Ginsenoside Rk1 prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease via activating silence information regulator 3-mediated Nrf2/HO-1 signaling pathway

Abstract

Objectives: Ginsenoside Rk1, a novel ginsenoside isolated from red ginseng, has anti-inflammatory and anti-tumor activities. This study was designed to elucidate the role of RK1 in an in vitro 1-methyl-4-phenylpyridinium (MPP⁺) cell model and an in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) of Parkinson's disease (PD).

Methods: The grasping test, pole-climbing test, and rotarod test were performed to measure the effects of RK1 on MPTP-induced motor disorders. The expression of tyrosine hydroxylase (TH) and IBA-1 were evaluated by western blotting. CCK-8 and flow cytometry  assays were utilized to assess cell viability and apoptosis. Reactive oxygen species (ROS), Lactate dehydrogenase (LDH), and superoxide dismutase (SOD) were detected to analyze the effects of RK1 on oxidative stress. The levels of inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA).

Results: The results showed that RK1 allayed motor deficit elicited by MPTP in a mouse model. RK1 administration augmented tyrosine hydroxylase (TH) expression in the brain striatum and substantia nigra (SN) of MPTP-treated mice. Moreover, RK1 pretreatment promoted viability and suppressed apoptosis in MPP⁺-induced PC-12 cells. Further, RK1 also attenuated MPP⁺-stimulated oxidative stress and inflammatory response in PC-12 cells. Besides, RK1 augmented the level of SIRT3, and SIRT3 deletion counteracted RK1-induced repression on MPP⁺-elicited apoptosis, oxidative stress, and inflammatory response in PC-12 cells via modulating the Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)  pathway.

Conclusions: RK1 might exert neuroprotective effects against MPP⁺/MPTP-induced neurotoxicity via activating SIRT3-mediated Nrf2/HO-1 signaling. RK1 might be a promising candidate against PD.

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disorder that primarily affects middle-aged and elderly individuals. The prevalence rate of PD in people over 65 years old in China is estimated to be around 1.7%.^1,2 PD is defined as the loss of dopaminergic (DA) neurons in the substantia nigra (SN) of the midbrain, ³ which is clinically manifested as motor symptoms and non-motor symptoms such as tremor, rigidity, movement disorders, and postural instability. ⁴ Despite the pathogenesis of PD is not fully understood, many studies have elucidated that the overproduction of reactive oxygen species (ROS), oxidative stress, neuroinflammation, and mitochondrial dysfunction may be responsible for the loss of DA neurons and neuronal apoptosis.^5,6 Current dopamine replacement therapies only relieve symptoms but are not able to interrupt the neurodegenerative process. ⁷ Therefore, the search for effective neuroprotectants and the exploration of their mechanisms against the loss of DA neurons remains important challenges in PD treatment.

Ginseng, derived from the radix of Panax ginseng Meyer, commonly known as Korean ginseng, is a highly valued herb in traditional medicine with a long history of use. ⁸ It is renowned for its various pharmacological activities, including anti-inflammatory, antihypertensive, antidiabetic, anticancer, antioxidant, and neuroprotective effects.^9–11 Ginsenosides from Ginseng are known for induction of various biological activities. Ginsenosides Rb1, Rb2, Rc, Rd, Rg1, and Re are the major constituents of ginseng, while Rg3, Rg5, Rg6, Rh1, Rh2, Rk1, Rs3, and F4 are unique constituents of red ginseng. ¹² Multiple evidence has demonstrated that RK1 was beneficial in the treatment of various diseases by ameliorating inflammation and oxidative stress. For instance, Hu et al. manifested that RK1 protected against acute liver injuries in mice via repressing inflammation, oxidative stress, and apoptosis. ¹³ Li et al. highlighted that RK1 exerted an antidepressant function in the mouse model of Lipopolysaccharide (LPS)-triggered depression via facilitating brain-derived neurotrophic factor (BDNF) signaling and inhibiting the neuroinflammatory response. ¹⁴ Ginsenoside Rg5 and RK1 can reverse memory dysfunction and play neuroprotective functions against excitotoxicity. ¹⁵ These discoveries raised the possibility that RK1 might contribute to the treatments for neurodegenerative diseases. Nevertheless, the role of RK1 in PD remains elusive.

