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العنوان
Study on potential protective effect of acetaminophen in experimentally
induced Parkinson’s disease in rats /
المؤلف
Mahmoud, Aya Yassin Mohamed.
هيئة الاعداد
باحث / آية ياسين محمد محمودآية ياسين محمد محمود
مشرف / ماريان جورج تادرس
مشرف / ريم نبيل أبو النجا
مناقش / الشيماء أمين زكي البهي
تاريخ النشر
2021.
عدد الصفحات
208 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
الصيدلة ، علم السموم والصيدلانيات (المتنوعة)
تاريخ الإجازة
1/1/2021
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - قسم الادوية والسموم
الفهرس
Only 14 pages are availabe for public view

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from 203

Abstract

Parkinson’s disease (PD) is the second most common neurodegenerative disease, following Alzheimer’s disease. Clinically, the disease is mainly characterized by motor impairments including: resting tremors, muscle rigidity, and aberrant gait, and posture. PD is characterized by two main pathological hall marks: 1) the progressive degeneration of the dopaminergic neurons originating from the substantia nigra pars compacta (SNpc) resulting in the depletion of dopamine (DA) output to the striatum, and 2) the accumulation of intra-neuronal proteinaceous inclusion called Lewy bodies (LB) containing -synuclein as the main component. Although the pathogenesis of PD is not yet clear, it is thought to involve several factors including: inflammatory processes, oxidative stress and apoptosis. Rotenone is a natural pesticide that can induce PD-like symptoms in rats. The chronic dosing of small doses of rotenone to rats is found to induce both motor features of PD.
Acetaminophen (ACM) is a widely-used analgesic and anti-pyretic. During the past two decades, several researches demonstrated the potential of other uses of this drug including neuroprotective and anxiolytic action. Furthermore in 2005, ACM was proven to undergo a two-step metabolism, where it is first deacetylated in the liver to give the intermediate 4-aminophenol. This is followed by its conjugation with arachidonic acid in the brain, through the action of fatty acid amide hydrolase enzyme (FAAH), to form the active metabolite, AM404. AM404 was found to modulate the endocannabinoid system (ECB). Following researches tried to investigate whether the actions of ACM were due to this metabolite. It was reported that the analgesic and anxiolytic activity of ACM is dependent upon its conversion to AM404; however, the hypothermic effect of ACM was discovered to be independent upon this conversion.
Accordingly, this study aimed to investigate the potential neuroprotective effect of ACM in a rotenone-induced rat model of PD, the potential mechanisms of this neuroprotection, and whether these actions are dependent upon its conversion to the active metabolite, AM404.
The main findings of the study can be summarized as follows:
Part I: The dose-response study
In the initial dose-response study, 30 rats were divided into 5 groups (n=6) where, the 1st group served as control (received vehicles; saline, i.p. and sunflower oil, s.c., for 4 weeks), the 2nd group served as the disease group (received rotenone 2 mg/kg/day, s.c. for 4 weeks), the 3rd, 4th, and 5th groups received different doses of ACM (25, 50, 100 mg/kg/day, i.p., respectively) followed by rotenone (2 mg/kg/day, s.c.) 20 minutes later, for the period of 4 weeks. Twenty four hours following the last dose, rats were tested for behavioral abnormalities through testing their locomotor activities and their latency to fall off the rotarod bar (Rotarod test), then they were sacrificed and their brains were dissected out and used for histopathological examination, immunohistochemical analysis of TH and -synuclein, and chromatographic assay of DA content.
A. The effects of rotenone on rats:
1. Rotenone administration (2 mg/kg/day) for 28 days induced motor deficiencies in rats shown by significant reduction in latency to fall off the rotarod and locomotor activity, as compared to the control group.
2. Histological examination of striata and midbrain of rotenone-treated rats showed several pyknotic nuclei and blood vessels congestion.
3. Significant reduction in TH immunoreactivity in rats’ midbrains, as compared to control group. Furthermore, rotenone significantly increased -synuclein immune-reactivity in rats’ midbrains and striata, as compared to the control group.
4. Rotenone induced a significant reduction in the striatal DA content, as compared to the control group.
B. The effects of different doses of ACM on rotenone-induced Parkinson’s disease in rats:
1. No significant improvements were observed in rotarod performance nor in locomotor activity upon the co-administration of ACM (25 mg/kg/day) for 28 days, as compared to the rotenone-treated group. In contrast, the locomotor activity was significantly improved upon the co-administration of ACM (50 mg/kg/day) for 28 days, as compared to the rotenone-treated group. However, no significant difference was observed in rotarod performance, as compared to the rotenone-treated group. Notably, both locomotor activity and rotarod performance were significantly improved with the co-administration of ACM (100 mg/kg/day) for 28 days, as compared to the rotenone-treated group.
2. The histological examination showed pyknotic nuclei and blood vessels congestion with ACM (25 or 50 mg/kg/day). Nevertheless, the co-administration of ACM (100 mg/kg/day) restored normal histological structure.
3. The co-administration of ACM (25 or 50 mg/kg/day) did not show any significant improvement from the rotenone-treated rats in TH immunoreactivity and -synuclein immunoreactivity. However, ACM (100 mg/kg/day) significantly reversed the rotenone-induced reduction of TH immunoreactivity and increase of -synuclein immunoreactivity.
4. The Striatal DA content was not increased with the co-administration of ACM (25 or 50 mg/kg/day), as compared to the rotenone-treated group, whereas, the co-administration of ACM (100 mg/kg/day) significantly increased rotenone-induced reduction of the striatal DA content.
