DFC7000T, Leica, Germany). Each group contains 3 animals. Count the TH-positive cells in the substantia nigra of each group with ImageJ (NIH, Bethesda, MD, USA, version 1.53) software.

[0056] 1.8 qRT-PCR

[0057] Total RNA was extracted from the midbrain tissues of mice by the Trizol method, and total RNA in MN9D cells was extracted using a cell RNA extraction kit (Accurate Biology, AG21024, China). Reverse transcription was performed using an ABclonal reverse transcription kit to obtain a cDNA template. Real-time fluorescence quantitative PCR was carried out using SYBR GREEN reagent, with β-actin as an internal reference, and the relative mRNA expression levels of genes were calculated according to the method. The primer sequences used for qRT-PCR are shown in Table 1:

[0058] Table 1 Primer sequences

[0059]

[0060]

[0061] 1.9 Glutamate (Glu) content analysis

[0062] A glutamate assay kit (Solarbio, BC1585, China) was used to measure the glutamate levels in mouse tissues and cells. 100 mg of tissue was taken or 5×106 MN9D cells were collected, and lysates were obtained according to the manufacturer's instructions. Fluorescence was measured using a microplate reader at OD 450 nm. Find a microplate reader to measure fluorescence.

[0063] 1.10 Reduced glutathione (GSH) content determination

[0064] The intracellular GSH levels were measured using a glutathione assay kit (Solarbio Life Sciences, BC1175, China). After collecting 5×10 6 treated MN9D cells, the GSH levels were measured according to the manufacturer's instructions.

[0065] 1.11 Cell culture and treatment

[0066] The MN9D cells were purchased from iCell Bioscience (China) and cultured in RPMI-1640 (Gibco, California, USA) containing 10% fetal bovine serum (Gibco, California, USA) and 1% penicillin / streptomycin / amphotericin B (Beyotime Biotechnology, Shanghai, China) in an incubator at 37 °C and 5% CO2. Once the MN9D cells reached 80% confluence, cell passage was performed using standard cell culture techniques. Specifically, for routine cell culture passage, 0.25% trypsin-EDTA (Beyotime Biotechnology, China) was used to detach the cells from the cell culture flask or plate until the logarithmic growth phase.

[0067] The MPP+ powder was prepared as a stock solution with a concentration of 1 mM using sterilized phosphate-buffered saline (PBS). When in use, the MPP + stock solution was diluted to 50 μM, 100 μM, 200, 400 μM with complete medium, and the cells were treated with different concentrations of MPP + for 24 hours. The NaB was first prepared as a 10 mM stock solution with PBS, and the cells were cultured with medium containing different concentrations of NaB (2 mM, 4 mM, 8 mM) for 24 hours before MPP+ treatment.

[0068] 1.12 Cell transfection

[0069] To construct the overexpression vector, the cDNA sequence of SLC25A22 (Genebank ID: NM_001177576.1) was cloned into the empty vector pcDNA3.1 from Beijing Tsingke Biotechnology. Cells were seeded in six-well plates at a concentration of 1×10 5 cells per well and waited until the cell density was about 60 - 80%. Transfection was carried out using Lipofectamine 2000 (Thermo Fisher, 11668019, USA) reagent according to the manufacturer's protocol.

[0070] 1.13 Cell viability assay

[0071] Cell viability was evaluated using the CCK-8 method. The treated cells were seeded at 5×10 4Cells were seeded at a density of [number of cells] into 96-well plates and placed in an incubator for 8 - 12 hours. Subsequently, 10 μL of CCK-8 solution was added to each well according to the manufacturer's instructions (Beyotime Biotechnology, C0042, China) and incubated for 2 hours. The absorbance of the blank well (Ab), sample well (As), and control well (Ac) was measured using a microplate reader at a test wavelength of 450 nm. The cell viability rate (%) was equal to (As - Ab) / (Ac - Ab) × 100%.