The present work aimed at uncovering the neuroprotective effects of RK1 in PD and its specific molecular mechanism. The results indicated that RK1 protected against MPTP/MPP⁺-triggered neurotoxicity in vivo and in vitro through activating silence information regulator 3 (SIRT3)-mediated Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling.

Materials and methods

Establishment of the murine model of PD

This study was permitted by the Ethics Committee of the First People's Hospital of Changzhou, the Third Affiliated Hospital of Soochow University. 6–8-week-old male C57/BL6 mice were obtained from SLAC Laboratory Animal Co., Ltd (Shanghai, China). Mice were randomly divided into three groups (n = 8 per group) as follows: Sham, injected with the vehicle saline; MPTP, injected with MPTP (Sigma, 10 mg/kg, i.p.) for 7 days; MPTP + RK1 group, injected with RK1 (10 mg/kg, 20 mg/kg, i.p.) commencing 7 days before MPTP administration and lasting for 21 days. For mouse brain tissue, the striatum and SN were isolated as previously described. ¹⁶

Behavioral tests

The grasping test, pole-climbing test, and rotarod test were performed according to the previous reference to evaluate the motor function of PD mice. ¹⁷

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from cells (2 × 10⁶ cells) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized by a cDNA Synthesis Kit (Thermo Scientific). Total RNA was reverse transcribed into cDNA using the iScript™ cDNA Synthesis kit (Bio-Rad Laboratories). qPCR was performed using the SYBR-Green Master mix (Thermo Fisher Scientific, Inc.). GAPDH was used as the internal control. Relative gene expression was calculated using the 2^−ΔΔCq method. ¹⁸

Western blotting

Total protein was extracted from brain tissue or PC-12 cells with RIPA lysis buffer. Next, the protein was subjected to 10% SDS-PAGE and transferred onto PVDF membranes. Blocking was performed with 5% skim milk. Then, the membranes were joined with the primary antibodies at 4°C overnight and probed with secondary antibodies for 1 h at room temperature. Finally, protein bands were visualized by the ECL kit (Pierce). ¹⁹

Cell culture and treatment

Rat adrenal pheochromocytoma cell lines (PC-12) were acquired from Shanghai Institutes of Cell Biological Sciences. Cells were maintained in RPMI1640 medium (10% FBS; 5% horse serum; 1% penicillin–streptomycin) at 37°C with 5% CO₂. PC-12 cells (2 × 10 ⁵ cell/well) were seeded in a 6-well plate, and then incubated with RK1 (10, 20, and 30 μM) for 2 h, followed by stimulation with MPTP (500 μM) for 24 h. Finally, cells were collected for further analysis. ²⁰

Cellular viability via cell counting kit-8 (CCK-8)

PC-12 cell viability was assessed by CCK-8 assay. ²¹ PC-12 cells were plated in 96-well plates with 5 × 10⁴ cells for 24 h and pretreated with RK1 for 4 h. Then, 10 μL of CCK-8 solution (Sangon Biotech) was added and incubated for 2 h at 37°C. The OD value was recorded with a microplate reader at 450 nm.

Flow cytometry

PC-12 cell apoptosis was assessed with an Annexin-V-FITC/PI apoptosis detection kit (Solarbio, Beijing, China) following the manufacturer's instruction. In brief, treated PC-12 cells were rinsed in PBS and resuspended in a buffer. Then, PC-12 cells were incubated with Annexin V-FITC and PI (5 µL) for 15 min in darkness. Finally, apoptotic PC-12 cells were analyzed using a flow cytometer and quantified using FlowJo software. ¹⁷

Lactate dehydrogenase (LDH) release, reactive oxygen species and superoxide dismutase (SOD) activity assay

The levels of LDH release, ROS, and SOD were determined by the LDH Assay Kit (Cytotoxicity) (ab65393, Abcam), ROS Assay Kit (S0033, Beyotime), and SOD Assay Kit (S0109, Beyotime), respectively, referring to the directions of manufacturers. The absorbance of samples at 450 nm, 490 nm, and 530 nm was tested to reflect SOD, LDH, and ROS activity via a microplate reader, respectively.

Enzyme-linked immunosorbent assay (ELISA)

Treated PC-12 cells were lysed to obtain cell supernatant. Then, Tumor Necrosis Factor α (TNF-α) (ab46070, Abcam), Interleukin-1β (IL-1β) (ab100768, Abcam), and Interleukin-6 (IL-6) (ab234570, Abcam) levels were gauged using an ELISA kit as recommended by manufacturers.

