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.

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 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.

[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 and levodopa co-administration protects against 6-hydroxydopamine-induced damage in parkinsonian rat mesencephalon by regulating autophagy: down-expression Beclin-1 and light chain 3B and up-expression P62.

In this study, we investigated Beclin-1, light chain (LC)3B, and p62 expression in 6-hydroxydopamine (6-OHDA)-induced parkinsonian rats after ß-asarone and levodopa (l-dopa) co-administration. Unilateral 6-OHDA injection into the medial forebrain bundle was used to create the models, except in sham-operated rats. Rats were divided into eight groups: sham-operated group; 6-OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l-dopa group (60 mg/kg, p.o.); ß-asarone group (15 mg/kg, p.o.); ß-asarone + l-dopa co-administered group (15 mg/kg + 60 mg/kg, p.o.); 3-methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin-1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin-1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l-dopa, ß-asarone, and co-administered groups when compared with the 6-OHDA model. Beclin-1 and LC3B expression in the ß-asarone and co-administered groups were less than in the madopar or l-dopa groups, whereas p62 expression in the ß-asarone and co-administered groups was higher than in the madopar or l-dopa groups. In addition, a significant decrease in autophagosome was exhibited in the ß-asarone and co-administered groups when compared with the 6-OHDA group. Our findings indicate that Beclin-1 and LC3B expression decreased, whereas p62 expression increased after co-administration treatment. In sum, all data suggest that the co-administration of ß-asarone and l-dopa may contribute to the treatment of 6-OHDA-induced damage in rats by inhibiting autophagy activity.

[Effects of Total Ginsenosides and Volatile Oil of Acorus tatarinowii Co-Administration on Ability of Learning and Memory and Apoptosis in Alzheimer's Disease Mice Model Induced By D-Galactose and Aluminium Chloride].

OBJECTIVE: To observe the effects of the co-administration of total ginsenosides and volatile oil of Acorus tatarinowii on the ability of learning and memory and apoptosis in Alzheimer's disease (AD) mice model induced by D-galactose and aluminium chloride. METHODS: 50 Kunming (KM) mice were randomly divided into normal group, model group, Aricept group (1 mg/kg), Ding Zhi Wan group (10 g/kg) and co-administration of total ginsenosides and volatile oil of Acorus tatarinowii group (co-administered group, the doses of volatile oil of Acorus tatarinowii and total ginsenosides were 30 mg/kg and 150 mg/kg, respectively). In addition to normal group, mice in other groups were given D-galactose 150 mg/ (kg x d), ip, and aluminium chloride 5 mg/kg, ig, once daily for 40 days. At the same time, mice in the treated groups were administrated with the corresponding drug from the 20th day after the modeling, once daily for 40 days. Water maze and avoiding darkness experiments were used to test learning and memory abilities; Aß1-42 and BCL-2 content in cortex and hippocampus were detected by ELISA; the vitalities of acetyl cholinesterase ( AChE) and acetylcholine transferase (ChAT) were detected by ultraviolet spectrophotometry. Superoxide dismutase (SOD) vitalities were detected by a water-soluble tetrazolium salt (WST-1) method; the content of malondialdehyde ( MDA) in cortex and hippocampus were detected by the thiobarbituric acid (TBA) method; senile plaque on Aß1-42 precipitation were observed by immunohistochemistry; brain tissues were observed by hematoxylin-eosin staining (HE). RESULTS: As compared with model group, in the co-administered group, the time of AD mice swimming, the numbers of blind area and electric shock reduced significantly (P < 0.05), and the latent period was prolonged (P < 0.05); AChE activity and levels of Aß1-42 and MDA in cortex and hippocampus were decreased significantly (P < 0.05 or P < 0.01); ChAT and SOD activities as well as BCL-2 content were increased significantly (P < 0.05 or P < 0.01) the formation of senile plaque was decreased and brain tissue morphology was improved. CONCLUSION: Total ginsenosides and volatile oil of Acorus tatarinowii co-administration has an effect on improving the ability of learning and memory and inhibiting apoptosis.

Coadministration of ß-asarone and levodopa increases dopamine in rat brain by accelerating transformation of levodopa: a different mechanism from Madopar.

