Effect of rifampicin pre- and post-treatment on rotenone-induced dopaminergic neuronal apoptosis and alpha-synuclein expression**☆○
Yuanlin Sun1, Guohua Zhang1, Jie Xu1, Shiwen Chen1, Enxiang Tao1, Changqing Xu2,          M. Catherine Bennett○2

1Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
2Blanchette Rockefeller Neurosciences Institute, Rockville, MD  20850, USA

Yuanlin Sun☆, Studying for doctorate, Associate professor, Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China

Corresponding author: Enxiang Tao, Doctor, Professor, Doctoral supervisor, Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China   
taoenxiang@yahoo.com.cn

Supported by: the Natural Science Foundation of Guangdong Province, No. 04009355*; Science and Technology Planning Project of Guandong Province, China, 05B33801003*

Abstract
BACKGROUND: Rifampicin inhibits the formation of α-synuclein multimer and protects against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyritine (MPTP)-induced PC12 cell apoptosis.
OBJECTIVE: To compare the effect of rifampicin pre- and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson’s disease.
DESIGN, TIME AND SETTING: A randomized, controlled experiment was performed at the Ex-perimental Animal Center of Sun Yat-sen University North Campus (China) from November 2006 to October 2008.
MATERIALS: Rifampicin was purchased from MD, USA; rotenone was purchased from Sigma, USA; mouse anti-rat α-synuclein monoclonal antibody was purchased from B&D, USA; and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon, USA.
METHODS: A total of 72 male, Sprague Dawley rats, aged 8 weeks, were randomly assigned to 5 groups: blank control (n = 12), rifampicin (n = 12), rotenone (n = 16), rifampicin pre-treatment (n = 16), and rifampicin post-treatment (n = 16). Parkinson’s disease model rats were established via a subcutaneous injection of rotenone (1.5 mg/kg per day) in the three treatment groups, once a day for 3 successive weeks. Rifampicin (30 mg/kg per day) was intragastrically administered in the rifam-picin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction. Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day ri-fampicin in the rifampicin group, once a day for 3 successive weeks in total.
MAIN OUTCOME MEASURES: Prior to treatment and in the end of the 3rd week after treatment, the rats were evaluated using the modified neurological severity score. The substantia nigra from the rats was extracted for hematoxylin-eosin staining. Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression.
RESULTS: Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group, in addition to neurodegradation, hypopigmentation, and pyknosis. In the rifampicin pre-treatment and post-treatment groups, the number of dopaminergic neurons was significantly increased compared with the rotenone group (P < 0.01), with slight neu-ronal damage. Compared with the rotenone group, substantia nigral tyrosine hydroxylase expres-sion was significantly increased in the rifampicin pre-treatment and post-treatment groups  (P < 0.01), but α-synuclein expression and modified neurological severity scores were significantly de-creased (P < 0.01). In addition, the effect of rifampicin in the pre-treatment group was superior to the post-treatment group. There was no significant difference in tyrosine hydroxylase and α-synuclein expression, or in the modified neurological severity scores, between the blank control and rifampicin groups (P > 0.05).
CONCLUSION: Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone. Moreover, rifampicin enhanced tyrosine hydroxylase expression, but inhibited α-synuclein expression. The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.
Key Words: rifampicin; rotenone; Parkinson’s disease; α-synuclein; dopaminergic neurons

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