Tetramethyl pyrazine exerts anti-apoptotic and antioxidant effects in a mouse model of MPTP-induced Parkinson's disease via regulation of the expressions of Bax, Bcl-2, Nrf2 and GCLC

Purpose: To investigate the effect of tetramethyl pyrazine (TMP) on MPTP)-mediated neuronal apoptosis and oxidative imbalance in mice, and the mechanism of action involved. Methods: Forty-five mice were assigned evenly to blank control, MPTP and TMP groups. The protein concentrations of Bax, Bcl-2, cytochrome C (Cyt c), Nrf2, GCLC and cleaved caspase-3; and levels of glutathione (GSH) and thiobarbituric acid reactive products (TBARS) were evaluated and compared amongst the groups. Results: Cyt c, Bax, and cleaved caspase-3 protein levels in TMP group were significantly lower than those in MPTP group, while Bcl-2 protein expression was higher in TMP group than in MPTP mice (p < 0.05). Furthermore, TBARS was lower in TMP group than in MPTP group, while GSH level increased, relative to MPTP mice. The levels of Nrf2 and GCLC were significantly higher in TMP group than in MPTP group (p < 0.05). Conclusion: Tetramethyl pyrazine exerts anti-apoptotic and antioxidant effects on MPTP-mediated Parkinsonism via regulation of the expressions of Bax, Bcl-2, Nrf2 and glutamate-cysteine ligase catalytic subunit. Thus, TMP has potential for use in the treatment Parkinson’s disease.


INTRODUCTION
Parkinson's disease (PD) is predominant in middle-aged and elderly people. The disease manifests in symptoms such as static tremor, enhanced muscular tone, and delayed movement, all of which seriously affect the patient's quality of life [1]. Epidemiological reports show that, as a result of the increase in ageing population in China, the incidence of PD is increasing year by year. Thus, Parkinson's disease has become a serious health problem in the field of chronic diseases of the elderly in China [2].
At present, the pathogenesis of PD is not fully understood. Thus, clinically, there is no effective radical cure for the disease. Usually, drug intervention is used to delay the progression of the disease. However, long-term drug intervention may cause many adverse reactions, in addition to the fact that efficacy of the longterm treatment is not satisfactory [3]. Therefore, it is important to identify and develop new drugs with ideal clinical efficacy and high reliability for treatment of PD patients.
Tetramethyl pyrazine (TMP) is extracted from the rhizome of Ligusticum chuanxiong Hort. It is used in the treatment of systemic diseases such as diseases of the heart, brain and central nervous system. In addition, studies have shown that it exerts anti-tumor and anti-fibrotic effects [4,5].
Previous investigations have shown that the expression level of Bax in substantia nigra neurons is significantly increased in PD [6]. Other studies have confirmed that Bax ablation and Bcl-2 overexpression protect the neurons from MPTP-induced damage [7]. It is known that Nrf2 performs effective anti-oxidative stress functions in a wide range of living organisms [8]. Studies have demonstrated that glutamate-cysteine ligase catalytic subunit (GCLc) ameliorates oxidative stress-induced liver injury due to various factors [9]. However, at present, there are limited reports on the effect of TMP on levels Bax, Bcl-2, Nrf2, and GCLc in PD. The present study was carried out to investigate the effect of TMP on MPTP-induced neuronal cell apoptosis and oxidative stress in a mouse model of PD, and the involvement of Bax, Bcl-2, Nrf2, and GCLc in the process.

EXPERIMENTAL Materials
Forty-five male Wistar mice were purchased from Changsha Tianqin Biotechnology Co. Ltd (batch number: SCXK 2019-0005). The mice were raised in a greenhouse and allowed ad libitum access feed and drinking water. All protocols used in this study were approved by Animal Protection and Use Committee of The Fourth Military Medical University (Wuhan, China, approval no. 201903891) and conduct of the studies followed the guidelines of "Principles of Laboratory Animal Care" (NIH publication no. 85-23, revised 1985) [10].

