ß-asarone inhibits autophagy by activating the PI3K/Akt/mTOR pathway in a rat model of depression in Parkinson's disease.

OBJECTIVE: It is unclear whether ß-asarone has a good antidepressant effect and what is the main mechanism in Depression in Parkinson's disease (DPD) model rats. METHODS: In this study, DPD model rats were screened from 6-OHDA induced rats by sucrose preference test (SPT) and forced swimming test (FST). DPD model rats were divided into eight groups: model group, pramipexole group, ß-asarone low-dose group (ß-asarone 7.5 group), ß-asarone medium-dose group (ß-asarone 15 group), ß-asarone high-dose group (ß-asarone 30 group), 3-MA group, rapamycin group, and PI3K inhibitor group. 28 days after the end of treatment, open field test (OFT), SPT and FST were conducted in rats. The level of α-synuclein (α-syn) in the striatum was determined by enzyme-linked immunosorbent assay (ELISA). The expression of Beclin-1, p62 in the striatum was determined by western blot. The expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, Beclin-1, and p62 in the hippocampus was determined by western blot. The spine density of neurons in the hippocampus was detected by golgi staining. RESULTS: The results showed that 4-week oral administration of ß-asarone improve the motor and depressive symptoms of DPD model rats, and decrease the content of α-syn in the striatum. ß-asarone inhibited the expression of autophagy in the striatum of DPD model rats. Furthermore, ß-asarone decreased the levels of Beclin-1 protein, increased the expression of p62, p-PI3K, p-AKT, and p-mTOR, and improved the density of neuron dendritic spine in the hippocampus. CONCLUSIONS: We concluded that ß-asarone might improve the behavior of DPD model rats by activating the PI3K/Akt/mTOR pathway, inhibiting autophagy and protecting neuron.

ß-Asarone improves learning and memory in Aß1-42-induced Alzheimer's disease rats by regulating PINK1-Parkin-mediated mitophagy.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that is characterized by the extracellular accumulation of ß-amyloid (Aß). Many studies have shown a close relationship between autophagy and the formation of Aß. As AD develops and progresses, mitophagy diminishes insoluble Aß, and mitochondrial dysfunction seems to be a determining factor in the pathogenesis of AD. In our previous study, we showed that ß-asarone pharmacological effects in APP/PS1 transgenic mice, reducing Aß expression. However, the specific mechanism of this effect remains unclear. In this study, AD model rats induced by intracerebroventricular injection of Aß1-42 were randomly divided into nine groups, and medical intervention was applied to the animals for 30 days. Subsequently, spatial learning and memory were evaluated by the water maze test. Bcl-2 levels in the hippocampus were determined by western blotting (WB). The protein expression of Aß1-42, Beclin-1, p62, PINK1, and Parkin was assessed by WB and immunohistochemistry (IHC). The data showed that after ß-asarone treatment, the learning and memory of the AD rats were clearly improved compared with those of the model group. Moreover, ß-asarone decreased Aß1-42, Bcl-2, and p62 levels but increased Beclin-1 levels compared with those in the model group. In addition, we treated a group of rats with CsA to inhibit mitophagy. ß-Asarone increased PINK1 and Parkin expression compared with that in the model group. The results showed that ß-asarone can improve the learning and memory of rats with Aß1-42-induced AD by effectively promoting PINK1-Parkin-mediated mitophagy. Taken together, these results suggest that ß-asarone may have the capacity to become a pharmaceutical agent for the treatment of AD in the future.

ß-Asarone Regulates ER Stress and Autophagy Via Inhibition of the PERK/CHOP/Bcl-2/Beclin-1 Pathway in 6-OHDA-Induced Parkinsonian Rats.

