Deprivation-induced PC12 Cell Research Articles

Two new terpenoids from the branches and leaves of Rhododendron dauricum L. with neuroprotective activity

Two new terpenoids were isolated from the branches and leaves of Rhododendron dauricum L., named as rhodayritions A (1) and B (2), together with five known compounds which were identified litseachromolaevane A (3), 11-αH-dihydrodehydrocostus lactone (4), (+)-9β-hydroxyeudesma-4,11(13)-dien-12-al (5), macrostachyoside B (6) and aglaiabbreviatin E (7), respectively. The structures of isolated compounds were determined by UV, HR-ESI-MS, NMR analysis and X-Ray. Their neuroprotective activity was studied on serum deprivation-induced PC12 cells by the MTT method, compounds 1, 6, and 7 exhibited significant neuroprotective activity at 20 μΜ.

RETRACTED ARTICLE: Lycium barbarum polysaccharide alleviates oxygen glucose deprivation-induced PC-12 cells damage by up-regulating miR-24

We, the Editors and Publisher of the journal Artificial Cells, Nanomedicine, and Biotechnology, have retracted the following article: Shiqing Song, Faliang Lin, Pengyan Zhu, Changyan Wu, Shuling Zhao, Qiao Han & Xiaomei Li (2019) Lycium barbarum polysaccharide alleviates oxygen glucose deprivation-induced PC-12 cells damage by up-regulating miR-24. Artificial Cells, Nanomedicine, and Biotechnology, 47(1), 3994–4000, DOI: 10.1080/21691401.2019.1673767 Since publication, concerns have been raised about the integrity of the data in the article. When approached for an explanation, the authors have been unable to verify their original data. We also have concerns regarding the integrity of the authorship, as one author has stated they did not consent to being listed as an author. We are therefore retracting this article and the corresponding author listed in this publication have been informed. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Rearranged iridal-type triterpenoids from Iris tectorum

Three new iridal-type triterpenoids (1–3) featuring a rearranged homofarnesylside chain were isolated from the rhizomes of Iris tectorum. Compounds 2 and 3 were found to be a pair of epimers. Their structures were elucidated on the basis of comprehensive spectroscopic analysis. A possible biosynthetic pathway for them was postulated. Moreover, the mixture of compounds 2 and 3 exhibited moderate neuroprotective activity against serum deprivation-induced PC12 cell death.

The mechanism of neuroprotective effect of Viola odorata against serum/glucose deprivation-induced PC12 cell death.

Objective:Oxidative stress is associated with the pathogenesis of brain ischemia and other neurodegenerative disorders. Previous researches have shown the antioxidant activity of Viola odorata L. In this project, we studied neuro-protective and reactive oxygen species (ROS) scavenging activities of methanol (MeOH) extract and other fractions isolated from V. odorata in PC12 cell line in serum/glucose deprivation (SGD) condition.Materials and Methods:The PC12 neuronal cells were pretreated for 6 hr with MeOH extract and fractions of V. odorata (1 to 25 μg/ml) followed by 24 hr incubation under SGD condition. Cell viability was measured by Alamar Blue® assay. The level of ROS was calculated using DCFH-DA. Also, Bax/Bcl-2 protein ratio was analyzed by western blot assay.Results:SGD condition significantly decreased cells viability (p<0.001). Pretreatment with EtOAc (12.5 and 25 µg/ml), BuOH (12, 25, 50 µg/ml) and CH2Cl2 (1.5 µg/ml) fractions of V. odorata reduced SGD-induced cytotoxicity. MeOH extract could not increase the viability significantly. All four semi polar fractions (EtOAc, BuOH, CH2Cl2 and MeOH) decreased SGD-induced ROS production and changed Bax/Bcl-2 ratio.Conclusion: V. odorata showed promising effects against SGD condition; further mechanistic and clinical studies are warranted before application of V. odorata as a neuro-protective agent.

Helioscopianoids A–Q, bioactive jatrophane diterpenoid esters from Euphorbia helioscopia

The EtOH extracts of the whole plants of Euphorbia helioscopia afforded 17 new jatrophane diterpenoid esters, helioscopianoids A–Q (117), along with eight known compounds (18–25). Their structures were elucidated by extensive spectroscopic methods and Mo2(OAc)4-induced ECD analysis, and the structures of compounds 1, 2, and 7 were confirmed by X-ray crystallography. Compounds 1–17 were evaluated for inhibitory effects on P-glycoprotein (P-gp) in an adriamycin (ADM)-resistant human breast adenocarcinoma cell line (MCF-7/ADR) and neuroprotective effects against serum deprivation-induced and rotenone-induced PC12 cell damage. Compounds 8 and 16 increased the accumulation of ADM in MCF-7/ADR cells by approximately 3-fold at a concentration of 20 μmol/L. Compound 8 could attenuate rotenone-induced PC12 cell damage, and compounds 2, 8, and 12 showed neuroprotective activities against serum deprivation-induced PC12 cell damage.

Protective effects of glucosamine and its acetylated derivative on serum/glucose deprivation-induced PC12 cells death: Role of reactive oxygen species.