Animal studies
Forty-five (45) mice were assigned to blank control, MPTP and TMP groups, each with 15 mice. Animals in TMP group received intraperitoneal (i.v.) injection of TMP at a dose of 20 mg/kg once per day for 7 consecutive days. Mice in the TMP group were injected with MPTP (1 μmol MPTP dissolved in 2 μL of normal saline) in the substantia nigra on day 7, and then TMP administration was done for 7 days. Mice in blank control and MPTP groups were given equivalent volume of normal saline i.v. once daily for 7 consecutive days. The MPTP group received MPTP injection in the substantia nigra for 7 days. After 14 days, mice in each group were sacrificed and their striata and substantia nigra were isolated for subsequent studies.
Flow cytometry was employed to measure the apoptotic ability of substantia nigra cells in each group. The substantia nigra cells were routinely digested with 0.25% trypsin and centrifuged at 1000 r/min for 10 min. After discarding the supernatant, the cells were resuspended in PBS solution and adjusted to a concentration of 1×10 6 cells/mL, followed by centrifugation again at 1000 r/min for 5min, and resuspension of the cells in binding buffer. Thereafter, 5μL of Annexin V-FITC was added, followed by thorough mixing and incubation at laboratory temperature for 30 min. After centrifugation, the supernatant was discarded, and the cells were resuspended in binding buffer. Then, 10 μL of propidium iodide was added to mix well, placed at room temperature (25 ℃) for 30 min, and finally subjected to flow cytometry. Apoptosis (A) was computed as shown in Eq 1.
Relative protein amounts of Bax, Bcl-2, Cyt c, Nrf2, GCLc and cleaved caspase-3 in the substantia nigra of mice in each group were detected with western blot assay. Total protein of substantia nigra cells was extracted and its concentration was determined with BCA protein quantitative kit. After separation by SDS gel electrophoresis, the protein was electrically transferred to PVDF membrane. After sealing and immune reaction, chemiluminescence chromogenic agent was added for color development.
Finally, the corresponding completed image was used for copy processing.
The levels of oxidative stress related indexes glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) in the substantia nigra of mice in each group were detected with colorimetric method and chemiluminescence method, respectively. Colorimetric method: test tubes were taken and labeled as measuring tube, non-enzyme tube, blank tube and duplicating tube respectively. 0.2mL of GSH was added to all tubes except blank control group. 0.2mL of serum to be detected was added to the measuring tube, and 0.2mL of double distilled water was added to the other tubes. After mixing well, the tubes were placed in a water bath for 5 min at 37℃. Then, 0.1mL of H2O2 was added to all tubes except the blank tube. After incubating in a water bath for 5min, 0.8mL of phosphoric acid precipitant was added to the blank tube, and 2mL of phosphoric acid precipitant was added to the other tubes. After centrifugation, supernatants were taken, 1.25mL of disodium hydrogen phosphate and 0.25mL of DTNB were added, respectively, and detected at 422nm wavelength. Chemiluminescence method: 1% sample was placed in the measuring tube, and equal amount of distilled water was put into the control measuring tube. 1mL of reagent 1, 0.1mL of reagent 2, 0.1mL of reagent 3 and 0.1mL of reagent 4 were put into each tube respectively. After fully mixing, the sample was placed in a constant temperature water bath at 37℃ for 20min. 2mL of chromogenic reagent was added to each measuring tube, mixed well and put it at room temperature for 20min. The measuring tubes were placed in a 1cm light-diameter colorimetric cup, adjusted to zero with distilled water, and colorimetric reaction was carried out at 550nm. The content of TBARS in each group was calculated according to the kit instructions.

Statistical analysis
Levels of TBARS and GSH in mice in each group are presented as mean ± SD. Two-group comparison was carried out with Student's t-test, while single factor ANOVA was used for multigroup comparisons. All statistical analyses were done with SPSS version 23.0, and significant difference was assumed at p < 0.05.

Effect of TMP on apoptosis of PD mice
The percentage apoptosis was markedly increased in MPTP group, relative to control, but percentage apoptosis was markedly decreased in TMP group, relative to MPTP group. These results are presented on Table 1.

Effect of TMP on apoptosis-related proteins in PD mice
The relative protein concentrations of Cyt c, Bax, and cleaved caspase-3 in MPTP group were significantly higher than the corresponding expression levels in blank control group, while Bcl-2 protein was lower in MPTP group than in control.
However, there were lower concentrations of these proteins in TMP group than in MPTP group, while the expression level of Bcl-2 was markedly higher in TMP group. These results are presented on Table 2 and Figure 1.