ß-Asarone (1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene) is an essential component of Acorus tatarinowii Schott volatile oil. Previous research has observed that ß-asarone effectively attenuated symptoms in parkinsonian rats and improved their performance, but the mechanism of this effect remains unclear. Other research has shown that endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of Parkinson's disease (PD). The protein kinase RNA-like endoplasmic reticulum kinase (PERK) was observed in the nigrostriatal dopaminergic neurons of patients with PD. However, our group observed that ER stress and autophagy occurred in 6-hydroxy dopamine (6-OHDA)-induced parkinsonian rats, and ER stress might induce autophagy. We assume that the protective role of ß-asarone in parkinsonian rats is mediated via the ER stress-autophagy pathway. To support this hypothesis, we investigated the expressions of glucose regulated protein 78 (GRP78), PERK phosphorylation (p-PERK), C/EBP homologous binding protein (CHOP), Bcl-2 and Beclin-1 in 6-OHDA-induced parkinsonian rats after ß-asarone treatment. The results showed that the ß-asarone group and PERK inhibitor group had lower levels of GRP78, p-PERK, CHOP and Beclin-1 while having higher levels of Bcl-2. We deduced that ß-asarone might regulate the ER stress-autophagy via inhibition of the PERK/CHOP/Bcl-2/Beclin-1 pathway in 6-OHDA-induced parkinsonian rats.

Kai Xin San ameliorates scopolamine-induced cognitive dysfunction.

Kai Xin San (KXS, containing ginseng, hoelen, polygala, and acorus), a traditional Chinese herbal compound, has been found to regulate cognitive dysfunction; however, its mechanism of action is still unclear. In this study, 72 specific-pathogen-free male Kunming mice aged 8 weeks were randomly divided into a vehicle control group, scopolamine group, low-dose KXS group, moderate-dose KXS group, high-dose KXS group, and positive control group. Except for the vehicle control group and scopolamine groups (which received physiological saline), the doses of KXS (0.7, 1.4 and 2.8 g/kg per day) and donepezil (3 mg/kg per day) were gastrointestinally administered once daily for 2 weeks. On day 8 after intragastric treatment, the behavioral tests were carried out. Scopolamine group and intervention groups received scopolamine 3 mg/kg per day through intraperitoneal injection. The effects of KXS on spatial learning and memory, pathological changes of brain tissue, expression of apoptosis factors, oxidative stress injury factors, synapse-associated protein, and cholinergic neurotransmitter were measured. The results confirmed the following. (1) KXS shortened the escape latency and increased residence time in the target quadrant and the number of platform crossings in the Morris water maze. (2) KXS increased the percentage of alternations between the labyrinth arms in the mice of KXS groups in the Y-maze. (3) Nissl and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining revealed that KXS promoted the production of Nissl bodies and inhibited the formation of apoptotic bodies. (4) Western blot assay showed that KXS up-regulated the expression of anti-apoptotic protein Bcl-2 and inhibited the expression of pro-apoptotic protein Bax. KXS up-regulated the expression of postsynaptic density 95, synaptophysin, and brain-derived neurotrophic factor in the cerebral cortex and hippocampus. (5) KXS increased the level and activity of choline acetyltransferase, acetylcholine, superoxide dismutase, and glutathione peroxidase, and reduced the level and activity of acetyl cholinesterase, reactive oxygen species, and malondialdehyde through acting on the cholinergic system and reducing oxidative stress damage. These results indicate that KXS plays a neuroprotective role and improves cognitive function through reducing apoptosis and oxidative stress, and regulating synapse-associated protein and cholinergic neurotransmitters.

Endoplasmic reticulum stress induced autophagy in 6-OHDA-induced Parkinsonian rats.

