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.

Inhibitory effects of ß-asarone on lncRNA BACE1-mediated induction of autophagy in a model of Alzheimer's disease.

The primary aim of this study was to examine the correlation between the formation of Aß plaques and autophagy, which is regulated by ß-asarone and the lncRNA BACE1-AS. Additionally, the study sought to explore potential targets of the drug in inhibiting the deposition of toxic AD-related proteins and restoring impaired mitochondrial and autophagic functions. SHY5Y cells were utilized to construct a stable Alzheimer's disease (AD) model, followed by the utilization of interference and overexpression lentiviruses targeting BACE1-AS to establish a cell model. The cells were categorized into five groups, including a normal group, siRNA/BACE1 group, and ß-asarone group. The fluorescence quantitative PCR technique was employed to assess the disparity in BACE1 mRNA expression, while changes in immunofluorescence (IF) were observed to determine the stable interference titre and action time of the lentiviruses. Additionally, western blotting (WB) and fluorescence quantitative PCR were employed to evaluate the expression of proteins and mRNAs associated with AD and autophagy. The findings demonstrated a significant elevation in BACE1 expression levels in brain tissue among individuals with AD compared to those without the condition. Moreover, the results indicated that the introduction of ß-asarone led to an increase in the expression of the BACE1-AS gene in the cell group transfected with plasmid H12732. Furthermore, it was observed that ß-asarone enhanced the expression levels of shRNA and BACE1 after 72 h. In contrast, ß-asarone suppressed the expression of PS1, Aß, BACE1, APP, and p62, while promoting the expression of syn, LC3 I/II, and Beclin-1. Based on these findings, it can be concluded that ß-Asarone exerts a comprehensive influence on the expression of proteins associated with AD and synaptic function. ß-Asarone exhibits the potential to mitigate Aß deposition by impeding the expression of lncBACE1, thereby facilitating autophagy through the suppression of BACE1's inhibitory impact on autophagy. This complements the self-enhancing effect of autophagy.

Development and Validation of a Clinical-Based Severity Scale for Patients with Cerebral Venous Thrombosis.

Introduction: Cerebral venous thrombosis (CVT) is a rare subtype of stroke. However, existing scales were insufficient to evaluate the overall severity of CVT. The aim of this study is to develop and validate a CVT severity scale. Methods: Items 1-11 were directly derived from NIHSS. New items were generated from a literature review and focus group discussion. A total of 170 CVT patients were prospectively recruited from 26 top tertiary hospitals in China Mainland from January 2021 to May 2022 to validate the CVT severity scale. The CVT severity scale, NIHSS, mRS and GCS were rated at admission. The lumbar puncture opening pressure was also recorded. Twenty randomly selected CVT patients were rated with the CVT severity scale again 24 hours later. The clinical outcome of CVT was evaluated by mRS at 6 months after baseline. Results: We successfully established a CVT severity scale with 18 items. Exploratory factor analysis showed that 18 items were attributed to factor 1 (focal neurological deficits), factor 2 (diffuse encephalopathy), factor 3 (intracranial hypertension) and factor 4 (cavernous sinus syndrome). CVT severity scale was positively correlated with ICP, NIHSS and mRS, and negatively correlated with GCS at baseline. CVT severity scale >3 or factor 3 >2 indicated intracranial hypertension. CVT severity scale >10 indicated poor clinical outcome at 6 months of follow-up. Meanwhile, CVT severity scale showed high internal consistency and test-retest reliability. Conclusion: The CVT severity scale included 18 items encompassing 4 domains of focal neurological deficits, diffuse encephalopathy, IH and cavernous sinus syndrome. CVT severity scale correlated well with ICP, NIHSS, mRS and GCS. Patients with CVT severity scale >10 can be defined as severe CVT. The CVT severity scale may serve as a valid and reliable tool for measuring the overall severity of CVT.

Relationship Between Weight-Adjusted Waist Index and Osteoporosis in the Senile in the United States from the National Health and Nutrition Examination Survey, 2017-2020.