Effect of TMP on oxidative stress in PD mice
As shown in Table 3, the TBARS level in TMP group was markedly raised, relative to control, while GSH was markedly reduced in the control group. In contrast, TBARS level was significantly higher in the TMP group than in the MPTP group while GSH level was lower.

Effect of TMP on protein expression levels of Nrf2 and GCLc in PD mice
The protein expression levels of Nrf2 and GCLc in PD mice were raised in MPTP group, relative to blank control. However, protein expression levels of Nrf2 and GCLc in PD mice were lower in TMP group than in MPTP group. These results are presented in Table 4 and Figure 2.

DISCUSSION
Previous reports have shown that TMP exerts neuroprotective effects in vivo and in vitro. In addition, TMP has been shown to play a protective role against ischemic brain injury, and to accelerate cell proliferation and differentiation [11]. It is known that MPTP is a neurotoxin that induces Parkinson's disease via its effect on neurons, and it has been widely used in the establishment of animal models of the disease [12]. However, there are no extant studies on the potential of TMP to block apoptosis of neurons in PD through anti-oxidant effects. This formed the basis of the present study. The Bax protein is apoptosis-inducing. In contrast, Bcl-2 is an anti-apoptosis protein that blocks Bax translocation and dimer production and inhibits Cyt c release, thereby blocking apoptosis [13,14]. Several studies have shown that caspase-3 plays a key role in apoptosisrelated processes. In this study, apoptosisrelated changes in the substantia nigra was determined, and it was revealed that Cyt c and Bax protein expressions were significantly raised in MPTP group, while Bcl-2 protein expression was significantly downregulated when caspase-3 was cleaved. Thus, the MPTP-induced changes in the above indices were effectively blocked by TMP. These results suggest that TMP effectively inhibits MPTP-induced apoptosis of rat brain substantia nigra cells [15]. Clinical studies have demonstrated that oxidative damage plays a key role in osteoporotic and dopamine-neuron attenuation-related processes which accelerate mitochondrial apoptosis, thereby aggravating oxidative damage and triggering "oxidative stress-cell apoptosis" cycle.
Other reports have shown that MPTP accentuates release free radicals, while triggering attenuation of dopamine neurons and oxidative stress response. Many drugs inhibit MPTP-induced neurotoxicity by eliminating free radicals. The release of TBARS reflects the degree of oxidative damage in vivo. Reduced glutathione (GSH) is an important reductant that participates in resistance to oxidative damage in the brain. It participates in the scavenging of reactive oxygen radicals and parent-child electronic reagents, which is of great significance in metabolic processes. The levels of GSH are significantly reduced in PD patients and MPTPinduced animal models [16].
In the present study, the effect of TMP and MPTP on redox balance in the substantia nigra of mice was determined, and it was shown that TBARS levels were significantly decreased, while GSH levels were significantly increased in the MPTP group, indicating the occurrence of oxidative damage. However, TMP intervention effectively blocked the increase in TBARS and decrease in GSH. In vivo, Nrf2 is the strongest anti-oxidative stress regulatory transcription factor which activates antioxidant enzymes, including GCLc in cell redox regulation, thereby protecting the cells from damage by reactive oxygen species.
Finally, the potential molecular mechanism involved in the protective effect of TMP against MPTP-induced redox imbalance was investigated. The MPTP-induced downregulations of Nrf2 and GCLc were effectively blocked after TMP intervention. This suggests strongly that TMP blocks MPTP-induced oxidative stress injury in substantia nigra of PD mice.

CONCLUSION
The results obtained in this investigation indicate that TMP exerts anti-apoptotic and anti-oxidative effects on MPTP-damaged neurons in a mouse Parkinsonism model through regulation of expression levels of Bax, Bcl-2, Nrf2, and GCLc. Therefore, TMP can potentially be developed for the management of Parkinson's disease.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was performed by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. Bibo Tang designed the study, supervised the data collection, and analyzed the data. Lixing Dai interpreted the data and prepared the manuscript for publication. Ruiqing Diao, Jinghua Zhang, Mengying Cao and Hongli Gao supervised the data collection, analyzed the data and reviewed the draft of the manuscript.

Open Access
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