The aim of the present study was to investigate the effect of coadministration of ß-asarone and levodopa (l-dopa) on increasing dopamine (DA) in the striatum of healthy rats. Rats were randomly divided into four groups: (i) a normal group, administered normal saline; (ii) a Madopar group, administered 75 mg/kg Madopar (l-dopa : benserazide, 4 : 1); (iii) an l-dopa group, administered 60 mg/kg l-dopa; and (iv) a group coadministered 15 mg/kg ß-asarone and 60 mg/kg l-dopa. All drugs (or normal saline) were administered intragastrically twice a day for 7 days. Then, plasma and striatum concentrations of DA, l-dopa, 5-hydroxytryptamine (5-HT), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), tyrosine hydroxylase (TH), catechol-O-methyltransferase (COMT) and monoamine oxidase B (MAO-B) were determined. In the group coadministered ß-asarone and l-dopa, there was a decline in plasma and striatal concentrations of l-dopa; however, DA and DOPAC concentrations increased in the striatum and plasma and plasma HVA concentrations increased, whereas there was no significant change in striatal levels. Concentrations of 5-HT in the striatum and plasma were similar in the coadministered and Madopar-treated groups. In addition, plasma and striatal COMT levels decreased after coadministration of ß-asarone and l-dopa, whereas there were no significant differences in MAO-B concentrations among groups. Furthermore, coadministration of ß-asarone and l-dopa increased plasma TH concentrations. Altogether, ß-asarone affects the conversion of l-dopa to DA by modulating COMT activity and DA metabolism. The mechanism of coadministration is different from that of Madopar in Parkinson's disease (PD) treatment. Thus, the coadministration of ß-asarone and l-dopa may be beneficial in the treatment of PD.

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.

Dynamic expressions of Beclin 1 and tyrosine hydroxylase in different areas of 6-hydroxydopamine-induced Parkinsonian rats.

Beclin 1, a regulator of the autophagy pathway, plays an important role in Parkinson's disease (PD). However, the crucial mechanism of Beclin 1 in PD remains unclear. Therefore, we investigated dynamic expressions of Beclin 1 and tyrosine hydroxylase (TH) in different brain areas of 6-OHDA-induced rats. Beclin 1 and TH expressions were analyzed by flow cytometry and immunohistochemistry, respectively. The results showed that Beclin 1 expressions were low in the sham group, but rose significantly after 6-OHDA injection. In the striatum and cortex, Beclin 1 increased at 3 h, peaking at 12 h, while in the hippocampus, it increased at 3 h and peaked at 24 h, then it declined slowly and remained steady at 72 h. Beclin 1 expression in the striatum and cortex areas was higher than that of the hippocampus area at 12 h. In addition, the time-course of TH expression in the striatum was similar to that in the mesencephalon. TH expression declined dramatically between 0 and 12 h. Pearson analysis showed significant negative correlations between TH and Beclin 1 expression in the areas we analyzed. While TH expression declined gradually between 12 and 72 h, significant positive correlations between TH and Beclin 1 were detected during that interval. This indicated that activation of Beclin 1-dependent autophagy may inhibit the loss of TH-positive neurons.

Ethanolic extract of propolis inhibits atherosclerosis in ApoE-knockout mice.

BACKGROUND: The present study was undertaken to investigate the effects and underlying mechanism of ethanolic extract of propolis (EEP) on the development of atherosclerotic lesions in ApoE-/- mice. METHODS: Eight-week-old male ApoE-/- mice fed a high-fat diet were treated with EEP (160 mg/kg/d) or vehicle (the same dose) respectively for 14 weeks. The serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were determined by enzymatic methods. Non-HDL-C was calculated as TC minus HDL-C. Serum interleukin-6 (IL-6), interleukin-17 (IL-17), endothelin (ET), inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) were determined with enzyme-linked immunosorbent assay methods. Nitric oxide (NO) content was measured with an enzymatic nitrate reductase assay. Analyses of atherosclerotic lesions in whole aorta and aortic root sections were performed with plaque staining using Oil Red O. RESULTS: Compared with the vehicle-treated group, serum contents of total cholesterol (TC), triglycerides (TG) and non-HDL-C reduced significantly by 31.88%, 21.01%, and 27.11% respectively in the EEP-treated group. Administration of EEP decreased the level of IL-6 and increased the level of IL-17 in ApoE-/- mice with a high-fat diet. Compared with the vehicle-treated group,EEP significantly reduced the levels of ET and VEGF,and showed a trend to increase NO and inhibit iNOS. In the ApoE-/- mice fed a high-fat diet, EEP significantly reduced atherosclerotic lesion development in the aortic root and whole aorta. CONCLUSION: EEP can inhibit atherosclerotic lesion formation in ApoE-/- mice fed a high-fat diet possibly through modulating cholesterol, regulating inflammatory reaction,inhibiting ET and VEGF, and protecting vascular endothelial cells.

