| Beta-asarone is the main active ingredients of traditional Chinese medicine (Acorus tatarinowii), which has a wide range of pharmacological effects, especially in terms of the role of the central nervous system. Early research group found that β-asarone can significantly improve cerebral ischemia-reperfusion injury and β-amyloid protein induced damage cells. Also we found that β-asarone had a protective role by regulating autophagy pathway. As a second procedural manner of death, autophagy is closely linked to neurodegenerative diseases, which is the current hot research in biological sciences. However, whether β-asarone have the role of improving behavior test and neurotransmitters in Parkinson’s disease model? Whether β-asarone could protect dopamine neurons via autophagy pathway? What is mechanism of β-asarone? Currently, there is no relevant reports.The experiment was mainly composed of four parts, part I:the brains were divided into four regions:the midbrain, striatum, cortex, hippocampus, and the dynamic changes of Beclin1and Tyrosine hydroxylase (TH) were studied in the four regions of6-OHDA induced rats;Part II:the brains were divided into three regions:the striatum, cortex, hippocampus, and the dynamic change of monoamine neurotransmitters were studied in the three regions of6-OHDA induced rats;Part III:The pharmacodynamic effect of β-asarone on behavioral teat, monoamine neurotransmitters, TH and a-synuclein(a-syn) of6-OHDA induced rats were explored. Part IV:To investigate whether β-asarone could improve the damage of6-OHDA rats through autophagy pathway.1.1ObjectiveBeclin1played a significant role in PD. Mice with the conditional deletion of Atg7in the dopamine neurons of the SNpc and other regions could result in the age-related loss of dopaminergic neurons and corresponding loss of striatal dopamine, the accumulation of low-molecular-weight a-syn, and the presence of ubiquitinated protein aggregates, recapitulating many of the pathologic features of PD. Tyrosine hydroxylase (TH) is the main regulator and rate-limiting enzyme of dopamine synthesis. The decrease of substantia nigra TH content and resultant dopamine insufficiency were believed to trigger PD. But the time-course of Beclin1in striatum, hippocampus and cortex areas of6-OHDA-induced rats remained unknown. Also the time-course of TH in striatum and mesencephalon areas of6-OHDA-induced rats remained unknown. And the relationship between TH and Beclin1remained unclear.1.2MethodsTo determine time-course and relationships of Beclin1and TH expressions in6-OHDA-induced rats, rats were divided into seven groups:sham group and six groups treated with6-OHDA lesion (sacrifice time=3,6,12,24,48and72h). And we divided brain into three areas:striatum, hippocampus and cortex areas, and then analyzed the Beclin1in the three areas. Meanwhile TH expressions in mesencephalon and striatum areas were analyzed. Beclin1and TH analysis were performed by flow cytometry and immunohistochemistry respectively.1.3Results1.3.1Beclin1levels of6-OHDA induced rats among different timesSpecifically, Beclin1expression increased at3h, peaking at24h, after that it declined slowly and remained stable at72h in hippocampus. Compared to Beclin1in group D (sacrifice time=24h), it showed a significant decrease in other groups (sham group; sacrifice time=3,6,12,48and72h)(P<0.05). While in cortex and striatum areas, it also increased at3h, peaking at12h, after that it declined slowly and remained stable at72h.1.3.2Beclin1levels of6-OHDA induced rats in hippocampus, cortex and striatum areasIn group C (sacrifice time=12h), Beclin1level in striatum and cortex areas (23.6±3.46%;19.96±4.19%) were significant higher than that of hippocampus area (7.11±0.81%)(P<0.05). Besides, in group D (sacrifice time=24h), Beclin1levels in hippocampus area (15.56±1.84%) were significant higher than that of cortex area (13.02±1.94%)(P<0.05).1.3.3TH expressions of6-OHDA induced rats in different timesIn mesencephalon and striatum, TH expressions were high in sham group, but it declined significantly after6-OHDA injection. Compared to TH expression in sham group, it showed a significant decrease in other times (sacrifice time=3,6,12,24,48and72h)(P<0.05). The TH expressions were almost similar with each other in two areas.1.3.4TH expressions of6-OHDA induced rats in mesencephalon and striatum areasIn sham and other groups, there were no significant differences for TH expressions between mesencephalon and striatum areas (P>0.05). However, in group C and D (sacrifice time=12and24h), mesencephalon TH expressions showed a significant decline compared with that of striatum area (P<0.05).1.3.5Correlations between Beclin1and TH expressions of6-OHDA induced ratsPearson analysis showed that correlations between TH and Beclin1expressions were significant negative from0to12h (P<0.05), while their correlations were significant positive from12to72h (P<0.05).1.4ConclusionsIn conclusion, our studies could provide the time-course and relationship of Beclin1and TH expressions in different areas of6-OHDA-induced rats, and our data could offer great potential of Beclin1dependent autophagy regulation for future studies regarding new potential