DMC (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone) enhances exercise tolerance via the AMPK-SIRT1-PGC-1α pathway in mice fed a high-fat diet.

Obesity is caused by an imbalance between energy intake and energy expenditure. This study aimed to determine the effects and mechanisms of 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) on exercise tolerance in high-fat diet (HFD)-fed mice. Male C57BL/6J mice were randomly divided into two categories (7 groups [n = 8]): sedentary (control [CON], HFD, 200 mg/kg DMC, and 500 mg/kg DMC) and swimming (HFD, 200 mg/kg DMC, and 500 mg/kg DMC). Except the CON group, all other groups were fed HFD with or without DMC intervention for 33 days. The swimming groups were subjected to exhaustive swimming (three sessions/week). Changes in swimming time, glucolipid metabolism, body composition, biochemical indicators, histopathology, inflammation, metabolic mediators, and protein expression were assessed. DMC combined with regular exercise improved endurance performance, body composition, glucose and insulin tolerance, lipid profile, and the inflammatory state in a dose-dependent manner. Further, DMC alone or combined with exercise could restore normal tissue morphology, reduce fatigue-associated markers, and boost whole-body metabolism and the protein expression of phospho-AMP-activated protein kinase alpha/total-AMP-activated protein kinase alpha (AMPK), sirtuin-1 (SIRT1), peroxisome-proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), and peroxisome proliferator-activated receptor alpha in the muscle and adipose tissues of HFD-fed mice. DMC exhibits antifatigue effects by regulating glucolipid catabolism, inflammation, and energy homeostasis. Furthermore, DMC exerts a synergistic exercise-related metabolic effect via the AMPK-SIRT1-PGC-1α signaling pathway, suggesting that DMC is a potential natural sports supplement with mimicked or augmented exercise effects for obesity prevention.

Icariin attenuates ischaemic stroke through suppressing inflammation mediated by endoplasmic reticulum stress signalling pathway in rats.

Ischaemic stroke possesses the characteristics of high incidence, high disability and high mortality. Icariin (ICA) is a flavonoid extracted from the traditional Chinese medicine Epimedium. The protective effect of ICA on ischaemic stroke is worthy of further study. In this study, male Sprague-Dawley rats were randomly divided into the following seven groups: sham, model, ICA low-dose (10 mg/kg), ICA medium-dose (20 mg/kg), ICA high-dose (40 mg/kg), positive control drug (12 mg/kg nimodipine) and endoplasmic reticulum stress induction (0.16 mg/kg tunicamycin) groups. The model of cerebral ischaemia-reperfusion injury in the rats, including 2 h ischaemia and 24 h reperfusion, was accomplished by applying the method of transient middle cerebral artery occlusion (MCAO). At 24 h reperfusion, neurological deficits, brain water content, pathological damage of brain tissues, the expression of inflammation-related targets, and the signal pathway-related proteins were explored. Compared with the model group, ICA significantly improved neurological deficits, brain oedema and pathological damage after MCAO. In addition, ICA increased neuron survival, reduced microglial activation and expression of IL-1ß, alleviating the inflammatory damage caused by ischaemic stroke. Moreover, ICA suppressed the expressions of glucose-regulated protein 78 (GRP78), inositol requiring enzyme-1 α (IRE1α), phospho-IRE1α (p-IRE1α), protein kinase RNA-like ER kinase (PERK), phospho-PERK (p-PERK), spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), thioredoxin-interacting protein (TXNIP), NLRP3, and caspase-1. These results suggested that ICA offers neuroprotection against ischaemic stroke by inhibiting ER stress-mediated inflammation.

Mulberry Leaf Extract Alleviates Staphylococcus aureus-Induced Conjunctivitis in Rabbits via Downregulation of NLRP3 Inflammasome and Upregulation of the Nrf2 System and Suppression of Pro-Inflammatory Cytokines.