[0072] 1.14 Detection of Reactive Oxygen Species (ROS) Content

[0073] According to the manufacturer's instructions, the ROS level was determined using a fluorescence microscope with a reactive oxygen species detection kit (Beyotime Biotechnology, S0033S, China). Briefly, the cells were detached and washed twice with PBS, resuspended in 300 μL of binding buffer containing 3 μL of FITC-DCFH-DA, and incubated at room temperature for 20 minutes. The fluorescence intensity was measured using a fluorescence microscope (DFC7000T, Leica, Germany).

[0074] 1.15 Determination of NADP+ / NADPH

[0075] After treating MN9D cells according to the experimental requirements, [number of cells] were collected, and the NADP+ / NADPH level was determined using a NADP+ / NADPH detection kit (Beyotime Biotechnology, S0179, China) according to the manufacturer's instructions. The absorbance at 450 nm was measured. Finally, a BCA assay was performed to determine the protein concentration. 6 After treating MN9D cells according to the experimental requirements, [number of cells] were collected, and the NADP+ / NADPH level was determined using a NADP+ / NADPH detection kit (Beyotime Biotechnology, S0179, China) according to the manufacturer's instructions. The absorbance at 450 nm was measured. Finally, a BCA assay was performed to determine the protein concentration.

[0076] 1.16 Determination of Glutamate Dehydrogenase (GDH) Activity

[0077] After treating the cells according to the experimental requirements, the cells were harvested, and the absorbance was measured according to the manufacturer (Solarbio, BC1465, China) to estimate the activity of GDH.

[0078] 1.17 Determination of α-Ketoglutarate Dehydrogenase (α-KDGH) Activity

[0079] According to the manufacturer's instructions, a microplate α-KGDH detection kit (Solarbio, BC7015, China) was used to measure the absorbance to estimate the activity of α-KGDH.

[0080] 1.18 Detection of α-Ketoglutaric Acid (α-KG) Content

[0081] According to the manufacturer's instructions, the ROS level was determined using a fluorescence microscope with a FITC Dichrofuorescin Dictate (FITC-DCFH-DA) detection kit (Beyotime Biotechnology, China). Briefly, cells were isolated and washed twice with PBS, resuspended in 300 μL of binding buffer containing 3 μL of FITC-DCFH-DA, and incubated at room temperature for 20 minutes. Fluorescence intensity was measured with a fluorescence microscope (DFC7000T, Leica, Germany).

[0082] 1.19 ATP content detection

[0083] First, the culture medium was aspirated, and then 5 x 10 6 cells were collected. The ATP content in MN9D cells was detected using an enhanced ATP assay kit (Beyotime Biotechnology, S0027, China) according to the manufacturer's protocol.

[0084] 1.20 Mitochondrial membrane potential (MMP) detection

[0085] The mitochondrial membrane potential was measured using a JC-1 probe detection reagent kit (Beyotime Biotechnology, C2006, China) to determine the mitochondrial membrane potential. Cells were stained with JC-1 dye (concentration: 5 μM; red: EX / EM = 535 / 590 nm; green: ex / em = 485 / 530 nm) for 30 minutes. Then, fluorescence intensity was measured with a fluorescence microscope (DFC7000T, Leica, Germany).

[0086] 1.21 Transmission electron microscopy (TEM) analysis

[0087] MN9D cells were perfused with 2.5% glutaraldehyde. The specimens were fixed in 1% osmium tetroxide, stained in an aqueous acetate solution, then dehydrated and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and examined with a transmission electron microscope. Healthy mitochondria have a clear membrane structure. The inner membrane folds to form normal mitochondrial cristae, including lamellar, tubular, and vesicular cristae. Mitochondrial damage is characterized by the disappearance of the double-membrane structure and mitochondrial cristae, a decrease in internal density, an increase in blank areas, and blurred edges. Three to five fields of view were randomly selected for each group to take pictures, and the percentage of mitochondrial damage was calculated.