Statistical analysis

Statistical analysis was performed as described in our previous studies. ²² In brief, each experiment was performed 3 times. Data were presented as mean ± SD. The differences were analyzed by Student’s t-test or one-way analysis of variance via SPSS 20.0 software. p < .05 was deemed statistically significant.

Results

RK1 alleviates movement dysfunction in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice

To evaluate the neuroprotective effects of RK1 on the MPTP-induced mouse model of PD, we first performed behavior function tests to measure motor function. The results showed that MPTP injection increased the time in the Pole-climbing test and decreased the time in the grasping test (Figure 1(a) and (b)) or rotarod test (Figure 1(c)), confirming that the PD mice model was established successfully. However, RK1 administration significantly attenuated the changes. These results indicated that RK1 could alleviate the adverse impacts of MPTP on motor function.

Figure 1.

RK1 alleviates movement dysfunction in MPTP-induced PD mice. Mice were divided into Control, MPTP, MPTP + RK1 (10 mg/kg), and MPTP + RK1 (20 mg/kg) groups. (a) Pole-climbing test (a), grasping test (b) and rotarod test (c) were carried out to examine the motor deficits of PD mice. *p < .05; **p < .01; ***p < .001.

RK1 suppresses DA neuron degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated PD mice

PD is typically characterized by the loss of DA neurons; hence, we studied whether RK1 can protect DA neurons in MPTP-treated mice. The amount of TH, a key enzyme of DA, was tested via western blotting. Results revealed that RK1 heightened TH protein level in the brain striatum and SN of MPTP-injected mice (Figure 2(a) and (b)). Furthermore, MPTP distinctly raised the protein expression of IBA-1 (a marker of activated microglia) in the striatum and SN, while RK1 administration diminished IBA-1 expression in MPTP-treated PD mice, determining that RK1 suppressed microglial activation (Figure 2(c) and (d)). Our findings suggested that RK1 attenuated DA neuron loss and microglial activation.

Figure 2.

RK1 suppresses DA neurons degeneration in MPTP-treated PD mice. (a) and (b) Western blotting for TH protein levels in striatum and SN of PD mice. (c) and (d) IBA-1 protein level in striatum and SN. *p < .05; **p < .01; ***p < .001.

RK1 mitigates 1-methyl-4-phenylpyridinium-induced apoptosis

To verify the neuroprotective effect of RK1 in vitro, PC-12 cells were treated with RK1 (10, 20, and 30 μM) before exposure to 500 μM of MPP⁺ for 24 h. CCK-8 results displayed that RK1 pretreatment concentration-dependently reversed MPP⁺-triggered inhibition in PC-12 cell viability (Figure 3(a)). In addition, flow cytometry assay found that MPP⁺ treatment increased PC-12 cell apoptosis, which was attenuated by RK1 pretreatment (Figure 3(b)). Simultaneously, RK1 markedly reversed MPP⁺-induced increase levels of Caspase-3 and Bax, and decrease Bax expression (Figure 3(c)). Collectively, these results showed that RK1 restrained MPP⁺-triggered apoptosis in PC-12 cells.

Figure 3.

RK1 mitigates MPP⁺-induced apoptosis. PC-12 cells were assigned into Control, MPP⁺, MPP⁺+RK1 (10 µM), MPP⁺+RK1 (20 µM), and MPP⁺+RK1 (30 µM) groups. (a) CCK-8 assay for cell viability. (b) Flow cytometry for cell apoptosis. (c) Western blotting for Bax, Bcl-2 and caspase-3 levels. *p < .05; **p < .01; ***p < .001.

RK1 alleviates 1-methyl-4-phenylpyridinium-stimulated oxidative stress and inflammatory responses in PC-12 cells

As depicted in Figure 4(a)–(c), MPP⁺ induction raised the relative activities of LDH and ROS levels while decreased SOD level in PC-12 cells, which was dose-dependently reversed by RK1 pretreatment. Moreover, levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in MPTP stimulation PC-12 cells were apparently augmented, while these changes were reversed by RK1 pretreatment (Figure 4(d)–(f)). These data demonstrated that RK1 attenuated oxidative stress and inflammatory response in MPP⁺- disposed PC-12 cells.