Both endoplasmic reticulum (ER) stress and autophagy involve in the pathological process of Parkinson's disease (PD). But the relationship between them is not clear in PD. A 6-OHDA-induced parkinsonian rat is recognized as a standard model for many years, and it can be used in experimental study. The glucose regulated protein 78 (GRP78) is a master regulator of ER stress, and the C/EBP homologous binding protein (CHOP) is an indicator of the UPR signaling. Besides, the Beclin-1 is also well known as a regulator of autophagy, and P62 is a specific marker to monitor autophagy. Therefore, we investigated the expressions of GRP78, CHOP, Beclin-1 and P62 in 6-OHDA-induced parkinsonian rat. Unilateral 6-OHDA injection into medial forebrain bundle was used except sham-operated rats. The rats were randomly divided into 6 groups: a sham-operated group; a model group; a 3-methyladenine (3-MA) group, administered 3-MA---autophagy inhibitor; a rapamycin group, administered rapamycin---autophagy inducer; a 4-phenylbutyric acids (4-PBA) group, administered 4-PBA---ER stress inhibitor; a tunicamycin (TM) group, administered TM---ER stress inducer. The results showed that the expressions of GRP78, CHOP and Beclin-1 increased, P62 decreased in model group; the expressions of GRP78 and CHOP were unchanged in 3-MA group and rapamycin group; but the expression of Beclin-1 decreased and P62 increased in 4-PBA group, while the expression of Beclin-1 increased and P62 decreased in TM group. These data suggest that ER stress and autophagy occurred in 6-OHDA-induced parkinsonian rat, and ER stress might induce autophagy. The result is important for the pathological mechanism of PD.

Ligustilide Ameliorates Memory Deficiency in APP/PS1 Transgenic Mice via Restoring Mitochondrial Dysfunction.

Ligustilide, the main lipophilic component of Radix angelicae sinensis, has been shown to ameliorate cognitive dysfunction in a few Alzheimer's disease mouse models, but its mechanism is not fully understood. In this study, we employed 7-month-old APP/PS1 mice to explore whether LIG is able to protect against Alzheimer's disease progression. The Morris water maze and Y-maze test results showed that eight weeks of intragastric administration of LIG (10 mg/kg, 40 mg/kg) every day improved memory deficit in APP/PS1 mice. The thioflavin-S staining and Western blot results (Aß1-42 monomer/oligomer, APP, ADAM10, SAPPα, and PreP) showed that LIG reduced Aß levels in the brain of APP/PS1 mice. Transmission electron microscopy analysis showed that LIG reduced the mitochondria number and increased the mitochondrial length in the hippocampal CA1 area of APP/PS1 mice. A reduced level of Drp1 (fission) and increased levels of Mfn1, Mfn2, and Opa1 (fusion) were found in APP/PS1 mice treated with LIG. An increased ATP level in the brain and increased activities of cytochrome c oxidase (CCO) and succinate dehydrogenase (SDH) in mitochondrion separated from the hippocampus and cortex revealed that LIG alleviated mitochondrial dysfunction. LIG exerts an antioxidation effect via reducing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) and increasing the activity of Mn-SOD in the brain. Elevated levels of PSD-95, synaptophysin, and synapsin 1 in both the hippocampus and cortex indicated that LIG provided synaptic protection. These findings show that treatment with LIG ameliorates mitochondrial dynamics and morphology issues, improves mitochondrial function, reduces Aß levels in the brain, restores the synaptic structure, and ameliorates memory deficit in APP/PS1 mice. These results imply that LIG may serve as a potential antidementia drug.

ß-asarone induces cell apoptosis, inhibits cell proliferation and decreases migration and invasion of glioma cells.

Glioma is the most common primary brain tumor Despite the availability of adjuvant therapies, malignant glioma grows fast and metastasizes via cerebrospinal fluid after tumorectomy or cerebrospinal fluid shunt placement, and the prognosis for patients with glioma remains poor. Our previous study demonstrated that ß-asarone has anti-tumor effects on several kinds of cancer cells, especially for glioma cells. In this study, human glioma U251 cells and rat glioma C6 cells were treated with different concentrations of ß-asarone. Cultured them for 24 h, 48 h, 72 h and evaluated the IC50 with the results of Counting Kit-8 assay. Then, cell apoptosis and cell DNA cycles were evaluated with flow cytometry. Apoptosis related mRNA and protein were analyzed In addition, cell migration and invasion were also detected with wound healing and transwell assays, respectively. What is more, glioma specific proteins: GFAP, NRP-1 and NSE an enzyme-linked immunosorbent assay. The corresponding CCK-8 results showed that ß-asarone altered cell morphology and inhibited cell proliferation. ß-asarone can also induced cell apoptosis, decreased the expression of BCL-2 mRNA and blocked the DNA cycle at the G0/G1 phase for all the two cells. In addition, ß-asarone inhibited cell migration and invasion by reducing the expression of GFAP, NRP-1 and NSE. Co-administration with TMZ showed a more pronounced effect. In summary, ß-asarone induces cell death and inhibits cell migration and invasion in Glioma U251 and C6 cells.