[Inhibitory effect of quercetin preconditioning on tunicamycin-induced apoptosis in macrophages and its mechanism].

The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 µmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 µmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.

Change of Beclin-1 dependent on ATP, [Ca(2+)](i) and MMP in PC12 cells following oxygen-glucose deprivation-reoxygenation injury.

Autophagy is usually up-regulated to provide more ATP in response to starvation or OGD (oxygen-glucose deprivation), but the relationship between autophagy and ATP, [Ca2+]i (intracellular free Ca2+ concentration) or MMP (mitochondrial membrane potential) during reoxygenation is not yet fully clear. The role of autophagy is unknown in PC12 cells subjected to 2 h OGD with different time points of reoxygenation. In the present study, we showed that Beclin-1 was up-regulated beginning at 0 h reoxygenation peaking at 24 h and lasting for 48 h. Cell viability was decreased from 0 to 48 h reoxygenation, reaching its minimum at 10 h reoxygenation. ATP was decreased from 0 to 10 h reoxygenation, reaching its minimum at 4 h reoxygenation. A significant negative correlation was observed between ATP and Beclin-1 (r = -0.61, P<0.05) at 0 h reoxygenation, but ATP was not significant related (r = 0.24, P>0.05) to Beclin-1 at 24 h reoxygenation. Besides, Nimodipine, a calcium antagonist, significantly reduced [Ca2+]i and Beclin-1, but increased MMP in OGD/R-treated cells. At 24 h reoxygenation, Beclin-1 expression reached its maximum, cell viability continued to increase, and ATP was higher than that before OGD. These results suggest that energy metabolism dysfunction can induce autophagy during OGD in PC12 cells. Increased [Ca2+]i and decreased MMP may induce autophagy during reoxygenation in PC12 cells. Autophagy may be a protective effect on PC12 cells treated with different time points of reoxygenation after 2 h OGD.

ß-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy.

AIM: To explore the effects of ß-asarone from Acorus Tatarinowii Schott on autophagy in an ischemic stroke model of PC12 cells. METHODS: The ischemic stroke model of PC12 cells was made by OGD/R (2 h oxygen-glucose deprivation followed by 24 h reperfusion). Drug administration was started 1 h before OGD and last for 3 h. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 24 h. After the treatments, Beclin-1, intracellular free calcium concentration ([Ca(2+)](i)) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Cell viability was measured using MTT assay. Cell morphology was studied under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope. RESULTS: Pretreatment with ß-asarone (20, 30, or 45 µg/mL) or the calcium channel antagonist nimodipine (10 µmol/L) significantly increased the cell viability and MMP, and decreased Beclin-1 expression and [Ca(2+)](i) in OGD/R-treated PC12 cells. Under inverted phase contrast microscope, pretreatment with ß-asarone or nimodipine dramatically increase the number of cells and improved the cellular morphology. Autophagosomes were found in OGD/R-treated PC12 cells as well as in drug plus OGD/R-treated PC12 cells. CONCLUSION: ß-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin-1-dependent autophagy caused by decreasing [Ca(2+)](i) and increasing MMP.

Inhibitory effect of the combination therapy of simvastatin and pinocembrin on atherosclerosis in ApoE-deficient mice.