INTRODUCTION: Mulberry (Morus alba L.) leaves are widely used in traditional Chinese medicine for their antioxidant, anti-inflammatory, antibacterial, anti-obesity, antidiabetic, antiatherosclerotic, and anticancer properties. The current study aimed to investigate the effect of mulberry leaf extract (MLE) on Staphylococcus aureus (S. aureus)-induced conjunctivitis (5 × 109 colony-forming units, 0.5 mL/eye) in a rabbit model. METHODS: Rabbits were treated with MLE (5 mL/kg·d-1 and 10 mL/kg·d-1), 0.9% saline, pearl bright eye (PBE) drops, or erythromycin eye ointment (EEO) group for 5 days. The ocular infection symptoms, bacterial negative conversion rate, and conjunctival histopathological changes of rabbits in each group were observed. The expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, NOD-like receptor leucine-rich pyrin domain-containing protein 3 (NLRP3), interleukin (IL)-18, IL-6, IL-1ß, TNFα, Keap1, and nuclear factor erythroid 2-related factor 2 (Nrf2) in conjunctival tissue of rabbits were detected by quantitative real-time reverse transcription PCR and/or Western blot analysis. RESULTS: The results showed that MLE treatment significantly reduced the clinical sign scores of conjunctivitis, alleviated clinical signs, and decreased bacterial load, and histological damage in a time- and dose-dependent manner was compared to that in the control group. The antibacterial and anti-inflammatory activities of MLE (10 mL/kg·d-1) were similar to those of the positive control drug PBE and EEO. In addition, MLE significantly decreased the levels of pro-inflammatory cytokines, downregulated the NLRP3 inflammasome, and upregulated the Nrf2 system. CONCLUSIONS: MLE is effective in alleviating S. aureus-induced conjunctivitis in rabbits, and this mechanism is associated with the inhibition of the NLRP3 inflammasome and activation of the Nrf2 system to regulate pro-inflammatory signaling.

Icariin inhibits the expression of IL-1ß, IL-6 and TNF-α induced by OGD/R through the IRE1/XBP1s pathway in microglia.

CONTEXT: Icariin (ICA), a flavonol glycoside extracted from Epimedium brevicornum Maxim (Berberidaceae), has been proven to inhibit inflammatory response in ischaemic rats in our laboratory's previous work. However, its underlying mechanism is still unclear. OBJECTIVE: This study investigates the effects of ICA on endoplasmic reticulum (ER) stress mediated inflammation induced by cerebral ischaemia-reperfusion (I/R) injury in vitro. MATERIALS AND METHODS: The primary cultured microglia were treated with oxygen-glucose deprivation (OGD) for 2 h followed by a 24 h reoxygenation. ICA (0.37, 0.74 and 1.48 µmol/L) administration was performed 1 h prior OGD and acting through 2 h OGD. The control group was cultured in normal conditions. At 24 h after reoxygenation, the expression of IRE1α, XBP1u, XBP1s, NLRP3 and caspase-1 was detected by western blotting (WB) and quantitative real-time (qRT) PCR; the expression of p-IRE1α was examined by WB; the expression of IL-1ß, IL-6 and TNF-α was measured by WB and enzyme-linked immunosorbent assay (ELISA). RESULTS: ICA (0.37, 0.74 and 1.48 µmol/L) reduced the ratio of p-IRE1α/IRE1α, the mRNA level of IRE1α, the expression of XBP1u, XBP1s, NLRP3, caspase-1 at both the mRNA and protein level expression of IL-1ß, IL-6 and TNF-α in OGD/R injured microglia. Overexpression of IRE1 significantly reversed the effects of ICA. DISCUSSION AND CONCLUSIONS: These results suggested that ICA might decrease the expression of IL-1ß, IL-6 and TNF-α by inhibiting IRE1/XBP1s pathway. The anti-inflammatory effect of ICA may provide a potential therapeutic strategy for the treatment of brain injury after stroke.

Icariin protects neurons from endoplasmic reticulum stress-induced apoptosis after OGD/R injury via suppressing IRE1α-XBP1 signaling pathway.

Icariin (ICA), a flavonol glycoside isolated from Epimedium, has been considered as a potential alternative therapy for ischemic stroke. However, the protective mechanisms of ICA on cerebral ischemia-reperfusion (I/R) are not fully illuminated yet. The effects of ICA on ER stress and inflammatory response which were involved in the pathological process of cerebral I/R were investigated in vitro. Microglia and neurons were subjected to OGD/R. ICA was administrated to microglia 1 h before OGD and maintained 2 h throughout OGD. At 24 h after reoxygenation, the protein expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia was measured using ELISA assay; neuronal apoptosis was assessed by TUNEL staining; and cell viability was detected using CKK-8 assay; the expression of IRE1α, XBP1u, XBP1s, and cleaved caspase-3 in neurons was examined by western blotting and qRT-PCR; the expression of p-IRE1α in neurons was detected by western blotting. We found that OGD/R induced the expression of IL-1 ß, IL-6, TNF-α in the supernatant of microglia; OGD/R and these proinflammatory cytokines promoted the mRNA as well as protein expression of XBP1u, XBP1s and cleaved caspase-3, increased the ratio of p-IRE1α/IRE1α, as well as apoptosis, and decreased cell viability in primary cortical neurons, while ICA reversed the levels of the above factors. IRE1 overexpression enhanced ER stress as well as apoptosis, and impaired the protective effects of ICA. These results suggested that ICA can inhibit apoptosis in neurons after OGD/R through IRE1/XBP1 signaling pathway beside its anti-inflammatory effect.