treatments for PD.2.1ObjectiveThe main cause of PD motor symptoms lies in the degeneration of dopaminergic (DAergic) neurons in the SNpc, leading to a reduction of DA in the caudate putamen complex. However,damage to the noradrenergic and serotonergic systems may also influence motor and non-motor symptoms of PD, such as cognitive function and mood. Furthermore, many brain areas other than SNpc and striatum are also involved in PD. Studies mostly focus on the neurotransmitter changes of6-hydroxydopamine (6-OHDA) models after weeks. However, the immediate neurotransmitters change of unilateral MFB6-OHDA injection rats remains unknown.2.2MethodsIn this study, we investigated dynamic neurotransmitters change in striatum, cortex and hippocampus areas of6-OHDA-induced rats. Rats were divided into seven groups:sham group and six groups treated with6-OHDA (sacrifice time=3,6,12,24,48and72h). And then, brain was divided into three areas and analyzed time-course of DA, NE,5-HT, HVA and5-HIAA.2.3Results 2.3.1Dynamic changes of DA, NE,5-HT, HVA and5-HIAA in6-OHDA-induced ratsIn striatum,6-OHDA injection caused a sharp decline in DA levels in the first12hours followed with a further reduction between12and48hours. In contrast, HVA level was more stable in the first12hours and showed a marked reduction between12and24hours.5-HT and5-HIAA levels decreased linearly for72hours, whereas NE level showed a slight reduction in the first48hours, and returned back to normal afterwards.In hippocampus,6-OHDA injection caused a sharp decline in DA levels in the first24hours followed with a further reduction between48and72hours. However, HVA level showed a gradual reduction for72hours.5-HT level showed a sharp reduction in the first6hours followed by a gradual reduction between6and72hours. In contrast,5-HIAA levels showed a stable level in the first12hours followed by a slight reduction between12and48hours until could not be analyzed. NE level showed a slight reduction in the first6hours, and returned back to normal at last.In cortex,6-OHDA injection caused a sharp decline in DA levels in the first6hours followed with a further reduction between12and72hours. However, HVA level showed a gradual reduction for72hours.5-HT level also showed a sharp reduction in the first6hours followed with a stable level between12and72hours. In contrast,5-HIAA levels showed a slight reduction in the first24hours until could not be analyzed. NE level showed a slight reduction in the first12hours followed by returning back to normal at last.2.3.2Correlations between neurotransmitters in hippocampus, cortex and stri-atum of6-OHDA induced ratsPearson analysis showed significant positive correlations among the neurotransmitters, but there was no significant difference between NE and DA.2.4ConclusionsIn conclusion, our study showed that DA,5-HT, HVA, NE and5-HIAA levels in hippocampus, cortex and striatum areas of6-OHDA induced rats were both affected in the early phase of6-OHDA induced rats. Furthermore, it showed that there were significant positive correlations between DA and5-HT, while there were significant negative correlations between NE and DA. This indicated the dopaminergic, noradrenergic and serotonergic systems involved in the PD have interactions with each other due to the6-OHDA injection.3.1Objective Our previous studies suggested that β-asarone can attenuate ischemia-reperfusion brain brain injury and improve the β-amyloid protein induced damage cells, and we found that β-asarone had a protective role by regulating autophagy pathway. As a second procedural manner of death, autophagy are closely linked to neurodegenerative diseases, which is the current hot research in biological sciences. However, whether β-asarone have the role of improving the behavior test and neurotransmitters in Parkinson’s disease models? Whetherβ-asarone could protect dopamine neurons via autophagy pathway? What is mechanism of β-asarone? Currently, there are no relevant reports.In this study, the pharmacodynamic effect of β-asarone on behavioral tests, monoamine neurotransmitters, TH and α-syn of6-OHDA induced rats were explored.3.2MethodsThe6-OHDA model was adopted in our study. Rates were randomized into groups of10animals. The treatment was as follows:group â… (sham group),2mL/kg water orally per day for4weeks; group â…¡ (model control),2mL/kg water orally per day for4weeks; group â…¢ (low dose),10mg/kg of β-asarone orally per day for4weeks; group IV (medium dose),20mg/kg of β-asarone orally per day for4weeks; and group V (high dose),40mg/kg of β-asarone orally per day for4weeks;group VI,75mg/kg of madopar orally per day for4weeks. At1hr after the last administration, rats were anesthetized with3%chloral hydrate by intraperitoneal injection (350mg/kg). Before sacrifice, the animals were investigated by open field test, rotational behaviour test, initiation time and stepping time. The monoamine neurotransmitters in striatum were detected by HPLC-FD, and the TH and a-syn of6-OHDA induced rats were explored by immunosorbent assay and ELASA, respectively. Finally, the Beclin1and LC3B levels were evaluated by western blot. Additionally, the transmission electron microscope was employed to observe the