BACKGROUND: Some studies suggested obesity may be beneficial in preventing bone loss through the negative relationship between body mass index (BMI) and osteoporosis in senile. However, using BMI to measure obesity is unconvincing due to confounding factors such as muscle mass were not taken into account, and few articles have yet taken a better way to evaluate the relationship between obesity and osteoporosis. METHODOLOGY: Using a cross-sectional sample of 1,979 participants aged ≥65 years from the National Health and Nutrition Examination Survey (NHANES) 2017 to 2020, we evaluated the relation of weight-adjusted waist index (WWI) with osteoporosis. WWI was calculated as waist (cm) divided by the square root of body weight (kg). Diagnosis of osteoporosis was described as follows: according to the updated reference for calculating bone mineral density T-Scores, we marked the BMD value as X, using the formula T femoral neck= (X g/cm2-0.888 g/cm2)/0.121 g/cm2, T lumbar spine= (X g/cm2- 1.065 g/cm2)/0.122 g/cm2, and defined those with a final T femoral neck <-0.25. T lumbar spine<-0.25 or patients with previously diagnosed OP in other hospitals as osteoporosis. RESULTS: All the 1,979 participants were between 65 and 80 years, there were 379 (21.1%) with osteoporosis, 608 (30.7%) with WWI exceeding 12 (cm/√kg) (range 8.85-14.14), and 955 (48.3%) women. Furthermore, the relationship between WWI and osteoporosis was nonlinear with a threshold effect point. Odds of OP significantly increased with the increase of WWI (OR 2.33, 95% CI 11.48-3.38, P = 0.0001) at the right side of the threshold point (WWI≥12) according to the threshold effect study. CONCLUSIONS: Found a significant positive relationship between WWI and osteoporosis. Body fat management in the senile may be good to prevent osteoporosis if confirmed by other prospective studies analyzing the longitudinal risk of osteoporosis with obesity.

13 C NMR quantification of polyamine syntheses in rat prostate.

Currently, many prostate cancer patients, detected through the prostate specific antigen test, harbor organ-confined indolent disease that cannot be differentiated from aggressive cancer according to clinically and pathologically known measures. Spermine has been considered as an endogenous inhibitor for prostate-confined cancer growth and its expression has shown correlation with prostate cancer growth rates. If established clinically, measurements of spermine bio-synthesis rates in prostates may predict prostate cancer growth and patient outcomes. Using rat models, we tested the feasibility of quantifying spermine bio-synthesis rates with 13 C NMR. Male Copenhagen rats (10 weeks, n = 6) were injected with uniformly 13 C-labeled L-ornithine HCl, and were sacrificed in pairs at 10, 30, and 60 min after injection. Another two rats were injected with saline and sacrificed at 30 min as controls. Prostates were harvested and extracted with perchloric acid and the neutralized solutions were examined by 13 C NMR at 600 MHz. 13 C NMR revealed measurable ornithine, as well as putrescine-spermidine-spermine syntheses in rat prostates, allowing polyamine bio-synthetic and ornithine bio-catabolic rates to be calculated. Our study demonstrated the feasibility of 13 C NMR for measuring bio-synthesis rates of ornithine to spermine enzymatic reactions in rat prostates. The current study established a foundation upon which future investigations of protocols that differentiate prostate cancer growth rates according to the measure of ornithine to spermine bio-synthetic rates may be developed.

Effectiveness of Acupuncture for Anxiety Among Patients With Parkinson Disease: A Randomized Clinical Trial.

Importance: One of the ordinary manifestations of Parkinson disease (PD) is anxiety, which remains untreated. Anxiety is closely associated with the accelerated progression of PD. Efficacy of acupuncture for anxiety has been reported. However, to date, there are no data on acupuncture's effectiveness on anxiety for patients with PD. Objective: To investigate the effect of acupuncture vs sham acupuncture for treating anxiety in patients with PD. Design, Setting, and Participants: This is randomized, double-blinded, clinical trial enrolled patients between June 20, 2021, and February 26, 2022. Final follow-up was April 15, 2022. Patients with Parkinson disease and anxiety were allocated randomly (1:1) to receive acupuncture or sham acupuncture for 8 weeks. Acupuncture operators, outcome measures evaluators, and statistical analysts were blinded to the grouping of patients. Patients were blinded to their own grouping during the study. This study took place in the Parkinson clinic of a hospital in China. Interventions: Real acupuncture or sham acupuncture for 8 weeks. Main Outcomes and Measures: Primary outcome was Hamilton Anxiety Scale (HAM-A) score. Secondary outcomes were scores on the Unified Parkinson Disease Rating Scale (UPDRS), 39-item Parkinson Disease Questionnaire (PDQ-39), and serum levels of the adrenocorticotropic hormone (ACTH) and cortisol (CORT). Results: Seventy eligible patients were enrolled, including 34 women (48.5%) and 36 men (51.4%). Sixty-four patients (91%) completed the intervention and the 8-week follow-up, including 30 women (46.9%) and 34 men (53.1%) with a mean (SD) age of 61.84 (8.47) years. At the end of treatment, the variation of HAM-A score was 0.22 (95% CI, -0.63 to 1.07; P = .62) between the real acupuncture and sham acupuncture groups. At the end of follow-up, the real acupuncture group had a significant 7.03-point greater (95% CI, 6.18 to 7.88; P < .001) reduction in HAM-A score compared with the sham acupuncture group. Four mild adverse reactions occurred during the study. Conclusions and Relevance: This study found acupuncture to be an effective treatment for anxiety in patients with PD. These findings suggest that acupuncture may enhance the wellbeing of patients who have Parkinson disease and anxiety. Trial Registration: Chinese Clinical Trial Registry: ChiCTR2100047253.