ß-asarone inhibited cell growth and promoted autophagy via P53/Bcl-2/Bclin-1 and P53/AMPK/mTOR pathways in Human Glioma U251 cells.

Glioma is the most common type of primary brain tumor and has an undesirable prognosis. Autophagy plays an important role in cancer therapy, but it is effect is still not definite. P53 is an important tumor suppressor gene and protein that is closely to autophagy. Our aim was to study the effect of ß-asarone on inhibiting cell proliferation in human glioma U251 cells and to detect the effect of the inhibition on autophagy through the P53 signal pathway. For cell growth, the cells were divided into four groups: the model, ß-asarone, temozolomide (TMZ), and co-administration groups. For cell autoghapy and the P53 pathway, the cells were divided into six groups: the model, ß-asarone, 3MA, Rapa, Pifithrin-µ, and NSC groups. The counting Kit-8 assay and flow cytometry (FCM) were then used to measure the cell proliferation and cycle. Electron microscopy was used to observe autophagosome formation. Cell immunohistochemistry/-immunofluorescence, FCM and Western blot (WB) were used to examine the expression of Beclin-1 and P53. The levels of P53 and GAPDH mRNA were detected by RT-PCR. Using WB, we determined autophagy-related proteins Beclin-1, LC3-II/I, and P62 and those of the P53 pathway-related proteins P53, Bcl-2, mTOR, P-mTOR, AMPK, P-AMPK, and GAPDH. We got the results that ß-asarone changed the cellular morphology, inhibited cell proliferation, and enhanced the expression of P53, LC3-II/I, Beclin-1, AMPK, and pAMPK while inhibiting the expression of P62, Bcl-2, mTOR, and pmTOR. All the data suggested that ß-asarone could reduce the cell proliferation and promote autophagy possible via the P53 pathway in U251 cells.

ß-asarone and levodopa coadministration increases striatal levels of dopamine and levodopa and improves behavioral competence in Parkinson's rat by enhancing dopa decarboxylase activity.

Levodopa (L-dopa) is the key component in Parkinson's disease (PD) treatment. Recently, we demonstrated that ß-asarone improves the motor behavior of rats with unilateral striatal 6-hydroxydopamine lesion. Striatal level of dopamine (DA) and L-dopa increased after ß-asarone and L-dopa co-administered treatment in healthy rat. Since its effects and mechanisms on PD rats are still unclear, we investigated whether coadministration could help treat PD rats. Here, PD rats were randomly divided into seven groups (n=10/group): an untreated group, a Madopar-treated group, a L-dopa-treated group, a ß-asarone-treated group, and groups receiving low, medium or high doses of ß-asarone respectively plus the same dose of L-dopa. The sham-operated group rats were injected with saline. Treatments were administered to the rats twice per day continuously for 30days. The behavioral tests were assessed. Neurotransmitters, dopa decarboxylase (DDC), tyrosine hydroxylase (TH), catechol-O-methyltransferase (COMT), monoamine oxidase B (MAO-B) and dopamine transporter (DAT) levels were detected. The pathological characteristics of liver and kidney and ultrastructure of dopaminergic neurons were observed. The behavior of PD rats improved significantly after co-administered treatment compared with the untreated group. In addition, our results also showed that co-administered treatment increased L-dopa, DA, DOPAC, HVA and 5-HT levels, enhanced the MAO-B, COMT, TH and DAT levels, reduced creatinine level, decreased the amount of lysosome and mitochondria and showed no liver and kidney toxicity. These findings suggest that co-administered treatment could elevate striatal levels of L-dopa and DA and improve the behavioral abilities in PD rats by regulating the DDC, TH, MAO-B, COMT and DAT levels.

