Tanshinone IIA protects against dopaminergic neuron degeneration via regulation of DJ-1 and Nrf2/HO-1 pathways in a rodent model of Parkinson’s disease

Purpose: To study the potential neuroprotective effects of tanshinone IIA, a diterpene quinone, in an experimental model of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson disease (PD). Methods: Mice (C57BL/6) were administered freshly-prepared MPTP at a dose of 20 mg/kg body weight intraperitoneally, 4 times at 2-h intervals, to induce PD. Doses of 12.5, 25 and 50 mg/kg tanshinone IIA were administered to the mice as treatments for PD. Pole and Rota-rod tests were carried out to assess muscular coordination and bradykinesia. Protein expressions, reactive oxygen species (ROS) and malonaldehyde and other parameters were evaluated. Results: Tanshinone IIA at doses of 12.5, 25 and 50 mg/kg reduced deficits in muscular coordination and improved learning ability of MPTP-treated mice. It also reduced loss of tyrosine hydroxylase (TH)positive neurons following MPTP-induction. Tanshinone IIA regulated apoptotic pathway proteins, i.e., Bax and Bcl-2, and inhibited the translocation of Cyt C to the mitochondria. Oxidative stress induced by MPTP was significantly inhibited by tanshinone IIA via up-regulation of DJ-1/Nrf2 /HO-1 expression and reduction of ROS and MDA levels. Brain tissue total glutathione content was increased by tanshinone IIA treatment. Conclusion: Tanshinone IIA effectively improves antioxidant status and reduces neuronal loss following MPTP treatment. These results indicate that tanshinone IIA exerts protective effects in MPTPinduced PD in mice. Thus, tanshinone IIA has a good potential for use as a therapy for PD.


INTRODUCTION
Parkinson's disease (PD) is a chronic progressive neurodegenerative disorder characterized by postural instability, tremors, muscular stiffness and bradykinesia [1]. The disease is prevalent in the aged population (60 years and above) [2]. The etiology of PD is complex and yet to be unravelled completely. Pathological observations in PD include the presence of Lewy bodies, degeneration of dopaminergic neurons, gliosis in the substantia nigra pars compacta (SNpc), and proteinaceous cytoplasmic inclusions [1]. Oxidative stress is critically associated with neurodegeneration in PD [3]. Data from autopsy studies in brain tissues of PD patients reveal oxidative modifications and lipid peroxidation of DNA, and protein oxidation [4]. Oxidative stress due to loss of redox balance of the cell leads to excessive generation of reactive oxygen species (ROS). The brain is highly susceptible to oxidative damage by ROS owing to high oxygen consumption, low antioxidant levels and higher levels of oxidizable polyunsaturated fatty acids. Aging brains are more affected by oxidative stress than younger brains, resulting in neuronal damage [5].
Protein deglycase DJ-1 or Parkinson's disease protein 7 (PARK7) is one of the key proteins involved in the cell survival process by regulating antioxidant defence mechanisms [6]. The protein prevents cellular apoptosis under oxidative stress conditions, regulates the synthesis of key antioxidant molecule i.e. reduced glutathione (GSH) [6], and enhances the transcription of MnSOD which scavenges superoxide radicals and prevents cell damage [7]. Nuclear factor erythroid 2-related factor (Nrf2), a transcription factor is also upregulated, thereby modulating the Nrf2-dependent detoxification pathways [8].
Cellular apoptotic dysfunction is regarded as a major event that leads to neuronal cell death in PD. Elevated expressions of caspase 3 and caspase 8 were noticed in brain tissues of PD patients, suggesting activation of the caspase pathway [10]. Therefore, identification of compounds capable of activating DJ-1/Nrf2 and down-regulating apoptotic cascades would be potentially beneficial in PD treatment.
Recent studies have demonstrated the potential benefits of plant-derived compounds in the management of PD [11]. Tanshinone II A is a diterpene quinone isolated from the roots of Salvia miltiorrhiza. It possesses several beneficial properties such as neuroprotective [12], anti-cancer and antioxidant effects [13]. In this study, the protective effect of tanshinone IIA against MPTP-induced PD was investigated in a mouse model of PD.

Animals
For this study, male C57BL/6 mice (n = 80, 7-8 weeks, 25 -30 g) procured form Changchun University animal centre were used. The mice were held under standard in-house conditions of 12-h day /12-h night cycle, temperature between 22 -24 °C, and relative humidity of 55 ± 10 %. They were permitted unrestricted access to animal feed and water. The mice were kept in sterile acrylic cages (6 per cage), and were acclimatised to laboratory environment for 5 days prior commencement of the study. They were maintained with care in accordance with the Animal Care and Use Guidelines of the Institution/Hospital, and in line with National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals [14]. This study was approved by the ethics committee of Affiliated Hospital of Changchun University of Traditional Chinese Medicine (no. TY20160311).