Suppression of abnormal α-synuclein expression by activation of BDNF transcription ameliorates Parkinson's disease-like pathology.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs) in the brain. LBs are mainly composed of phosphorylated and aggregated α-synuclein (α-Syn). Thus, strategies to reduce the expression of α-Syn offer promising therapeutic avenues for PD. DNA/RNA heteroduplex oligonucleotides (HDOs) are a novel technology for gene silencing. Using an α-Syn-HDO that specifically targets α-Syn, we examined whether α-Syn-HDO attenuates pathological changes in the brain of mouse models of PD. Overexpression of α-Syn induced dopaminergic neuron degeneration through inhibition of cyclic AMP-responsive-element-binding protein (CREB) and activation of methyl CpG binding protein 2 (MeCP2), resulting in brain-derived neurotrophic factor (BDNF) downregulation. α-Syn-HDO exerted a more potent silencing effect on α-Syn than α-Syn-antisense oligonucleotides (ASOs). α-Syn-HDO attenuated abnormal α-Syn expression and ameliorated dopaminergic neuron degeneration via BDNF upregulation by activation of CREB and inhibition of MeCP2. These findings demonstrated that inhibition of α-Syn by α-Syn-HDO protected against dopaminergic neuron degeneration via activation of BDNF transcription. Therefore, α-Syn-HDO may serve as a new therapeutic agent for PD.

Regulation of BDNF transcription by Nrf2 and MeCP2 ameliorates MPTP-induced neurotoxicity.

Mounting evidence suggests the key role of brain-derived neurotrophic factor (BDNF) in the dopaminergic neurotoxicity of Parkinson's disease (PD). Activation of NF-E2-related factor-2 (Nrf2) and inhibition of methyl CpG-binding protein 2 (MeCP2) can regulate BDNF upregulation. However, the regulation of BDNF by Nrf2 and MeCP2 in the PD pathogenesis has not been reported. Here, we revealed that Nrf2/MeCP2 coordinately regulated BDNF transcription, reversing the decreased levels of BDNF expression in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Repeated administration of sulforaphane (SFN, an Nrf2 activator) attenuated dopaminergic neurotoxicity in MPTP-treated mice through activation of BDNF and suppression of MeCP2 expression. Furthermore, intracerebroventricular injection of MeCP2-HDO, a DNA/RNA heteroduplex oligonucleotide (HDO) silencing MeCP2 expression, ameliorated dopaminergic neurotoxicity in MPTP-treated mice via activation of Nrf2 and BDNF expression. Moreover, we found decreased levels of Nrf2 and BDNF, and increased levels of MeCP2 protein expression in the striatum of patients with dementia with Lewy bodies (DLB). Interesting, there were correlations between BDNF and Nrf2 (or MeCP2) expression in the striatum from DLB patients. Therefore, it is likely that the activation of BDNF transcription by activation of Nrf2 and/or suppression of MeCP2 could be a new therapeutic approach for PD.

The Combination of ß-Asarone and Icariin Inhibits Amyloid-ß and Reverses Cognitive Deficits by Promoting Mitophagy in Models of Alzheimer's Disease.

