Neuroprotective Effects Of Tetramethylpyrazine Against Dopaminergic Neuron Injury In A Rat Model Of Parkinson’s Disease Induced By MPTP

Objective:Parkinson’s disease(PD) is the second most prevalent progressive neurodegenerative disease. Although several hypotheses have been proposed to explain the pathogenesis of PD, apoptotic cell death and oxidative stress are the most preva lent mechanisms. Tetramethylpyrazine(TMP) is a biological component that has been extracted from Ligusticum wallichii Franchat(C huan Xiong), which exhibits anti-apoptotic and antioxidant roles. In the current study, we aimed to investigate the possible protective effect of TMP against dopaminergic neuron injury in a rat model of Parkinson’s disease induced by MPTP and to elucidate probable molecular mechanisms. Methods: 1. Animals and Experimental designRats were distributed randomly into 4 groups with 10 replicates in each group: the control group(Con, treated with vehicles), MPTP(MPTP-lesioned group and injection of vehicles) group, TMP group(injected with TMP) and TMP +MPTP group(injected with TMP plus MPTP).TMP(20 mg/kg/d per rat, dissolved in 3% DMSO, i.p.) was administered for 7 days. On day 7, the animals received bilateral stereotaxic injection into the substantianigra(SN) with MPTP(Sigma, USA; 1 μmol in2 μl of saline, 0.35 μl/min) through a 30-gage needle according to the following coordinates: AP:- 5.0 mm, ML: ±2.1 mm, DV:- 7.7 mm from the bregma,midline, and skull surface after 1 h of administration. The method of brain surgery has been reported previously [17]. Following brain lesioning, the rats continued to receive TMP for another 7 days. Ladder walking was conducted at the 14 th day at 14:00–18:00 h. At the end of these experiments, the rats were sacrificed and the striatum and SN were dissected for other experiments. 2. Ladder walkingThe ladder walking test was conducted to estimate the ability of rats to coordinate hindlimb and forelimb. 3. Immunostaining.After the treatment, immunostaining was conducted as previously reported [18]. In brief, rats were perfused through the heart under deepanesthesia with 150-200 ml of 4% paraformaldehyde in phosphate buffer, p H7.4. Then brains were removed, and cryoprotection was conducted in 30% sucrosephosphatebuffer, p H 7.4. After that, the brains were frozen at-20℃, and serial sections(5 μm thick) were cut and mounted on glass slides. Nonspecific binding was blocked and cells were incubated overnight at 4 oC with specific antibodies, and then incubated with Cy3-conjugated antibody. Specimens were examined with a confocal microscopy(Nikon AR1). 4. Measurement of dopamine and its metabolitesIn striata, the levels of dopamine and its metabolites were detected by HPLC analysis as previously described [19]. The results were calculated and expressed as ng/mg tissue weight. 5. Measurement of TBARS and GSHThiobarbituric acid(TBA) reactive substances(TBARS) and glutathione(GSH) content in substantianigra(SN) homogenate were determined by commercial kits according to manufacturer’s protocol. The results were normalized to protein content. 6. Western blot Results: 1. Effect of TMP on MPTP-induced motor deficits.To detect the deficits in limb coordination and limb placing and evaluate the rat’s ability to navigate across a run way with irregularly spaced rungs, we conduc ted the ladder walking test. As shown in Fig. 1, rats in the MPTP group markedly spent longer time in crossing the run way than those of control, confirming that MPTP induced motor deficits. Compared with those of MPTP group, rats in TMP + MPTP group spent shorter time in crossing the run way. The results demonstrated that TMP prevented against MPTP-induced motor deficits. 2. Effect of TMP on MPTP-induced dopaminergic neuron damage.In order to measure the protective effect of TMP on dopaminergic neurons, immunostaining was conducted using an antibody against tyrosine hydroxylase(TH). As shown in Fig. 2, we observed significant decrease of TH- immunoreactivity in MPTP-treated rats, compared with that of control. As expected, the administration of TMP could significantly prevent MPTP- induced decrease of TH- immunoreactivity. Moreover, western blot was used to measure the protein expression of TH. we figured out that, compared with control groups, the expression of TH was decreased after the exposure to MPTP. TMP treatment notably inhibited MPTP- decreased TH protein expression, reflected by the results of western blot.Furthermore, we detected dopamine release in SN. Dopamine and DOPAC levels were assayed by HPLC. MPTP injections in SN resulted in a significant reduction of DA and DOPAC. Injection of TMP prevented MPTP- induced reduction of DA and DOPAC. The results demonstrated that TMP could prevent MPTP-induced dopaminergic neuron damage. 3. Effect of TMP on MPTP-induced apoptosis.In order to evaluate apoptosis-related alterations, we detected protein expression of Bax and Bcl-2. in SN, MPTP injection increased Bax expression and decreased Bcl-2 expression. MPTP- induced changes of Bax and Bcl-2 expression were prevented by TMP administration. Moreover, we de termined cytochrome c release in cytoplasm. MPTP injection in SN increased cytoplasmic cytochrome c expression, which was inhibited significantly by TMP. Furthermore, we measured the cleavage of caspase 3 in SN. we figured out that the injection of MPTP induced evident cleavage of caspase 3 and TMP administration significantly decreased the expression of cleaved caspase 3 in SN. These data demonstrated that TMP could prevent MPTP-induced apoptosis in SN. 4. Effect of TMP on MPTP-induced oxidative stress.Next, we evaluated the effect of TMP and MPTP on redox balance in SN. In Fig. 5A, the results showed that in SN, MPTP significantly increased the level of TBARS, indicating the occurrence of oxidative injury. The administration of TMP prevented the increase of TBARS level. Moreover, GSH content was examined and the results showed that MPTP injection reduced GSH level significantly, compared with that of control. In contrast, the administration of TMP could prevent MPTP- induced reduction of GSH content. Furthermore, we examined the molecular mechanisms underlying the effect of TMP and MPTP on redox balance. nuclear factor erythroid 2 p45-related factor 2(Nrf2) and glutathione cysteineligase catalytic subunit(GCLc) expression was reduced by MPTP, compared with that of control. As expected, TMP significantly prevented MPTP- induced reduction of Nrf2 and GC Lc expression. These results indicated that TMP could prevent MPTP-induced oxidative stress in SN. Conclusion:1. TMP could improve MPTP induced rat movement disorders and can significantly prevent MPTP induced dopaminergic neuron injury.2. Antiapoptotic and anti-oxidation are TMP possible protection mechanisms of neuronal damage.3. TMP’s antiapoptotic effect is through the inhibition of Bax expression, increase the Bcl- 2 expression and inhibiting the release of cytochrome c, inhibit caspase3 cracking to implement; Its antioxidant properties associated with regulating Nrf2 and GCLc.