ß-Asarone promotes Temozolomide's entry into glioma cells and decreases the expression of P-glycoprotein and MDR1.

Glioma is the most common primary brain tumor and has an undesirable prognosis due to the blood-brain barrier (BBB) and drug resistance. A thorough investigation of the changes in intracellular drug concentrations is important to observe therapeutic effects and cell resistance. P-glycoprotein (P-gp) is an essential protein of Multi-drug resistance 1 (MDR1). The over-expression of P-gp and MDR1 is associated with poor prognosis and drug-resistance in glioma. However, ß-asarone can pass through the BBB easily and increase the drug concentration in the rat brain. Our aim is to study the effect of ß-asarone on promoting the entry of temozolomide (TMZ) into human glioma U251 cells. The cells were divided into three groups: model group, TMZ group (300µM) and co-administration group (360µM ß-asarone; 300µM TMZ). We further detected P-gp and MDR1 expression in U251 and rat glioma C6 cells in four groups: model group (U251/C6), TMZ group (U251 300µM, C6 420µM), ß-asarone group (U251 360µM, C6 450µM) and co-administration group (ß-asarone 360µM, TMZ 300µM for U251; ß-asarone 450µM, TMZ 420µM for C6). Then, high performance liquid chromatography was used to determine the intracellular and extracellular levels of TMZ. Morphological changes in both cells were observed by the microscope. The Counting Kit-8 assay was used to measure the cell proliferation and toxicity. Cell immunohistochemistry/immunofluorescence, flowcytometry and western blot were synchronously used to examine the expression of P-gp. We also determined the levels of MDR1 mRNA by RT-PCR. The results showed that ß-asarone could promote the entry of TMZ into U251 cells through the membrane. The co-administration of ß-asarone and TMZ also decreased cell proliferation and the expression of P-gp and MDR1 better than single medication in U251 and C6 cells. All of the data suggest that ß-asarone might contribute to treatment by promoting TMZ's entry into glioma cells, thereby contributing to anti-cancer growth and inhibiting P-gp and MDR1 expression.

[Advanced glycated albumin induces macrophage apoptosis via activating caspase-12 pathway].

The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on the activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms of macrophage apoptosis. RAW264.7 macrophages were treated with AGE-alb (2, 4 and 6 g/L), control albumin (C-alb, 4 g/L), tunicamycin (TM, 4 mg/L), or pretreated with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then treated with AGE-alb (4 g/L). After incubation for 24 h, the cell viability and apoptosis were determined by using MTT assay and TUNEL detection kit, respectively. Lactate dehydrogenase (LDH) activity in media was determined by using an assay kit. The protein levels of caspase-12 were examined by Western blot analysis. The results showed that like TM (an ERS inducer), incubation with AGE-alb led to significant decrease in viability and increase in LDH activity in media and apoptotic rate in a dose-dependent manner. In addition, AGE-alb induced activation of caspase-12 especially at the concentration of 4 and 6 g/L (P < 0.01), which was similar to TM. However, PBA (an ERS inhibitor) protected RAW264.7 macrophages from AGE-alb-induced decrease in viability and increases in LDH activity and apoptosis. Moreover, PBA also inhibited the caspase-12 activation induced by AGE-alb (P < 0.05). These results suggest that AGE-alb may induce apoptosis in RAW 264.7 macrophages, and the mechanism may be related to the activation of ERS-associated apoptotic pathway mediated by caspase-12.