ß-Asarone is the main constituent of Acorus tatarinowii Schott and exhibits important effects in diseases such as neurodegenerative and neurovascular diseases. Icariin (ICA) is a major active ingredient of Epimedium that has attracted increasing attention because of its unique pharmacological effects in degenerative disease. In this paper, we primarily explored the effects of the combination of ß-asarone and ICA in clearing noxious proteins and reversing cognitive deficits. The accumulation of damaged mitochondria and mitophagy are hallmarks of aging and age-related neurodegeneration, including Alzheimer's disease (AD). Here, we provide evidence that autophagy/mitophagy is impaired in the hippocampus of APP/PS1 mice and in Aß1-42-induced PC12 cell models. Enhanced mitophagic activity has been reported to promote Aß and tau clearance in in vitro and in vivo models. Meanwhile, there is growing evidence that treatment of AD should be preceded by intervention before the formation of pathological products. The efficacy of the combination therapy was better than that of the individual therapies applied separately. Then, we found that the combination therapy also inhibited cell and mitochondrial damage by inducing autophagy/mitophagy. These findings suggest that impaired removal of defective mitochondria is a pivotal event in AD pathogenesis, and that combination treatment with mitophagy inducers represents a potential strategy for therapeutic intervention.

Efficacy of manual acupuncture versus placebo acupuncture for generalized anxiety disorder (GAD) in perimenopause women: study protocol for a randomized controlled trial.

BACKGROUND: Generalized anxiety disorder (GAD) is common among perimenopausal women. Acupuncture may be an effective treatment for GAD, but evidence is limited. The pathogenesis of GAD is not yet clear, but it is related to the hypothalamic-pituitary-adrenal axis and its excretion, cortisol (CORT), and adrenocorticotropic hormone (ACTH). The object of this study is to evaluate the efficacy of manual acupuncture (MA) versus placebo acupuncture (PA) for perimenopausal women with GAD. METHODS: This study is a single-center, randomized, single-blind clinical trial that will be conducted in the First Affiliated Hospital of Guangzhou University of Chinese Medicine. A total of 112 eligible GAD patients will be randomly assigned (1:1) to receive MA (n=56) or PA (n=56) three times per week for 4 weeks. The primary outcome measure will be the HAMA score. The secondary outcome measures will be the GAD-7 and PSQI scores and the levels of CORT and ACTH. The evaluation will be executed at baseline, 2 weeks, the end of the treatment, and a follow-up 3-month period. All main analyses will be carried out based on the intention-to-treat (ITT) principle. DISCUSSION: This study intends to compare the efficacy between MA and PA in the treatment of perimenopausal women with GAD and to further study the mechanisms underlying the effect. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2100046604 . Registered on 22 May 2021.

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.

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.

ß-Asarone Inhibits Amyloid-ß by Promoting Autophagy in a Cell Model of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most common types of dementia that causes memory, thinking, and behavior problems. The most important feature of AD is the gradual irreversible loss of cognitive ability through the formation of amyloid ß (Aß) plaques and neurofibrillary tangles composed of tau protein. The metabolism of Aß and tau proteins is closely related to and is affected by autophagy. Current research speculates that autophagy dysfunction leads to an increase in harmful proteins in AD. ß-Asarone is the main constituent of Acorus tatarinowii Schott and has important effects on the central nervous system. In this paper, we primarily explored the effects of ß-asarone on the clearance of noxious proteins and the associated potential mechanisms via autophagy in a PC12 cell AD model. A CCK-8 assay and LDH experiments were used to assess cell viability/toxicity, and SPiDER-ßGal was used to detect cellular senescence. The important proteins associated with the pathogenesis of AD including APP, PS1, Aß, BACE1, and SYN1 were analyzed by immunofluorescence (IF) and Western blot analysis. Antimycin A (A3) and cyclosporine A (CSA) were selected as the activators and inhibitors of autophagy, respectively. LC3, BECN, P62, PINK1, and Parkin protein expression were also examined by IF and Western blot analysis. The data showed that ß-asarone administration significantly dose-dependently increased cell proliferation and decreased cytotoxicity; moreover, ß-asarone inhibited SA-ßGal and improved cell senescence. The results further showed that, compared to the model, APP, PS1, Aß, BACE1, and p62 were reduced, while SYN1, BECN1, and LC3 were increased after treatment with ß-asarone. The results of Canonical Correlation Analysis (CCA) showed a highly significant relationship between the pathological factors of AD and the protein expression of autophagy. In conclusion, our study demonstrated that ß-asarone can inhibit Aß, and this effect may occur by promoting autophagy in a cell model of AD.

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 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.

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 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.