autophagy.3.3Results3.3.1Evaluation of β-asarone effects on behaviour testsIn open field test and rotational behaviour test, compared to the model control, the move times in low dose, medium dose, high dose and Madopar groups were significantly increased (P<0.05). However, no significant differences were found amomng different β-asarone doses. In the initiation time and stepping time test, compared to the model control, the initiation and stepping times in low dose, medium dose and high dose and Madopar groups were significantly decreased (P<0.05). However, no significant differences were found amomng differentβ-asarone dose groups.3.3.2Evaluation of the β-asarone effects on neurotransmittersNo significant differences of DA and5-HT levels were found in low dose, medium dose and high dose groups compared to the model control. However, HVA, Dopael and5-HIAA levels of low dose, medium dose and high dose groups were raised significantly (P<0.05). However, no significant differences were found between the medium and high dose groups.3.3.3Evaluation of the β-asarone effects on TH and α-synCompared to the model control, a-syn levels of low dose, medium dose and high dose groups were raised significantly (P<0.05). However, no significant differences were found amomng differentβ-asarone dose groups.Compared to the model control, TH levels of low dose, medium dose and high dose groups were raised significantly (P<0.05). However, there no significant differences between the groups.3.3.4Evaluation of the β-asarone effects on Beclin1and LC3BCompared to the model control, Beclin1and LC3B levels in low dose, medium dose and high dose groups were raised significantly (P<0.05). Beclin1and LC3B levels of low β-asarone groups were significantly raised compared to both the medium and high dose groups (P<0.05). However, no significant differences were found between the medium and high dose groups.Furthermore, we investigated the autophagy by Electron microscopy, and found the same results as above.3.4ConclusionsThe β-asarone can improve the performances of open field test, rotational behaviour test, initiation time and stepping time in6-OHDA induced rats.The β-asarone could increase HVA, Dopacl and5-HIAA levels in6-OHDA induced rats. Furthermore, the β-asarone can attenuate TH and a-syn injury in6-OHDA induced rats.The β-asarone can attenuate autophagy by decreasing Beclin1and LC3B levels in6-OHDA induecd rats. The rats with the low dose (10mg/kg) were with significant of attenuate autophagy.4.1Objective The above data suggested that β-asarone attenuated autophagy by decreasing Beclin1and LC3B levels in6-OHDA induecd rats. However, whether β-asarone could affect the autophagy has not been reported yet.And in this study, we analyzed its possible mechanism related to autophagy.In our previous study, the β-asarone of Acorus tatarinowii Schott can raise the c-Jun expression. And it could modulate the levels of JNK and Bcl-2. Therefore, we deduced that β-asarone may modulate the JNK, p-JNK, and Bcl-2and Beclin1levels.4.2MethodsThe rats were anesthetized with chloral hydrate (350mg/kg) by intraperitoneal injection, and then the rats were treated with6-OHDA. The treatment of rats:Rats were treated2mL water orally per day for4weeks in model control group; Rats were treated β-asarone orally per day for4weeks in β-asarone group(10mg/kg); Rats were treated JNK inhibitor intraperitoneally per day for4weeks in JNK inhibitor group (7.5mg/mL) per day for4weeks. The brains were harvested1hr after the last administration. We evaluated the JNK, p-JNK, Bcl-2and Beclin1levels.4.3Results The effects of β-asarone’s on the JNK, p-JNK, Bc1-2and Beclin1The Beclin1, JNK and p-JNK levels of β-asarone and JNK inhibitor groups were declined significantly compared to the model control group(P<0.05), however, the Bcl-2levels of β-asarone and JNK inhibitor groups were raised significantly (P<0.05).4.4ConclusionsThe mechanism of β-asarone on autophagy was related with JNK, p-JNK, Bcl-2and Beclin1levels. The β-asarone may firstly decrease the levels of JNK and p-JNK, and then Bcl-2levels were raised, at last the Beclin1was downregulated by the increase the Bcl-2.Final ConclusionsAt first, we found that the increased Beclin1may benefit the injury of TH positive cells in the dynamic changes of Beclin1and TH of6-OHDA induced rats. Then the dynamic change of monoamine neurotransmitters of the striatum, cortex and hippocampus were studied in6-OHDA induced rats, and we found that there were significant positive correlations between DA and5-HT, while there were significant negative correlations between NE and DA. And then we found β-asarone can improve the performances of behaviour test, neurotransmitter levels, TH and α-syn injury in6-OHDA induced rats. Meanwhile the β-asarone can attenuate autophagy by decreasing Beclin1and LC3B levels. Finally, we deduced that the β-asarone may attenuate autophagy in rat brains via modulating JNK, p-JNK, Bcl-2and Beclin1levels in6-OHDA induced rats. The β-asarone may firstly decrease the levels of JNK and p-JNK, and then Bcl-2levels were raised, at last the Beclin1was declined by the increase the Bcl-2. |
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