Based on the previous results, the highest dose of ACM (100 mg/kg/day) was selected for further investigations.
Part II: The mechanistic study
The mechanistic study involved 60 rats divided into 6 groups (n=10), where the 1st group served as the control group (received vehicles), the 2nd group received ACM alone (100 mg/kg/day, i.p., for 4 weeks), the 3rd group served received rotenone (2 mg/kg/day, s.c., for 4 weeks), the 4th group received ACM (100 mg/kg/day, i.p.) followed by rotenone (2 mg/kg/day, s.c.) 20 min later, for 4 weeks, the 5th group received the FAAH enzyme inhibitor, URB597 (0.15 mg/kg/day, i.p.), followed by rotenone (2 mg/kg/day, s.c.) 15 min later, for 4 weeks, the 6th group received URB597 (0.15 mg/kg/day, i.p.) followed by ACM (100 mg/kg/day, i.p.) 15 min later, then rotenone administration (2 mg/kg/day, s.c.) 20 min later, for 4 weeks. Twenty four hours after the last dose, the rats were sacrificed and brains were removed and used for the assessment of the following parameters: inflammatory markers (iNOS, COX-2, and NF-КB), Oxidative stress markers (MDA and GSH), apoptotic markers (cytochrome-c and caspase-3), and ECB receptors mRNA expression (CB1 and CB2 receptors).
A. The effects of rotenone on rats:
1. Increased immunoreactivity of the inflammatory markers: NF-КB, COX-2, and iNOS, in midbrain and striata of the rotenone-treated rats, as compared to the control group.
2. The oxidative stress markers were significantly altered in midbrain and striata of the rotenone-treated rats, indicated by the marked increase in the MDA levels as well as a significant reduction in the GSH levels, as compared to the control group.
3. The apoptotic markers (cytochrome-c and caspase-3) were significantly increased in both the midbrain and striatum of the rotenone-treated group, as compared to the control group.
4. The expression of CB1 and CB2 receptors was significantly reduced in both the midbrain and striatum of the rotenone-treated rats, as compared to the control group.
B. The effects of ACM on rotenone-induced Parkinson’s disease:
1. The inflammatory markers’ levels were significantly reduced in the midbrain and striatum upon the administration of ACM or URB597, as compared to the rotenone-treated group. Together, URB597 and ACM administration showed a further decrease in the immunoreactivity of the inflammatory markers, which was significant from the ACM- and the URB597-treated groups.
2. The oxidative stress markers showed significant changes with the administration of ACM or URB597, as showed by a significant reduction in the MDA levels and an increase in the GSH levels in the midbrain and striatum, as compared to the rotenone-treated group. Combined, URB597 and ACM administration induced further reduction in the MDA levels and further increase in GSH levels in both midbrain and striatum, as compared to the ACM- and URB597-treated groups.
3. The apoptotic markers, cytochrome-c and caspase-3, were significantly reduced upon the administration of ACM, as compared to the rotenone-treated group. The co-administration of URB597 significantly reduced the cytochrome-c levels in both the midbrain and striatum, as compared to the rotenone-treated group. Together, URB597 and ACM administration induced further reduction in the cytochrome-C levels in both the midbrain and striatum, as compared to the ACM- and URB597 treated groups. The co-administration of URB597 had no effect on caspase-3 activity neither when it was administered alone nor with ACM in the rotenone-treated rats.
4. The mRNA levels of CB1 receptor were significantly increased only in the striatum with the administration of ACM or URB597, as compared to the rotenone-treated group. The co-administration of both URB579 and ACM resulted in a further increase in CB1 receptor mRNA in the striatum, which was statistically significant from the ACM- and URB597-treated groups.
5. The CB2 receptor mRNA levels were significantly increased in the midbrain and striatum with ACM or URB597, as compared to the rotenone-treated group. The administration of URB597 prior to ACM resulted in a further increase in the CB2 receptor mRNA levels in the midbrain and striatum, as compared to the ACM- and URB597-treated groups.
In conclusion, rotenone successfully induced behavioral, histological, and biochemical features of PD in rats in the present study. A low dose of ACM (25 mg/kg) could not ameliorate the rotenone-induced effects in rats. The administration of ACM (50 mg/kg) before rotenone showed some improvement in the rats’ locomotor behavior; however, no improvement was observed in the histological or biochemical assessments. The administration of ACM (100 mg/kg) showed a significant improvement in the rotenone-induced rats’ behavioral abnormalities, altered TH and -synuclein expression, and the striatal DA content. A mechanistic study showed that ACM’s neuroprotective properties may be due to its anti-oxidant, anti-inflammatory, anti-apoptotic, and up-regulatory effect on cannabinoid receptors. Inhibiting ACM metabolism into AM404, using the FAAH inhibitor (URB597) did not prevent the neuroprotective actions of ACM, indicating that the neuroprotective actions of ACM are exerted independently of its conversion to the active metabolite, AM404. It is worth mentioning that inhibiting AM404 formation increased the ability of ACM to induce CB receptors expression. This means that ACM, rather than its metabolite, is responsible for the modulation of CB receptors expression. In addition, URB597 showed anti-oxidant, anti-inflammatory, anti-apoptotic, and modulatory actions of CB receptors, as compared to the rotenone-only treated rats. The current study sheds the light on the neuroprotective effects of ACM and its promising potentials in the management of PD. Indeed, future research is required to deeply investigate the benefits of combining ACM and FAAH inhibitors for the neuroprotection in PD.