Ezrin enhancer knockout inhibits the proliferation and migration of human esophageal carcinoma Eca-109 cells / 中国肿瘤生物治疗杂志

@#Objective: To investigate the effects of ezrin enhancer knockout on ezrin gene expression, cell proliferation and migration of human esophageal carcinoma Eca-109 cells. @*@#Methods: The CRISPR/Cas9 recombinant plasmids targeting upstream/downstream of human ezrin enhancer were co-transfected into human esophageal carcinoma Eca-109 cells, and the cell line Eca-C2 with ezrin enhancer knockout was screened by purinomycin. Then the expression levels of ezrin mRNAand protein in Eca-C2 cells were detected by Real-time quantitative PCR (qPCR) and Western blotting, respectively; The expression levels of MAPK-pathway-related proteins were detected by protein array technology; and the effects of ezrin enhancer knockout on the proliferation and migration of Eca-C2 cells were analyzed by WST-1 method and wound-healing assay, respectively. @*@# Results:The human esophageal carcinoma cell line Eca-C2 with stable ezrin enhancer knockout was established successfully. Compared with control cells, the mRNA and protein expressions of ezrin in Eca-C2 cells were significantly reduced (all P<0.05).Among the 17 detected MAPK pathway related proteins in Eca-C2 cells, 9 proteins (AKT, CREB, GSK3b, MKK6, mTOR, P38, P53, P70S6K and RSK1) were down-regulated, and the cell proliferation and migration were significantly inhibited (all P<0.05).@*@# Conclusion: ezrin enhancer knockout can significantly inhibit the cell proliferation and migration of human esophageal carcinoma Eca-109 cells.

Synergistic effect of phytochemicals on cholesterol metabolism and lipid accumulation in HepG2 cells.

BACKGROUND: Crocin (CRO), chlorogenic acid (CGA), geniposide (GEN), and quercetin (QUE) are all natural compounds with anti-obesity properties, in particular, hypolipidemic effects, which have been widely used for the treatment of obesity-related metabolic diseases. However, it is not yet known whether these compounds interact synergistically. Here, we investigated the effects and molecular mechanisms of CRO, CGA, GEN, QUE, and a combination of all four compounds (CCGQ), on lipid accumulation in human hepatoma (HepG2 cells). METHODS: The optimal concentration of CRO, CGA, GEN, QUE to stimulate HepG2 cells proliferation was determined using MTT assay. HepG2 cells were pretreated with 10 µmol/L simvastatin, 1 µmol/L CRO, 30 µmol/L CGA, 10 µmol/L GEN, 10 µmol/L QUE, and CCGQ (a combination of 1 µmol/L CRO, 30 µmol/L CGA, 10 µmol/L GEN, and 10 µmol/L QUE) for 24 or 48 h. Oil red O staining and extracellular TC and TG levels were detected. The RT-PCR was used to observe on cholesterol metabolism-related gene expression. Immunocytochemistry and western-blot assayed the 3-hydroxy-3-methylglutaryl-coenzyme (HMGCR) protein expression in HepG2 cells. RESULTS: Compared to those of control, we demonstrated that treating HepG2 cells for 48 h with CCGQ resulted in a strong synergistic effect, causing a marked decrease in lipid deposition in comparison to individual treatments, in both triglyceride and total cholesterol (CRO, 5.74- and 1.49-folds; CGA, 3.38- and 1.12-folds; GEN, 4.04- and 1.44-folds; QUE, 3.36- and 1.24-folds; simvastatin, 5.49- and 1.83-folds; and CCGQ, 7.75- and 2.20-folds), and Oil red O staining assays. In addition, CCGQ treatment increased ATP-binding cassette transporter (ABCA1), cholesterol 7α-hydroxylase (CYP7A1), and AMP-activated protein kinase 2α (AMPKα2) mRNA expression, while decreasing sterol regulatory element binding protein 2 (SREBP2), and liver X receptor alpha (LXRα) mRNA expression. Notably, CCGQ was more effective in decreasing HMGCR expression than the individual treatments. CONCLUSION: The CCGQ combination has potential, both as a complementary therapy for hyperlipemia, and in preventing further obesity-related complications.

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen-glucose deprivation-induced injury.