UNLABELLED: The present study was performed to investigate the effects of the combination therapy of pinocembrin and simvastatin on the atherosclerotic lesions development in the ApoE-/- mice. METHODS: Eight-week-old male ApoE-/- mice were fed high fat diet (HFD) and treated with simvastatin (10 mg/kg per day), pinocembrin (20 mg/kg per day), or the combination therapy (simvastatin 5 mg/kg per day and pinocembrin 20 mg/kg per day) for 14 weeks. The serum lipid levels, nitric oxide (NO), endothelin (ET), superoxide dismutase (SOD) and malondialdehyde (MDA) were determined with spectrophotometric measurement and ELISA assay. Vascular endothelial growth factor (VEGF) in serum and aortic root was detected. En face analyses of atherosclerotic lesion in whole aorta and aortic root sections were performed with plaque staining using oil red O. RESULTS: The combination treatment with simvastatin and pinocembrin resulted in significantly decreased levels of serum total cholesterol, triglycerides and low-density lipoprotein cholesterol, augmented NO levels and SOD activity, inhibited ET and VEGF expression. Immunohistochemistry of aortic valve sections revealed that the combination therapy also suppressed the expression of VEGF induced by HFD. In addition, HFD-induced arterial wall lipid disposition displayed by oil red O staining was reduced significantly in aortic root and whole aorta en face in the combination administrated mice. The effect of the combination was superior to simvastatin alone. CONCLUSION: The combination of simvastatin and pinocembrin synergistically inhibited atherosclerotic lesion development in ApoE-/- mice with hyperlipidemia, which is partially dependent on the protective of vascular endothelium.

Pharmacokinetics of beta-asarone in rabbit blood, hippocampus, cortex, brain stem, thalamus and cerebellum.

beta-Asarone has significant pharmacological effects on the central nervous system. As a potential therapeutic agent to manage brain diseases, analysis of the pharmacokinetics of beta-asarone in brain is necessary. We used cardio-perfusion method to exclude the beta-asarone in the brain blood. The brain was divided into five regions: hippocampus, cortex, brain stem, thalamus and cerebellum, and pharmacokinetic differences were investigated. We found that concentration-time profile of beta-asarone in blood, hippocampus, cortex, brain stem and cerebellum could be adequately described by a first-order equation, consistent with a linear two-compartmental model, but a first-order equation with a linear one-compartmental model in thalamus. The half lives of beta-asarone in blood, hippocampus, cortex, brain stem, thalamus and cerebellum were 1.3801, 1.300, 1.937, 7.142, 2.832 and 8.149 h, respectively. Gender differences do not significantly influence plasma pharmacokinetics of beta-asarone.

[Compound Xuanju capsule improves sex hormones and sex organ indexes in castrated male rats].

OBJECTIVE: To investigate the effects of Compound Xuanju Capsule on the levels of sex hormones and the weight of sexual organs in castrated male rats. METHODS: A randomized model control trail was performed in 60 young male SD rats of SPF grade, of which 12 were included in the normal control group, and the others were castrated and randomly divided into a model control group and a high-dose, a median-dose and a low-dose Xuanju group. The control groups received intragastric administration of normal saline, and the model groups solution of Compound Xuanju Capsule, all for 20 days. Then we determined by radioimmunoassay the levels of testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) in the peripheral blood of the rats, and measured the weights of the epididymis, preputial gland, seminal vesicle, prostate and levator ani muscle. RESULTS: The T levels were remarkably lower in the castrated groups than in the normal controls, and significantly higher in the three Xuanju groups than in the model controls (P < 0.01). Both LH and FSH levels were increased in the model control and Xuanju groups as compared with the normal control group, the former with statistically significant difference (P < 0.05) and the latter without. In comparison with the normal controls, the model control rats showed a marked reduction in the indexes of the preputial gland, seminal vesicle, prostate and levator ani muscle, and the high-dose Xuanju group exhibited a significant increase in the seminal vesicle index as compared with the model controls (P < 0.05). There were no statistically significant differences in the indexes of preputial gland, prostate and levator ani muscle among different dose groups (P > 0.05). CONCLUSION: Compound Xuanju Capsule can elevate T and LH levels in the peripheral blood of male SD rats and improve the indexes of their sex organs, which may be an important mechanism behind its effect on ED.

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.