ß-asarone improves learning and memory and reduces Acetyl Cholinesterase and Beta-amyloid 42 levels in APP/PS1 transgenic mice by regulating Beclin-1-dependent autophagy.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, and studies have suggested that ß-asarone has pharmacological effects on beta-amyloid (Aß) injected in the rat hippocampus. However, the effect of ß-asarone on autophagy in the APP/PS1 transgenic mouse is unreported. APP/PS1 transgenic mice were randomly divided into six groups (n=10/group): an untreated group, an Aricept-treated group, a 3-MA-treated group, a rapamycin-treated group, an LY294002-treated group, a ß-asarone-treated group. The control group consisted of wild-type C57BL/6 mice. All treatments were administered to the mice for 30 days. Spatial learning and memory were assessed by water maze, passive avoidance, and step-down tests. AChE and Aß42 levels in the hippocampus were determined by ELISA. p-Akt, p-mTOR, and LC3B expression were detected by flow cytometry. The expression of p-Akt, p-mTOR, Beclin-1, and p62 proteins was assessed by western blot. Changes in autophagy were viewed using a transmission electron microscope. APP and Beclin-1 mRNA levels were measured by Real-Time PCR. The learning and memory of APP/PS1 transgenic mice were improved significantly after ß-asarone treatment compared with the untreated group. In addition, ß-asarone treatment reduced AChE and Aß42 levels, increased p-mTOR and p62 expression, decreased p-Akt, Beclin-1, and LC3B expression, decreased the number of autophagosomes and reduced APP mRNA and Beclin-1 mRNA levels compared with the untreated group. That is, ß-asarone treatment can improve the learning and memory abilities of APP/PS1 transgenic mouse by inhibiting Beclin-1-dependent autophagy via the PI3K/Akt/mTOR pathway.

ß-asarone increases MEF2D and TH levels and reduces α-synuclein level in 6-OHDA-induced rats via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway: Chaperone-mediated autophagy activation, macroautophagy inhibition and HSP70 up-expression.

Inactive myocyte enhancer factor 2D (MEF2D) and alpha-synuclein (α-syn) aggregation will cause neuronal death. MEF2D or α-syn degradation is also associated with macroautophagy, chaperone-mediated autophagy (CMA) and heat-shock protein 70 (HSP70). We found that ß-asarone had positive effects on treating 6-hydroxydopamine (6-OHDA)-induced rats, but mechanisms of ß-asarone affecting on MEF2D and α-syn via regulating the HSP70/MAPK/MEF2D/Beclin-1 pathway remain unclear. Unilateral 6-OHDA injection into the medial forebrain bundle was used to create PD rats, which were divided into four groups and administered for 30days: 6-OHDA model group, MEF2D inhibitor-treated group (SB203580, 0.5mg/kg, i.p.), MEF2D activator-treated group (LiCl, 100mg/kg, i.p.), ß-asarone-treated group (15mg/kg, p.o.). Expressions of tyrosine hydroxylase (TH), α-syn, heat-shock cognate protein 70 (HSC70), lysosome-associated membrane protein type 2a (LAMP-2A), MEF2D, HSP70, Beclin-1, light chain 3B (LC3B) and p62 in the mesencephalon were measured after 30-day administration. α-syn, Beclin-1 and LC3B levels were higher in the 6-OHDA model group, while TH, MEF2D, HSC70, LAMP-2A, p62 levels were lower compared to the sham-operated group. Our results also showed thatß-asarone treatment reduced protein and mRNA levels of α-syn, Beclin-1 and LC3B, but increased HSP70, TH, MEF2D, HSC70, LAMP-2A and p62 levels compared to the 6-OHDA model group. Additionally, certain correlations among α-syn, TH, Beclin-1, LC3B, p62, HSP70, LAMP-2A and MEF2D were also discovered in this study. These findings suggested that ß-asarone treatment could increase MEF2D and TH as well as reduce α-syn to protect against 6-OHDA induced damage in PD rat mesencephalon via modulating the HSP70/MAPK/MEF2D/Beclin-1 pathway.

ß-Asarone Inhibits IRE1/XBP1 Endoplasmic Reticulum Stress Pathway in 6-OHDA-Induced Parkinsonian Rats.