CONTEXT: The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. OBJECTIVE: To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. MATERIALS AND METHODS: PC12 cells were treated with icariin (10-7, 10-6 or 10-5 mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10-7, 10-6, or 10-5 mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. DISCUSSION AND CONCLUSION: These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

In vitro evaluation of ruthenium complexes for photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a promising anti-tumor treatment strategy. Photosensitizer is one of the most important components of PDT. In this work, the anticancer activities of PDT mediated by six new ruthenium porphyrin complexes were screened. The mechanisms of the most efficacious candidate were investigated. METHODS: Photocytotoxicity of the six porphyrins was tested. The most promising complex, Rup-03, was further investigated using Geimsa staining, which indirectly detects reactive oxygen species (ROS) and subcellular localization. Mitochondrial membrane potential (MMP), cell apoptosis, DNA fragmentation, c-Myc gene expression, and telomerase activities were also assayed. RESULTS: Rup-03 and Rup-04 had the lowest IC50 values. Rup-03 had an IC50 value of 29.5±2.3µM in HepG2 cells and 59.0±6.1µM in RAW264.7 cells, while Rup-04 had an IC50 value of 40.0±3.8µM in SGC-7901 cells. The complexes also induced cellular morphological changes and impaired cellular ability to scavenge ROS, and accumulated preferentially in mitochondria and endoplasmic reticulum. Rup-03 reduced MMP levels, induced apoptosis, and repressed both c-Myc mRNA expression and telomerase activity in HepG2 cells. CONCLUSIONS: Among six candidates, Rup-03-mediated PDT is most effective against HepG2 and RAW264.7, with a similar efficacy as that of Rup-04-mediated PDT against SGC-7901 cells. Repression of ROS scavenging activities and c-Myc expression, which mediated DNA damage-induced cell apoptosis and repression of telomerase activity, respectively, were found to be involved in the anticancer mechanisms of Rup-03.

Icariin Attenuates OGD/R-Induced Autophagy via Bcl-2-Dependent Cross Talk between Apoptosis and Autophagy in PC12 Cells.

Icariin (ICA), an active component of Epimedium brevicornum Maxim, exerts a variety of neuroprotective effects such as antiapoptosis. However, the mechanisms underlying antiapoptosis of ICA in neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) are unclear. The B-cell lymphoma-2 (Bcl-2) protein family plays an important role in the regulation of apoptosis and autophagy through Bcl-2-dependent cross talk. Bcl-2 suppresses apoptosis by binding to Bax and inhibits autophagy by binding to Beclin-1 which is an autophagy related protein. In the present study, MTT result showed that ICA increased cell viability significantly in OGD/R treated PC12 cells (P < 0.01). Results of western blotting analysis showed that ICA increased Bcl-2 expression significantly and decreased expressions of Bax, cleaved Caspase-3, Beclin-1, and LC3-II significantly in OGD/R treated PC12 cells (P < 0.01). These results suggest that ICA protects PC12 cells from OGD/R induced autophagy via Bcl-2-dependent cross talk between apoptosis and autophagy.

Chlorogenic acid-enriched extract from Eucommia ulmoides leaves inhibits hepatic lipid accumulation through regulation of cholesterol metabolism in HepG2 cells.

CONTEXT: Eucommia ulmoides Oliver (Eucommiaceae) leaf exhibits beneficial lipid-lowering and anti-obesity effects. However, the mechanisms remain unknown. OBJECTIVE: The objective of this study is to investigate the lipid-lowering effects of chlorogenic acid (CGA)-enriched extract from this plant (CAEF) in human hepatoma HepG2 cells, focusing on cholesterol metabolism. MATERIALS AND METHODS: HepG2 cells were treated with CAEF (10, 20, 25, 40, 60, and 80 mg/L), CGA (0.3, 3, 30, 300, and 600 µmol/L), and simvastatin (0.1, 1, 10, 50, and 100 µmol/L) for 24 or 48 h. The cytotoxicity, Oil red O staining, total cholesterol, and triacylglycerol in supernatants were determined. The mRNA expression of genes involved in cholesterol metabolism was determined with RT-PCR. The protein expression of HMG-CoA reductase (HMGCR) was examined by immunocytochemistry and western-blot. RESULTS: The IC50 values were 59.2 mg/L for CAEF, 335.9 µmol/L for CGA, and 10.5 µmol/L for simvastatin. By treating cells with CAEF (25 mg/L), CGA (30 µmol/L), or simvastatin (10 µmol/L) for 48 h, the efflux of total cholesterol and triacylglycerol was increased (CAEF, 4.06- and 31.00-folds; CGA, 2.94- and 2.17-folds; and simvastatin, 3.94- and 24.67-folds), and the cellular lipid droplets were reduced in Oil red O staining. CAEF and CGA increased mRNA expression of ABCA1, CYP7A1, and AMPKα2, while CAEF and simvastatin decreased SREBP2. However, their effects on LXRα mRNA expression were variable. Importantly, all drugs significantly inhibited protein expression of HMGCR at mRNA and protein levels. DISCUSSION AND CONCLUSION: CAEF is a promising dietary supplement to prevent obesity and dyslipidemia and the effects appear to be due, at least in part, to regulating cholesterol metabolism through inhibition of HMGCR in HepG2 cells.

Establish a flow cytometric method for quantitative detection of Beclin-1 expression.

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 µg/µL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.

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.