Experimental design and drug administration
The animals were divided into 6 treatment groups (12 per group). Separate groups of mice were administered tanshinone IIA at doses of 12.5, 25 and 50 mg/kg/day via oral gavage for 10 days. Freshly-prepared MPTP in saline was injected intraperitoneally at a dose of 20 mg/kg body weight, 4 times at 2-h intervals on the 6 th day of tanshinone treatment [15]. The control mice received MPTP only. The control group received equivalent volume of saline in place of MPTP. The tanshinone IIA alone-treated group of mice were given 50 mg per day for 10 days, but were not administered MPTP.

Statistical analysis
The results are presented as mean ± standard deviation (SD, n = 6). The analyses were performed using SPSS software (version 22.0, SPSS Inc., Chicago, IL). The results obtained were compared with one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) post-hoc analysis. Values of p < 0.05 were considered statistically significant.

Tanshinone IIA improved the behaviour of PD mice
The effect of tanshinone IIA on MPTP-induced impairment of muscular coordination and bradykinesia was assessed by pole test and Rota-rod test. Experimental PD induction significantly (p < 0.05) reduced retention time in mice to 30.15 % of normal control (Figure 1 ac). Tanshinone IIA administration markedly (p < 0.05) increased the retention time, when compared with MPTP control. The retention time increased to 57, 69.9 and 86.2 % of normal control on tanshinone IIA treatment at dose of 12.5,25 and 50 mg, respectively. In the pole test, T-turn and T-LA were significantly (p < 0.05) longer than those in normal control ( Figure 1b). Interestingly, tanshinone IIA treatment at the three tested doses significantly (p < 0.05) shortened T-turn and T-LA. At 50 mg, tanshinone IIA decreased T-turn and T-LA to 98.2 and 99.10 %, respectively, almost to the same levels in the normal control group. Tanshinone IIA alonetreated animals exhibited retention time and Tturn and T-LA time similar to normal control.

Tanshinone IIA reduced MPTP-induced dopaminergic neuronal cell loss
Immunohistochemical analysis was done to determine the effects of tanshinone IIA on dopaminergic neuronal loss following MPTP injections, and TH-immunohistochemistry (TH-IHC) was conducted in the ST and SNpc tissues. Mice that were treated with MPTP exhibited markedly reduced number of TH-immunopositive neurons ( Figure 2). In the MPTP-treated mice, TH-immunopositive neurons were 35.05 %, relative to the control. However, administration of tanshinone IIA significantly (p < 0.05) prevented MPTP-induced neuronal loss. The TH-positive counts increased to 90.20 % with 50 mg tanshinone IIA. When administered alone, tanshinone IIA at 50 mg did not cause any neuronal loss in mice. Results are expressed as mean ± SD (n = 6); *p < 0.05, compared to control; #p < 0.05, compared to MPTP control; a-d represent means from unlike experimental groups that differ at p < 0.05 as determined by oneway ANOVA and DMRT

Protective effects of tanshinone IIA against MPTP-induced apoptotic pathway dysfunction
In the present study, MPTP caused a significant (p < 0.05) increase in cleaved caspase-3 levels. Interestingly, tanshinone IIA dose-dependently supressed caspase-3 expressions (Figure 3 a-c). The expressions were brought down to 107 % by treatment with 50 mg tanshinone IIA, as against 188% in MPTP control. The mitochondrial Bax Results are presented as mean ± SD (n = 6). *p < 0.05, relative to control; #p < 0.05, compared to MPTP control; a -f represent means from different experimental groups that differ at p < 0.05 as determined by one-way ANOVA and DMRT levels were markedly increased (p < 0.05), while Bcl-2 levels were decreased by MPTP exposure, when compared to control group (Figures 3 a -3c). However, tanshinone treatment markedly (p < 0.05) reduced Bax and improved Bcl-2 levels, thereby restoring the Bcl-2/Bax ratio. Furthermore, the MPTP-induced toxicity significantly increased (p < 0.05) cytosolic Cyt c level to 167.8 %, relative to normal control, indicating translocation. Tanshinone IIA significantly (p < 0.05) suppressed the translocation of Cyt c from mitochondria to cytosol (Figure 3c). These results indicate that tanshinone II A effectively inhibited the apoptotic cascade, thereby exerting protective effects.