Parkinson's disease (PD) is a neurodegenerative disease, with genetics and environment contributing to the disease onset. The limited pathological cognize of the disease restrained the approaches to improve the clinical treatment. Recently, studies showed that endoplasmic reticulum (ER) stress played an important role in the pathogenesis of PD. There was a neuroprotective effect partly mediated by modulating ER stress. ß-Asarone is the essential constituent of Acorus tatarinowii Schott volatile oil. Our team observed that ß-asarone could improve the behavior of parkinsonian rats; increase the HVA, Dopacl, and 5-HIAA levels; and reduce α-synuclein levels. Here we assumed that the protective role of ß-asarone on parkinsonian rats was mediated via ER stress pathway. To prove the hypothesis we investigated the mRNA levels of glucose regulated protein 78 (GRP78) and C/EBP homologous binding protein (CHOP) in 6-hydroxy dopamine (6-OHDA) induced parkinsonian rats after ß-asarone treatment. Furthermore, the inositol-requiring enzyme 1/X-Box Binding Protein 1 (IRE1/XBP1) ER stress pathway was also studied. The results showed that ß-asarone inhibited the mRNA levels of GRP78 and CHOP, accompanied with the delined expressions of phosphorylated IER1 (p-IRE1) and XBP1. We deduced that ß-asarone might have a protective effect on the 6-OHDA induced parkinsonian rats via IRE1/XBP1 Pathway. Collectively, all data indicated that ß-asarone might be a potential candidate of medicine for clinical therapy of PD.

ß-asarone and levodopa co-administration increase striatal dopamine level in 6-hydroxydopamine induced rats by modulating P-glycoprotein and tight junction proteins at the blood-brain barrier and promoting levodopa into the brain.

Levodopa (L-dopa) is widely considered as one of the most effective drug constituents in the treatment of Parkinson's disease (PD), but the blood-brain barrier (BBB) permeability of L-dopa is <5%, which causes low efficacy. Neuroprotective effects of ß-asarone on 6-hydroxydopamine (6-OHDA)-induced PD rats were demonstrated by our previous studies. Co-administration of ß-asarone and L-dopa has not been explored until being investigated on PD rats in this study. PD rats were divided into four groups: untreated, L-dopa-treated, ß-asarone-treated and co-administered-treated groups. All of the treatments were administered to the rats twice per day for 30 days. The L-dopa, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), S100ß and neuron-specific enolase (NSE) levels were subsequently determined. The P-glycoprotein (P-gp), zonula occludens-1 (ZO-1), claudin-5, occludin and actin expression was also assessed in cortex. Changes in BBB ultrastructure were observed using transmission electron microscopy. Our results showed that the co-administered treatment increased levels of L-dopa, DA, DOPAC and HVA in striatum, and S100ß in plasma, but down-regulated NSE, P-gp, ZO-1, occludin, actin and claudin-5 in cortex. Crevices were observed between capillary endothelial cells at intercellular tight junction of the striatum in co-administered-treated group, while the endothelial cells in untreated group were tightly jointing each other. In addition, the correlations of L-dopa or DA and P-gp or tight junction proteins respectively were significantly negative in co-administered- and ß-asarone-treated groups. These findings suggest that co-administered treatment may enhance the L-dopa BBB permeability and attenuate brain injury, which may be beneficial to PD treatment.

Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells.

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aß) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aß1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aß1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aß1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aß1-42. When 3-MA was applied this exacerbated the effect of Aß1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aß1-42 induced cytotoxicity and so may have a preventive function in AD.

Neuroprotective Effects of ß-Asarone Against 6-Hydroxy Dopamine-Induced Parkinsonism via JNK/Bcl-2/Beclin-1 Pathway.