[0088] 2. Experimental results

[0089] 2.1 NaB feeding alleviated the motor disorders, increase in α-syn protein, and decrease in dopaminergic neurons induced by MPTP in mice

[0090] To investigate the effect of NaB treatment on the motor function of MPTP mice, we conducted open field tests, pole climbing tests, and rotarod tests on the mice. Compared with the mice in the NC group, the MPTP group of mice showed obvious motor disorders, specifically manifested as a shorter total movement distance in the central area and a slower average movement speed, while the climbing time of the mice increased and the time staying on the rotarod decreased. After treatment with NaB feeding, the motor ability disorder of the mice was significantly improved( Figure 1 B-F).

[0091] To detect the effect of NaB feeding on α-synuclein aggregation and the reduction of dopaminergic neurons, we used WB to detect the protein levels of tyrosine hydroxylase (TH) and α-synuclein in the striatum of mice in each group. The results showed that compared with the NC group, the expression of TH protein in the striatum of MPTP-induced mice was down-regulated and the expression of α-synuclein protein increased, while after treatment with NaB feeding, the reduction of TH protein in the striatum of mice was improved and the increase in the expression of α-synuclein protein was alleviated. At the same time, treatment with NaB could rescue the death of DA neurons in the substantia nigra of MPTP-induced mice( Figure 1 G-K). These results indicate that NaB feeding can reduce MPTP-induced behavioral disorders, increase in α-synuclein, and loss of DA neurons.

[0092] 2.2 Differentially expressed genes in the midbrain of mice related to NaB treatment

[0093] We randomly selected the midbrain tissues of 3 mice in each of the NC group, MPTP group, and NaB group for RNA sequencing analysis. Using FC>1, P<0.05 as the standard, we found that compared with the NC group, there were 155 genes up-regulated and 86 genes down-regulated in the midbrain tissues of the MPTP group of mice. Compared with the MPTP group, we found that there were 139 genes up-regulated and 373 genes down-regulated in the midbrain tissues of the NaB group of mice after sodium butyrate feeding( Figure 2 A). To further explore the molecular mechanism of sodium butyrate feeding in the midbrain of MPTP mice, we intersected the down-regulated differentially expressed genes between the MPTP group and the NC group and the up-regulated differentially expressed genes between the NaB group and the MPTP group, and obtained 57 genes. These genes were subjected to KEGG enrichment analysis to obtain the signaling pathways that these genes might be enriched in. The analysis results showed that multiple genes were enriched in the tumor necrosis factor and glutathione metabolism signaling pathways( Figure 2B, C). Subsequently, in order to identify the key genes that play a role, we selected 6 candidate genes from these 57 DEGs with an absolute value of log2FC greater than or equal to 1 and a TPM value greater than 2.5. The sequencing results of RNA-seq were verified by qRT-PCR. The results showed that the expression changes of the mRNA levels of Slc25a22, Ump49, Crip3, and Sclm4 were consistent with the sequencing results.( Figure 2 D, E) Since the TPM value of the SLC25A22 gene was significantly higher than that of other genes, we further detected its changes at the protein expression level. The results showed that there was a significant downregulation of the Slc25a22 protein in the MPTP group (P < 0.001), and the downregulation of Slc25a22 was inhibited after treatment with NaB (P < 0.01)( Figure 2 F, G). It is suggested that Slc25a22 may be a key gene for treating MPTP mice.

[0094] 2.3 NaB feeding alleviated neuroinflammation in MPTP mice

[0095] Because KEGG enrichment analysis showed that the differential genes were enriched in the TNF signaling pathway, and the enhanced release of TNF-α would induce the activation of NF-κB, we used Western blot experiments to detect the protein levels of TNF-α and NF-κB in the striatum of mouse tissues. The results showed that compared with the NC group, the protein expressions of TNF and NF-κB were upregulated in the MPTP group, while the upregulation of protein expression was inhibited after treatment with NaB( Figure 3 A - C). It is indicated that sodium butyrate treatment can alleviate neuroinflammation in Parkinson's disease model mice.

