Tanshinone IIA prevents the loss of nigrostriatal dopaminergic neurons by inhibiting NADPH oxidase and iNOS in the MPTP model of Parkinson's disease

https://doi.org/10.1016/j.jns.2014.11.026Get rights and content

Highlights

Abstract

Tanshinone IIA is one of the major constituents of Salvia miltiorrhiza Bunge known as Danshen. Recent reports have shown that Tanshinone IIA has neuroprotective effects against cerebral ischemia/reperfusion injury and traumatic injury of the spinal cord in rats. However, whether Tanshinone IIA has any neuroprotective effect in Parkinson's disease remains unknown. In this study, we evaluated whether Tanshinone IIA promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and microglial activation as visualized by tyrosine hydroxylase and CD11b immunoreactivity. The results of Western blot and immunohistochemistry showed upregulation of NADPH oxidase and iNOS in the MPTP-treated substantia nigra pars compacta. Treatment with Tanshinone IIA prevented degeneration of nigrostriatal DA neurons and increased the level of striatal dopamine content. This neuroprotection afforded by Tanshinone IIA was associated with the suppression of microglial activation and reduced expression of NADPH oxidase and iNOS. The present findings show that Tanshinone IIA may possess anti-inflammatory and anti-oxidative properties and may have therapeutic value in the treatment of Parkinson's disease.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by abnormal motor behavior, which includes resting tremor, rigidity and bradykinesia [13], [25]. The main neuropathological feature of PD is the progressive loss of the nigrostriatal dopamine (DA) containing neurons, the cell bodies of which are in the substantia nigra pars compacta (SNc), and the nerve terminals are in the striatum (STR) [28]. Although decades of hard work have been done to uncover the profound mystery of PD, its pathogenesis is still unknown.

Most of PD cases are sporadic, only a few have family history [19]. Many studies suggest that microglial activation causing oxidative stress increases the risk of developing sporadic PD [10], [22]. In these studies, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD is widely used. In the MPTP mouse model, large amounts of microglial cells transform from resting state to active state, and secrete several harmful factors, among which, reactive oxygen species (ROS) and nitrogen species (RNS) are two important factors. The two reactive species are mainly produced by NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively. NADPH oxidase and iNOS are commonly considered to be derived from microglial cells, and in the MPTP mouse model, both are upregulated in the SNc, and may contribute to the death of DA neurons [15], [32], [33].

Tanshinone IIA (Tan IIA) is extracted from Salvia miltiorrhiza Bunge known as Danshen, a traditional Chinese medicine, which is used in the treatment of several diseases, such as cardiovascular diseases [14], [34], cerebrovascular diseases [21], [37] and postmenopausal syndromes [16]. The molecular formula of Tan IIA is C19H18O3, and its molecular weight is 294.34 (Fig. 1). Several studies have shown that Tan IIA has anti-inflammatory and anti-oxidative properties and has been used to prevent ischemia/reperfusion injury [8], [9] and alleviate traumatic injury of the spinal cord in rats [35]. However, the effects of Tan IIA in the nigrostriatal DA system in the context of PD are still unknown.

In the present study, we aim to estimate the effects of Tan IIA on PD. We used the neurotoxin, MPTP to reproduce a PD model, and investigated the following changes in the SNc, to analyze the effects of Tan IIA. In our research, we found that after MPTP injection, the number of DA neurons was seriously decreased in the SNc, microglial cells were widely activated, and the expression of NADPH oxidase and iNOS was upregulated in the SNc. After the administration of Tan IIA, we found that nigrostriatal DA neurons were largely rescued, microglial activation was inhibited, and the expression of NADPH oxidase and iNOS was also suppressed.

Section snippets

Reagents

MPTP·HCL (Sigma, USA) was dissolved in phosphate buffered saline (PBS). Tan IIA (purity 98%, HPLC) was obtained from Kailai Biotechnology Co. (Shaanxi, China), and dissolved in PBS including 1% dimethylsufoxide (DMSO) before experiments. Antibodies were purchased from as follows: monoclonal anti-tyrosine hydroxylase (TH) antibody (Chemicon, USA), polyclonal anti-p47-phox antibody (Bioworld, USA), polyclonal anti-iNOS antibody (Bioss, China), polyclonal anti-CD11b antibody (Boster, China), and

Tan IIA protects nigrostriatal DA neurons from MPTP-induced neurotoxicity in vivo

In the brain, MPTP is converted to 1-methyl-4-phenyl-pyridinium (MPP+) which is mainly responsible for MPTP neurotoxicity. Data indicates that MPTP is totally converted to MPP+ and largely cleared within 12 h after injection [17], [26]. As Tan IIA was administered 12 h after the last injection of MPTP for all the groups in this experiment, its effects could not be attributed to lessened metabolism of MPTP to MPP+, or reduced uptake of MPP+ into DA neurons.

Similarly to a previous report [20], MPTP

Discussion

In the present study, we demonstrated that Tan IIA effectively protected nigrostriatal DA neurons from the toxic effects of MPTP in vivo. We showed that Tan IIA inhibited microglial activation, reduced the expression of NADPH oxidase and iNOS, and led to the survival of nigrostriatal DA neurons in vivo, recovery of dopamine content in the STR and improvement of motor dysfunction. To our knowledge, this is the first study to show that Tan IIA prevents the loss of nigrostriatal DA neurons in the

Conflict of interest statement

The authors have no financial conflicts of interest.

Acknowledgments

This work was supported by the Sci-tech Research Development Program(09130202A-3-19) of Thangshan, China and supported by the Natural Science Foundation(c2004000689), the Doctoral Research Foundation(05547008D-4) and the Sci-tech and Social Research Development Program(04276135) of Hebei Province, China.

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