Tanshinone IIA reduced oxidative stress in PD mice
Reactive oxygen species (ROS) and MDA levels were determined in the SNpc tissues as indicators of oxidative stress. Significant elevations in ROS and MDA were produced by MPTP treatment (Figures 4 a and b). Tanshinone II A treatment prior to MPTP and post-MPTP exposure significantly (p < 0.05) decreased ROS and MDA levels (Figure 4 a -d). The ROS levels were reduced to 138.67, 55.81 and 11.44 % from 215.60 % on treatment with 12.5, 25 and 50 mg tanshinone IIA, respectively. Furthermore, tanshinone IIA administration (50 mg) markedly raised levels glutathione to 0.0564 µM/mg protein from 0.0295 µM/mg protein. Tanshinone Results are presented as mean ± SD, n = 6. *p < 0.05, compared to control; # p < 0.05, compared to MPTP control; a-e represent means of different experimental groups that differ at p < 0.05 as obtained by one-way ANOVA and DMRT IIA at the 3 tested doses improved the PDinduced alterations in GSH: GSSG ratio ( Figures  5 a and b). These observations indicate that tanshinone IIA reduced oxidative damage in PD.

Tanshinone IIA effectively activated DJ-1/Nrf2 pathway following PD induction
The MPTP-induced oxidative stress resulted in a noticeable decreases in the expressions of DJ-1, Nrf2, HO-1 and NQO1 (Figures 5a -5c). However, tanshinone IIA treatment significantly up-regulated DJ-1 expression in nuclear, mitochondrial and cytosolic fractions. In addition, Nrf2 expression was increased in the nuclear and cytosol fractions to 158 and 122 %, respectively on tanshinone treatment (50 mg/kg). Although the lower doses of 12.5 mg and 25 mg caused substantial elevation in DJ-1 and Nrf2 levels, 50 mg dose was observed to be most effective (Figures 6a -6c). The expressions of SOD-1, HO-1 and NQO1 were significantly (p < 0.05) upregulated by tanshinone treatment, which could be due to enhanced DJ-1 and activation of Nrf2. Compared with the MPTP control group, the tanshinone IIA-treated groups