[0096] 2.4 NaB feeding alleviated glutamate excitotoxicity in MPTP mice

[0097] TNF-α can act on neurons and glial cells and affect the release of glutamate. Studies have shown that TNF-α can activate astrocytes in a neuroinflammatory state, and activated astrocytes will release more cytokines and chemokines, further aggravating the inflammatory response. Therefore, we measured the protein expression and immunofluorescence of the activated astrocyte marker GFAP in the striatum of mice. The results showed that compared with the NC group, the number of activated astrocytes increased in the MPTP group, while NaB feeding reduced the number of activated astrocytes( Figure 4A, B, E, F). And in activated astrocytes, the function of glutamate transporters may be inhibited, resulting in a decreased ability to uptake glutamate in the synaptic cleft, causing glutamate to accumulate in the synaptic cleft, thus increasing the concentration of glutamate in the synaptic cleft and making it easier to trigger glutamate excitotoxicity. Therefore, we detected the protein expression of glutamate transporter EAAT2 by immunoblotting. We found that NaB treatment alleviated the MPTP-induced decrease in EAAT2 protein and the glutamate content in the substantia nigra region of mice ( Figure 4 A, C, D). These results suggest that NaB treatment can alleviate glutamate excitotoxicity in Parkinson's disease model mice.

[0098] 2.5 NaB feeding alleviated the decrease in GSH in the substantia nigra of MPTP mice

[0099] Studies have shown that a significant decrease in GSH levels in the substantia nigra of PD patients can lead to uncontrolled oxidative stress, mitochondrial damage, and α-synuclein aggregation. At the same time, TNF-α can exacerbate inflammation and oxidative stress by activating NF-κB, promoting the production of reactive oxygen species (ROS) and accelerating the depletion of glutathione (GSH). Previous studies have found that the downregulation of SLC7A11 protein expression in the substantia nigra of PD patients can lead to a decrease in GSH levels, and the activity of GPX4 depends on the supply of GSH. The decrease in GSH content leads to a decrease in GPX4 activity, exacerbating neuronal oxidative stress and increasing the loss of dopaminergic neurons. Therefore, we measured the expression of SLC7A11 and GPX4 proteins in the substantia nigra of mice by immunoblotting. The results showed that compared with the NC group, the protein levels of GPX4 and SLC7A11 in the MPTP group decreased, while NaB treatment restored the protein levels of GPX4 and SLC7A11 ( Figure 5 A - C). And we detected the GSH content in the substantia nigra of mice. The results showed that the GSH level was lower than that of the NC group after MPTP treatment, while the GSH level increased after NaB treatment ( Figure 5 D). These results indicate that NaB treatment can improve the decrease in GSH in PD model mice.

[0100] 2.6 SLC25A22 expression was decreased in DA neurons of MPTP mice and MPP+-treated MN9D cells

[0101] Because MPTP can induce the loss of DA neurons in the substantia nigra of mice, in order to explore whether there is also a downregulation of SLC25A22 expression in DA neurons of MPTP model mice, we detected the co-labeling of SLC25A22 and TH in the substantia nigra of mice by immunofluorescence experiments. The results showed that the number of DA neurons in the MPTP group decreased and the expression of SLC25A22 also decreased ( Figure 6A). In addition, to investigate whether there is also a downregulation of SLC25A22 in dopaminergic neurons in vitro, we selected MN9D cells, which are mouse dopaminergic neurons, treated the cells with MPP + and then added NaB, and measured cell viability, and the expression of TH and SLC25A22 proteins. The results of the CCK-8 assay showed that when MN9D cells were treated with 200 μM MPP + for 24 hours, the cell viability was about 50%, which was significantly lower than that of the NC group. Therefore, 200 μM MPP + was selected as the treatment concentration.( Figure 6 B). Then we treated MN9D cells with three concentrations of NaB, 2 mM, 4 mM, and 8 mM, for 24 hours. The results showed that there was no difference in cell viability, TH, and SLC25A22 protein expression between the NaB 2 group and the MPP + group. However, the cell viability increased in the NaB 4 and NaB 8 groups, and the expression of TH and SLC25A22 proteins was upregulated.( Figure 6 C-F). And the co-localization of SLC25A22 and Mitotracker was detected by immunofluorescence, indicating that SLC25A22 is located on mitochondria.( Figure 6 G). These results suggest that the expression of SLC25A22 is decreased in the Parkinson's disease model mice.