Figure 4.

RK1 alleviates MPP⁺-stimulated oxidative stress and inflammatory responses in PC-12 cells. PC-12 cells were assigned into Control, MPP⁺, MPP⁺+RK1 (10 µM), MPP⁺+RK1 (20 µM), and MPP⁺+RK1 (30 µM) groups. (A-C) ELISA assay was utilized for LDH, ROS and SOD. (D-F) TNF-α, IL-1β, and IL-6 levels. *p < .05; **p < .01; ***p < .001.

RK1 protects against 1-methyl-4-phenylpyridinium-evoked PC-12 cell damage in a silence information regulator 3-dependent manner

SIRT3 plays a pivotal function in the occurrence and development of multiple neurodegenerative diseases including PD.^23,24 We wondered if the protective impacts of RK1 were implicated in SIRT3 activation. Firstly, the mRNA and protein levels of SIRT3 were tested via RT-qPCR and western blotting. The results displayed that MPP⁺ stimulation observably reduced SIRT3 expression in PC-12 cells, which could be augmented following RK1 pretreatment (Figure 5(a)). Thus, we speculated that RK1-mediated protection in PD was related to SIRT3 activation.

Figure 5.

RK1 protects against MPP⁺-evoked PC-12 cell damage in a SIRT3-dependent manner. (a) RT-qPCR and western blotting for SIRT3 expression in PC-12 cells treated with MPP⁺, MPP⁺+RK1 (10 µM), MPP⁺+RK1 (20 µM), and MPP⁺+RK1 (30 µM). (b)–(f) PC-12 cells were assigned into Control, MPP⁺, MPP⁺+RK1 (10 µM), MPP⁺+RK1 (10 µM)+shSIRT3 groups. (b) Cell viability. (c) Apoptosis. (d) Western blotting for Bax, Bcl-2 and caspase-3 levels. (e) LDH, ROS and SOD. (f) TNF-α, IL-1β, and IL-6. *p < .05; **p < .01; ***p < .001.

To further validate our hypothesis, we then studied the function of RK1 in MPP⁺-triggered PC-12 cells with or without SIRT3 depletion. As expected, MPP⁺ stimulation hindered cell viability, promoted cell apoptosis and oxidative stress, and heightened the levels of TNF-α, IL-1β, and IL-6, all of which could be reversed by RK1 treatment. However, SIRT3 knockdown abrogated the protective effects of RK1 in MPP⁺-evoked PC-12 cells (Figure 5(b)–(f)), suggesting that RK1 exerted a neuroprotective impact on MPP+-evoked PC-12 cell damage in a SIRT3-dependent manner.

RK1 regulates the Nrf2/HO-1 pathway in a silence information regulator 3-dependent manner during PD

A recent study unveiled that microglial SIRT3 reduced the release of pro-inflammatory factors, thereby suppressing neuroinflammation response and neurological deficits by activating the Nrf2/HO-1 signaling. ²⁵ Herein, as depicted in Figure 6(a) and (b), RT-qPCR and western blotting showed that MPP⁺ stimulation lessened the expression of Nrf2 and HO-1 in PC-12 cells, which was reversed by RK1 treatment. Moreover, SIRT3 deletion neutralized the regulating impacts of RK1 on Nrf2/HO-1 signaling, manifesting that the neuroprotective properties of RK1 may be mediated by Nrf2/HO-1 pathway in a SIRT3-dependent manner.

Figure 6.

RK1 regulates the Nrf2/HO-1 pathway in a SIRT3-dependent manner during PD. (a) and (b) RT-qPCR and western blotting for Nrf2 and HO-1 protein levels in PC-12 cells treated with MPP+, MPP⁺+RK1 (10 µM), MPP⁺+RK1 (10 µM)+shSIRT3 groups. *p < .05; **p < .01; ***p < .001.

Discussion

PD is the most serious and common neurodegenerative disorder in the world, and its prevalence is expected to increase markedly as the population ages. ²⁶ Pharmacological references published have elaborated memory-enhancing and neuroprotective impacts of ginsenoside Rk1. ²⁷ However, whether RK1 contributes to the amelioration of PD progression is unclear. To verify the effects of RK1 in PD, a murine PD model was first established by subjecting mice to MPTP intraperitoneal injection. Results determined that RK1 could alleviate the motor disorders induced by MPTP in mice. In addition, RK1 treatment markedly reversed the MPTP-caused decrease in TH expression and increase in IBA-1 expression in the striatum and SN region, suggesting RK1 prevented the dopaminergic neuronal loss and the microglia activation. Taken together, RK1 exerted a neuroprotective effect in MPTP-induced PD mice.

