مطالعه اثر سیلیمارین بر کاتالپسی و اختلال حرکتی ناشی از 6-هیدروکسی دوپامین وسنجش پارامترهای آپوپتوتیک و التهابی مغز در موش های صحرائی نر

Abstract

OBJECTIVES Most chronic neurodegenerative diseases such as Parkinsons disease (PD) are accompanied by neuroinflammation which is associated with glial cells activation and production of different inflammatory cytokines. Additionally, apoptosis and oxidative stress has been shown to be associated with the development of PD. In the present study, we investigated the effect of silymarin (SM), on 6-hydroxydopami (6-OHDA)-induced motor-impairment, myeloperoxidase (MPO) activity, cerebrospinal fluid (CSF) levels of inflammatory cytokines, midbrain level of glutathione and midbrain level of apoptotic related factors in rats. Methods In pre-treatment phase, male Wistar rats were pre-treated with intraperitoneal (i.p.) injections of SM (100, 200 and 300 mg/kg) for 5 consecutive days. Then, Motor in-coordination and catalepsy were induced by unilateral injection of 6-OHDA (8إg/2إl/rat) into the central region of the substantia nigra pars compacta (SNc). After 3 weeks as a recovery period anti-parkinsonian effects of SM were investigated by rotarod and bar test. In treatment groups, firstly 6-OHDA was injected into the SNc and then, after 21 days lesioned rats that show motor-disturbances (catalepsy and motor in-coordination), were treated with SM for 15 consecutive days. Catalepsy and motor coordination were assessed 1, 5, 10 and 15 days after treatment beginning. CSF levels of inflammatory cytokines evaluated by ELISA, midbrains glutathione and MPO activity assessed by spectrophotometery and apoptotic related factors assessed by western blotting.Results The results showed a significant (p<0.001) increase in catalepsy and motor in-coordination of 6-OHDA-lesioned rats whereas; SM in both pre-treatment and treatment groups was able to decrease catalepsy and improve motor coordination significantly (p<0.001) in a dose dependent manner. The most anti-cateleptic and motor improving effect was observed at the dose of 300 mg/kg of silymarin (p<0.001). There was a significant (p<0.001) increase in MPO activity of 6-OHDA-lesioned rats whereas; in SM pre-treated and treated hemi-parkinsonian rats MPO activity were decreased markedly (p<0.001). Furthermore, there was a significant (p<0.001) increase in CSF levels of pro-inflammatory cytokines (TNF-, IL-6 and IL-1) in 6-OHDA-lesioned rats whereas; in both SM pre-treated and treated hemi-parkinsonian rats their levels were decreased markedly (p<0.001). Another key finding of the present study was a significant (p<0.001) decrease of reduced glutathione (GSH) and increase of oxidized glutathione (GSSG) level in midbrain of hemi-parkinsonian rats. SM in both pre-treatment and treatment phases, could increase GSH level and attenuate GSSG level in midbrain of hemi-parkinsonian rats, in a significant (p<0.001) manner. The results of Western blotting analysis for Bax and Caspase-3 showed that in the 6-OHDA-lesioned rats these proteins significantly (p <0.001) were up-regulated and following treatment with SM strongly down regulated. Also, SM pre-treatment inhibited up-regulation of these pro-apoptotic proteins. In 6-OHDA-lesioned rats the expression of Bcl-2 significantly (p<0.001) decreased and pre-treatment with SM prevented from down regulation of this protein so that remain its level about the normal rang. Additionally, long-term treatment with SM significantly (p<0.001) up-regulate anti apoptotic protein Bcl-2.Conclusion In summary, we found that pre-treatment and long-term treatment with SM could improve 6-OHDA-induced catalepsy and motor imbalance by attenuating midbrain MPO activity as marker of microglial function and subsequent decrease of TNF-, IL-6 and IL-1 concentration in CSF as pro-inflammatory cytokines as well as increase in midbrain glutathione content as most important anti oxidant defense. Furthermore, SM in both pre-treatment and treatment phases, attenuated content of apoptotic protein Bax and Caspase-3 and augmented level of anti apoptotic protein Bcl-2. According to potential prophylactic and therapeutic effects of SM observed in this study, we suggest that SM can be used as adjuvant therapy along with commonly used anti-parkinsonian drugs in PD patients. However, further basic and clinical trial studies should be carried out to prove this hypothesis.