[0102] 2.7 Overexpression of SLC25A22 can alleviate the loss of MN9D cells after MPP+ treatment

[0103] To determine the effect of SLC25A22 on the viability of MN9D cells, we overexpressed SLC25A22 in MN9D cells. From the results of the CCK-8 and immunoblotting assays, it could be seen that overexpression of SLC25A22 after treatment with MPP + could increase the viability of MN9D cells and reverse the decrease in the expression of TH protein.( Figure 7 A-D). In summary, it was shown that overexpression of SLC25A22 could rescue the loss of MN9D cells.

[0104] 2.8 Overexpression of SLC25A22 alleviates oxidative damage in MPP+-induced MN9D cells

[0105] SLC25A22 is a mitochondrial glutamate transporter. Some studies have found that overexpression of SLC25A22 helps to increase the radioresistance of GBM cells. Overexpression of SLC25A22 promotes the accumulation of cytoplasmic glutamate. The accumulated cytoplasmic glutamate enhances GSH synthesis, and the increased GSH can protect cells from the effects of reactive oxygen species (ROS) induced by ionizing radiation (IR). Therefore, we detected the protein expression levels of SLC7A11 and GPX4, as well as the contents of Glu and GSH in MN9D cells. The results showed that overexpression of SLC25A22 could restore the downregulation of SLC7A11 and GPX4 protein expression during + MPP+ treatment ( Figure 8 A-C); overexpression of SLC25A22 restored the GSH content in MPP+-induced cells but downregulated the Glu level in cells ( Figure 8 C, E). To explore the effect of overexpression of SLC25A22 on the Glu content, we detected the mRNA expressions of glutamate transporters SLC1A1 and SLC1A2 in MN9D cells. The results showed that the levels of SLC1A1 and SLC1A2 remained unchanged, indicating that overexpression of SLC25A22 increased the utilization of Glu in MN9D cells. Furthermore, we detected the mRNA levels of GSH synthase, and the results showed that overexpression of SLC25A22 increased the expression of GSH synthesis mRNA in cells ( Figure 8 F). A recent study showed that knockout of SLC25A22 inhibited the synthesis of aspartate produced by the tricarboxylic acid (TCA) cycle in colorectal cancer cells and reduced the production of NADPH. Therefore, we detected the content of NADPH in MN9D cells, and the results showed that overexpression of SLC25A222 could promote the increase and alleviation of NADPH in MN9D cells ( Figure 8 G). It is known that GSH has the ability to scavenge ROS accumulated in cells. We detected the content of ROS in cells, and the results showed that overexpression of SLC25A22 could reduce the accumulation of ROS induced by MPP + + ( Figure 8 H, I). This suggests that overexpression of SLC25A22 can increase the antioxidant capacity of MN9D cells.

[0106] 2.9 Overexpression of SLC25A22 can alleviate MPP+-induced mitochondrial dysfunction in MN9D cells by promoting the synthesis of α-AKG

[0107] Since SLC25A22 functions as a transporter, which is a mitochondrial glutamate / H(+) symporter responsible for transporting glutamate from the cytosol into the mitochondrial matrix, we inferred that SLC25A22-mediated glutamate import might play a role in mitochondrial glutamate metabolism. In mitochondria, glutamate can be converted into α-ketoglutarate by glutamate dehydrogenase, and α-ketoglutarate is an important intermediate in the tricarboxylic acid cycle. The level of α-ketoglutarate affects the activity of the key rate-limiting enzyme α-ketoglutarate dehydrogenase in the TCA cycle. Therefore, we detected the activities of glutamate dehydrogenase and α-ketoglutarate dehydrogenase in MN9D cells. The results showed that overexpression of SLC25A22 could restore the decrease in GDH and α-KDG activities in MPP + -induced MN9D cells ( Figure 9 A, B). And we detected the relative expression levels of the two α-KDG protein subunits OGDHL and the GDH protein subunit GLUD1 by immunofluorescence. The results showed that the expressions of OGDHL and GLUD1 in the SLC25A22 OE group were significantly upregulated compared with the MPP+ group ( Figure 9 C-E). And overexpression of SLC25A22 restored the decrease in intracellular α-KG levels caused by MPP + ( Figure 9 F). Many studies have found that in PD, the death of dopaminergic neurons is related to mitochondrial metabolism and mitochondrial dysfunction, among which the decrease in mitochondrial membrane potential and the depletion of ATP are significant characteristics of mitochondrial dysfunction. Our results showed that overexpression of SLC25A22 could alleviate the decrease in mitochondrial membrane potential and the depletion of ATP in MPP+-induced MN9D cells ( Figure 9 G-I). These results indicate that overexpression of SLC25A22 can increase the entry of Glu into mitochondria, promote the synthesis of α-KG, and relieve MPP + -induced mitochondrial dysfunction in MN9D cells.