ß-asarone, a major component of Acorus tatarinowii Schott, has positive effects in neurodegeneration disease, however, its effect on the Parkinson's disease (PD) remains unclear. In this study, the effects of ß-asarone on behavioral tests, neurotransmitters, tyrosine hydroxylase (TH), and α-synuclein (α-syn) were investigated in 6-hydroxydopamine (6-OHDA) induced rats. Furthermore, the JNK/Bcl-2/Beclin-1 autophagy pathway was also studied. The results showed that ß-asarone improved the behavioral symptoms of rats in the open field, rotarod test, initiation time, and stepping time. And it increased the HVA, Dopacl, and 5-HIAA levels in striatum but not the DA and 5-HT levels. After administration of ß-asarone, the TH level was elevated but the α-syn was declined in rats. It inhibited the expressions of LC3-II, but increased the p62 expression in SN4741 cells. Moreover, it affected the expressions of Beclin-1, Bcl-2, JNK, and p-JNK in vivo. We deduced that ß-asarone may firstly downregulate expressions of JNK and p-JNK, and then indirectly increase the expression of Bcl-2. And the function of Beclin-1 could be inhibited, which could inhibit autophagy activation. Collectively, all data indicated that ß-asarone may be explored as a potential therapeutic agent in PD therapy.

Dynamic changes of five neurotransmitters and their related enzymes in various rat tissues following ß-asarone and levodopa co-administration.

The aim of the present study was to investigate the dynamic changes of five neurotransmitters and their associated enzymes in the rat plasma and brain tissues following the co-administration of ß-asarone and levodopa (L-dopa). The rats were divided into five groups, including the control group and four treatment groups that were intragastrically co-administered ß-asarone and L-dopa and sacrificed at 1, 5, 18 and 48 h, respectively. Neurotransmitter levels in the brain tissues and plasma were detected using high performance liquid chromatograph and the related enzymes of dopamine (DA) were measured using an enzyme-linked immunosorbent assay. The results indicated that the striatal levels of L-dopa and 3,4-dihydroxyphenylacetic acid (DOPAC) peaked at 1 h and then returned to the normal levels, while the striatal levels of DA were stable within 48 h. In the cortex and hippocampus tissue, L-dopa, DA, DOPAC and homovanillic acid (HVA) levels peaked at 1 h and then returned to normal levels. In the plasma, L-dopa, DA, DOPAC and HVA levels peaked at 1 h. Compared with the control group, L-dopa, DA and HVA levels were higher between 18 and 48 h, whereas the DOPAC level was lower. By contrast, no statistically significant differences were observed in the serotonin (5-HT) levels among the plasma, hippocampus, cortex and striatum. Furthermore, the DA/L-dopa ratio in the brain tissues and plasma increased in the first 5 h, while (DOPAC + HVA)/DA ratios demonstrated a significant reduction. Striatal tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC) levels were higher compared with the control group; however, catechol-O-methyltransferase (COMT) and monoamine oxidase B levels were reduced. In the rat plasma, TH and COMT peaked at 1 h, while AADC peaked at 5 h. In conclusion, the results of the present study indicate that the co-administration of L-dopa and ß-asarone may be used to maintain a stable striatal DA level within 48 h. In addition, this treatment may promote DA generation by AADC and reduce the metabolism of DA by COMT.

Celery Seed Extract Blocks Peroxide Injury in Macrophages via Notch1/NF-κB Pathway.

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and injury is one of the major atherogenic factors. This study is aimed to investigate the protective effect of celery seed extract (CSE) on ox-LDL-induced injury of macrophages and the underlying signaling pathway. RAW264.7 macrophages were pre-incubated with CSE for 24 h, followed by stimulation with ox-LDL. Oil red O staining and enzymatic colorimetry indicated CSE significantly lessened lipid droplets and total cholesterol (TC) content in ox-LDL-injured macrophages. ELISA revealed that CSE decreased the secretion of inflammatory cytokine TNF-α and IL-6 by 12-27% and 5-15% respectively. MTT assay showed CSE promoted cell viability by 16-40%. Cell apoptosis was also analyzed by flow cytometry and laser scanning confocal microscope and the data indicated CSE inhibited ox-LDL-induced apoptosis of macrophages. Meanwhile, western blot analysis showed CSE suppressed NF-κBp65 and notch1 protein expressions stimulated by ox-LDL in macrophages. These results suggest that CSE inhibits ox-LDL-induced macrophages injury via notch1/NF-κB pathway.