DISCUSSION
Parkinson's disease affects mostly the elderly, and is characterized by postural instability, muscular rigidity, tremors in the limbs and bradykinesia [2]. Studies have revealed evidence of elevated ROS levels, reduced glutathione (GSH) levels and raised oxidative modification of major biomolecules -nucleic acids, lipids, and proteins [16]. Thus, compounds that efficiently counter oxidative stress could be beneficial in PD treatment. The neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP) is widely employed for induction of PD in experimental animal models [17], and for evaluation of novel neuroprotective agents in PD treatment. Administration of MPTP produces neurodegeneration and neural impairments close to those observed in PD patients [18]. Within the non-dopaminergic cells, MPTP is converted by brain monoamine oxidase B to active 1-methyl-4-phenylpyridinium (MPP+) which enters the dopaminergic neurons and causes neuronal intoxication [1]. The active metabolite, MPP+ accumulates in the substantia nigra pars compacta (SNpc) and obstructs the electron transport chain (complex I) in the mitochondria, thereby leading to ROS production [19]. Increased ROS alters the membrane permeability of the mitochondria and results in the translocation of cytochrome c from the mitochondria to the cytosol. Furthermore, ROS induce activation of caspase-3, resulting in upregulation of apoptosis cascade [20].
In the present study, the neuroprotective effects of tanshinone IIA was assessed in experimental PD model induced by MPTP. The rota-rod test and pole tests are frequently employed for measuring bradykinesia, postural balance and motor coordination in experimental animals [15]. Mice exposed to MPTP exhibited behavioural abnormalities and deficits in motor coordination as reflected by T-turn and T-LA values. However, tanshinone IIA administration significantly improved muscular coordination and motor learning function as evidenced by changes in Tturn, T-LA and retention times, relative to mice given MPTP alone.
The dopaminergic neurons are abundant in the SNpc region. Dopamine is responsible for signal transmission between SNpc and many other areas of the brain. The association between the striatum and SNpc is vital for undisturbed, decisive movement. Irregular nerve-firing in the brain as a result of dopamine depletion causes impaired muscular coordination and movements. In the present study, dopaminergic neuronal loss was evaluated by measuring the number of THpositive cells in SNpc. Tyrosine hydroxylase catalyses conversion of L-dopa to dopamine, the rate-limiting step in the synthesis of dopamine [21].
A substantial decrease in TH-positive cell counts following MPTP treatment was observed, indicating dopaminergic neuronal loss. It has been reported that MPTP-administered mice presented motor deficits and loss of TH-positive neurons [22]. In the present study, tanshinone IIA treatment dose-dependently improved THpositive counts. This increase in TH immunoreactivity could have also improved behaviour and muscular coordination observed in tanshinone treated mice, which reveals the neuroprotective efficacy of tanshinone.
Loss of dopaminergic neurons involves activation of the pro-apoptotic proteins and apoptotic cascade in PD [15. The anti-apoptotic protein Bcl-2 prevents the release of Cyt c to the cytosol, and suppresses mitochondria-mediated initiation of cell damage, while, Bax promotes Cyt c translocation to the cytosol [23]. Caspase-3 is activated by Cyt c leading to activation of the caspase cascade. In this study, MPTP increased Bax in the mitochondria and decreased Bcl-2 levels. The observed increase in Cyt c in the cytosol reflects increased Bax levels. Activation of caspase-3 is an indicator of activation of caspase pathway. Thus, level of cleaved caspase-3 is regarded as major marker of cellular apoptosis. Treatment with MPTP caused a marked increase in the activity of caspase-3 as reflected in raised cleaved caspase-3 levels in the cytosol. Enhanced activation of caspase-3 could have caused activation of the apoptotic pathway leading to neuronal loss in SNpc. Interestingly, on pre-treatment with tanshinone IIA, decreases in cleaved caspase-3 levels were observed.
Moreover, tanshinone caused significant reversal of events leading to apoptosis such as improved membrane integrity, restoration of the Bcl-2/Bax ratio, and reduced release of cyt-C from mitochondria, thereby preventing loss of dopaminergic neurons.
Reactive oxygen species (ROS) disturb the integrity of mitochondrial membrane causing raised levels of Cyt c in the cytosol, thereby activating caspase-3 [24]. Following MPTP injection, increased levels of ROS and MDA were observed, suggesting oxidative stress in the brain tissues. Furthermore, the decreased total glutathione content and altered GSH:GSSG ratio observed indicate redox imbalance. Reduced glutathione (GSH) is a major antioxidant in the brain [25]. Reduced levels of GSH in brain tissues of MPTP-induced PD model have been reported [26]. Tanshinone IIA treatment remarkably suppressed oxidative stress status by reducing ROS production and MDA levels, and improving GSH levels and GSH:GSSG ratio. Altered redox balance has been reported in PD [27]. Thus, restoration of the redox balance with enhanced antioxidant status would aid in PD treatment.
Studies have reported the neuroprotective effects of DJ-1, a multifunctional protein in various experimental models of oxidative stress-induced cell death [22]. A direct antioxidant property of DJ-1 as a scavenger of ROS has been reported as one of the major mechanisms for combating oxidative stress [28]. It regulates the expression of glutathione synthase under oxidative stress conditions and also regulates critical transcription factor-nuclear factor erythroid-2-related factor 2 (Nrf2) [6,8]. The Nrf2 is a master transcription factor implicated in regulation of oxidative stress responses. In the absence of oxidative stress, Nrf2 remains localized in the cytoplasm bound to Keap1, leading to its degradation by ubiquitination. Under oxidative stress, DJ-1 sequesters Keap1, resulting in translocation of Nrf2 from cytosol to nucleus causing activation of various antioxidative stress response genes such as HO-1, NAD(P)H: quinone oxidoreductase-1 (NQO1) and enzymes involved in GSH synthesis [29]. Decline in Nrf2 activity is considered a chief risk factor for PD [30]. Moreover, Nrf2 knockout mice exposed to MPTP exhibited a greater loss of dopaminergic neurons, when compared with wild type mice [31]. In the present investigation, MPTP caused similar significant decreases in DJ-1, Nrf2, HO-1 and NQO1 expressions. Tanshinone IIA-mediated up-regulation in the expressions of DJ-1 and Nrf2 could have been responsible for the reduction in MDA and ROS levels as observed. The enhanced expressions of SOD1, HO-1 and NQO could be due to enhanced DJ-1 and Nrf2 which is likely to have aided in reduction of MPTP-induced oxidative stress. Improvement in the levels of GSH suggests the activation of GSH synthesis enzymes. Furthermore, DJ-1 is known to positively regulate the expression of the TH gene [32]. Thus, tanshinone IIA-induced DJ-1 expression possibly increased TH-positive cells and reduced dopaminergic neuron degeneration. These observations in part could be via direct antioxidant scavenging effects of tanshinone IIA, or indirectly through activation of the DJ-1/Nrf2 pathways.

CONCLUSION
Tanshinone IIA significantly reduces MPTPmediated neuronal loss and improves muscular coordination and oxidative stress in mice. Thus, tanshinone IIA is a potential candidate drug for the treatment of PD. However, further experiments need to be conducted to explore the mechanisms involved.

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

Contribution of authors
We declare that this research study was executed by the author(s) -Jingzhou Zhang, Yahong Wang, Xingwang Ji and Zunhua Shuand all liabilities pertaining to claims relating to the content of this article will be borne by the authors". All authors equally contributed to this study. Jingzhou Zhang and Zunhua Shu designed this study, collected and analysed the data, and also prepared the manuscript. Yahong Wang and Xingwang Ji had contributed in the experimental works and also in statistical study.

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