[0108] 3. Analysis

[0109] Our results found that glutamate excitotoxicity was activated in the substantia nigra of PD mice. The activation of glutamate excitotoxicity might be due to the downregulation of glutamate transporters induced by neuroinflammation triggered by the TNF signaling pathway, reducing glutamate reuptake and leading to the accumulation of glutamate in the synaptic cleft. The reduced uptake of glutamate by neuronal cells results in a decrease in the intracellular glutamate content, which affects the de novo synthesis of glutathione. The decrease in glutathione levels in neurons reduces the antioxidant capacity of cells and exacerbates the loss of dopaminergic neurons. Consistent with previous reports, we also found that SLC25A22 is responsible for transporting glutamate from the cytoplasm to mitochondria in MN9D cells and can mediate the production of glutathione through NADPH synthesis.

[0110] In the research of the present invention, it was found that SLC25A22 can promote the utilization of glutamate, the synthesis of α-ketoglutaric acid and the TCA cycle in the mitochondria of MN9D cells, improve the reduction of ATP production, ROS accumulation and mitochondrial membrane potential decline in MPP+-induced MN9D cells, thereby alleviating the impairment of MN9D cell activity. Eventually, the loss of dopaminergic neurons was rescued. This suggests that SLC25A22 may be a new target for PD treatment. The research of the present invention proves that the mitochondrial glutamate transporter SLC25A22 in MN9D cells transports glutamate from the cytoplasm into the mitochondria, generates α-ketoglutaric acid to participate in the TCA cycle, restores cellular energy metabolism and promotes GSH production to improve cellular mitochondrial dysfunction and alleviate the loss of dopaminergic neurons.

Claims

1. Application of SLC25A22 as a target in screening drugs for the treatment of Parkinson's disease.

2. The use according to claim 1, characterized in that: The drug promotes the expression of SLC25A22 or increases the protein activity thereof.

3. The use according to claim 2, characterized in that: The drug is a preparation that overexpresses SLC25A22.

4. The use according to claim 3, characterized in that: The drug is a SLC25A22 overexpression plasmid.

5. Use of a preparation that promotes the expression of SLC25A22 in the preparation of a drug for treating Parkinson's disease.

6. The use according to claim 5, characterized in that: The drug is a SLC25A22 overexpression plasmid.

7. The use according to claim 4 or 6, characterized in that: The backbone vector of the overexpression plasmid is pcDNA3.1(+).

8. The use according to any one of claims 1 to 7, characterized in that: SLC25A22 promotes the utilization of glutamate, the synthesis of α-ketoglutarate and the TCA cycle in the mitochondria of MN9D cells, improves the reduced ATP production, ROS accumulation and decreased mitochondrial membrane potential in MN9D cells induced by MPP+, thereby alleviating the damage to MN9D cell activity and ultimately saving the loss of dopaminergic neurons.

9. The use according to claim 8, characterized in that: In MN9D cells, the mitochondrial glutamate transporter SLC25A22 transports glutamate from the cytoplasm to the mitochondria to generate α-ketoglutarate, which participates in the TCA cycle, restores cellular energy metabolism, promotes GSH production, improves cellular mitochondrial dysfunction, and alleviates the loss of dopaminergic neurons.

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