Mounting evidence suggested that PD is associated with multiple factors, such as inflammatory response, oxidative stress, and cell apoptosis. Moreover, RK1 was reported to protect against oxidative stress-associated damage in melanocytes via activating the PI3K/AKT/Nrf2/HO-1 signaling to accelerate cell viability and suppress cell apoptosis. ²⁸ Further, RK1 might be a novel component that contributed to the development of ginseng-based treatments for neurodegenerative diseases. ¹⁵ Herein, to confirm the neuroprotective effect of RK1 in vitro, a cellular PD model was established by treating PC-12 cells with MPP⁺. RK1 treatment accelerated the decreased cell viability while suppressed the increased cell apoptosis rate of PC-12 cells induced by MPP⁺ treatment in a dose-dependent manner. Meanwhile, RK1 dramatically reversed MPP⁺-induced changes in Bax, Bcl-2, and Caspase-3 levels. Moreover, RK1 dose-dependently relieved MPP⁺-evoked oxidative stress in PC-12 cells, as evidenced by the decreased LDH and ROS levels and the enhanced SOD expression. Besides, RK1 pretreatment also attenuated MPP⁺-evoked the release of pro-inflammatory cytokines. All the results confirmed that RK1 mitigated MPP⁺-triggered apoptosis, oxidative stress, and inflammatory reaction in PC-12 cells.

Silence information regulator 3 (Sirtuin 3), which is a class III histone deacetylase, is responsible for many cellular functions such as metabolic homeostasis, oxidative stress, and apoptosis.^29,30 Moreover, SIRT3 was reported to be linked to several neurodegenerative diseases. For instance, Geng et al. showed that SIRT3 expression was declined in SH-SY5Y cells after MPP⁺ treatment and miR-494-3p silence exerted a neuroprotective function in MPP⁺-evoked PD through regulating SIRT3. ³¹ Zhang et al. implied that SIRT3 depletion significantly worsened the rotenone-induced decline of cell viability, enhanced cell apoptosis, and augmented α-synuclein accumulation in Parkinson cell model. ³² In addition, Ginsenoside Rb1 antagonized high glucose-induced premature senescence of human umbilical vein endothelial cells (HUVECs) via the SIRT3/superoxide dismutase 2 (SOD2) signaling pathway. ³³ Ginsenoside Rg1 treatment attenuated isoflurane/surgery-induced neurocognitive disorders and SIRT3 dysfunction. ³⁴ Thus, we conjecture that RK1 might relieve MPTP-induced PD via modulating SIRT3. Here, we found that RK1 pretreatment could upregulate and activate SIRT3 in MPP⁺-stimulated PC-12 cells. Meanwhile, we also disclosed that SIRT3 deletion could abolish the protective impacts of RK1 on MPP⁺-evoked PC-12 cells, suggesting that RK1 prevented MPP⁺-induced PC-12 cells in a SIRT3-dependent manner. Moreover, it was demonstrated that SIRT3 restrained the formation of calcium oxalate-elicited kidney stones via the Nrf2/HO-1 signaling. ³⁵ Besides, a recent study showed pharmacological modulation of the Nrf2/HO-1 pathway by bioactive compounds is a therapeutic target of PD. ³⁶ In this study, RK1 could activate the Nrf2/HO-1 pathway in a SIRT3-dependent manner in MPP⁺-evoked PC-12 cells.

Conclusion

Our study illuminated that RK1 allayed motor disorders in MPTP-evoked PD mice and RK1 protected MPP⁺-evoked injury in PC-12 cells by reducing apoptosis, oxidative stress, and inflammatory responses via activating the Nrf2/HO-1 signaling in a SIRT3-dependent manner. These findings may offer insights for the future treatment of PD via the application of RK1. In addition, RK1 may contribute to synergistic effects through combination therapy with standard drugs at the earliest stages of PD, which needs further study regarding the effects and the safety through animal studies and clinical trials in the future.