Finding products with antiapoptotic activities has been one of the approaches for the treatment of neurodegenerative disorders. Serum/glucose deprivation (SGD) has been used as a model for the investigation of the molecular mechanisms of neuronal ischemia. Recent studies indicated that glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) have many pharmacological effects including antioxidant activities. The present study aimed to investigate the protective effects of GlcN and GlcNAc against SGD-induced PC12 cells injury. The PC12 cells were pretreated with GlcN and GlcNAc for 2 h, and then exposed to SGD for 6, 12 and 24 h. Cell viability was evaluated by MTT assay. The level of intracellular reactive oxygen species (ROS) was determined by flow cytometry using 2’,7’- dichlorofluorescin diacetate (DCFH-DA) as a probe. SGD condition caused a significant reduction in cell survival after 6, 12, and 24 h (P < 0.001). Pretreatment with GlcN and GlcNAc (0.6-20 mM) increased cell viability following SGD insult. A significant increase in cell apoptosis was observed in cells under SGD condition after 12 h (P < 0.001). Pretreatment with GlcN and GlcNAc (5-20 mM) decreased apoptosis following SGD condition after 12 h. SGD resulted in a significant increase in intracellular ROS production after 12 h. Pretreatment with both amino sugars at concentrations of 10 to 20 mM could reverse the ROS increment. Results indicated that GlcN and GlcNAc had a cytoprotective property against SGD-induced cell death via anti-apoptosis and antioxidant activities, suggesting that these aminosugers have the potential to be used as novel therapeutic agents for neurodegenerative disorders.

Synergistic and Defensive Properties of Emblica officinalis, Terminalia chebula, and Terminalia bellerica Extracts Against Serum/Glucose Deprivation-Induced PC12 Cells Death

Background: Triphala as a combination of three plant's fruits including Emblica officinalis Gaertn, Terminalia chebula Retz, and Terminalia bellerica Roxb is valued for its antioxidant properties. It has been routinely used in various medicinal studies. Objective: In this study, the protective effects of E. officinalis, T. chebula, T. bellerica, and Triphala methanolic extracts on cell viability and reactive oxygen species (ROS) generated in PC12 cells were studied under serum/glucose deprivation (SGD) induced cell injury. Synergistic activity of three extracts was also explored. Methods: Cells were seeded overnight and then exposed to SGD condition for 18 h. Next, they were pretreated with different concentrations of the four extracts (3 -250 μg/ml) for 4 h. Cell viability and ROS generation were evaluated by viability assay and flow cytometry, respectively. Synergistic activity of three extracts was analyzed with Compusyn software. Results: SGD could induce cell toxicity after 18 h (P < 0.001). Pretreatment with extracts reduced SGD toxicity in PC12 cells. A significant raise in ROS generation was seen following SGD-induced toxicity. Pretreatment with T. chebula reversed the increased ROS production following ischemic insult. Conclusion: These results demonstrated the neuroprotection of these extracts possibly by ROS decrement and synergistic activity of them against SGD-induced toxicity. © 2018, Jundishapur Journal of Natural Pharmaceutical Products.

Three new coumarin glycosides from the stems of Hydrangea paniculata

Three new coumarin glycosides (1-3), together with three known compounds (4-6), have been obtained from the stems of Hydrangea paniculata Sieb. Their structures were elucidated based on spectroscopic data and chemical evidence. In addition, compounds 1-3 were screened for their neuroprotective effects against serum deprivation-induced PC12 cell damage, hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells and their ability to inhibit LPS-induced nitric oxide production in the murine microglia BV2 cell line, but they were inactive.

Anthraquinone derivative exerted hormetic effect on the apoptosis in oxygen-glucose deprivation-induced PC12 cells via ERK and Akt activated Nrf2/HO-1 signaling pathway

There were accumulated evidences that agents may attenuate neurological disorders through a hormetic effect. This study was designed to investigate hormetic effect of BME on the oxygen-glucose deprivation (OGD)-induced mitochondrial apoptosis in NGF-differentiated PC12 cells. The effect of BME on the intracellular reactive oxygen species (iROS) formation and pro-survival signals mediated by ERK and Akt as well as transcription factor nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) pathways was also determined. The present results showed that, at low concentrations, pretreatment with BME triggered stress response by causing ROS production, then, activated survival-promoting signals via ERK and Akt activated Nrf2/HO-1 signaling pathway, resulting in decrease in cytotoxicity induced by the OGD. It may be accepted that mild pretreatment with BME stimulated transient and moderate ROS production, but activated hormetic signals and induced stress responsive genes. In contrast, high concentrations of BME displayed toxic action due to massive ROS production. These results suggested that the effect of BME on the OGD-induced PC12 cells may be hormetic mechanism including induction of oxidative stress and subsequent activation of stress response gene expression.

Raf kinase inhibitor protein regulates oxygen-glucose deprivation-induced PC12 cells apoptosis through the NF-κB and ERK pathways.

Raf-1 kinase inhibitory protein (RKIP) is a critical molecule for cellular responses to stimuli. In this study, we investigated whether RKIP is responsible for neural cell apoptosis induced by oxygen-glucose deprivation (OGD) and explored the role of NF-κB and ERK pathways regulated by RKIP under OGD stimuli. RKIP was overexpressed or knocked down using lentivirus in PC12 cells, which were then challenged by OGD. RKIP overexpression significantly increased the cell viability of OGD cells, and attenuated apoptosis, cell cycle arrest, and reactive oxygen species generation. RKIP knockdown induced reverse effects. Moreover, we found that RKIP interacted with TAK1, NIK, IKK, and Raf-1 and negatively regulated the NF-κB and ERK pathways. RKIP overexpression significantly inhibited IKK, IκBα, and P65 phosphorylation in NF-κB pathway and MEK, ERK, and CREB phosphorylation in ERK pathway, respectively. RKIP knockdown induced reverse effects. Furthermore, a NF-κB inhibitor BAY 11-7082 and a MEK inhibitor U0126 blocked the changes caused by RKIP down-regulation after OGD. In conclusion, these results demonstrate that RKIP plays a key role in neural cell apoptosis caused by OGD partly via regulating NF-κB and ERK pathways. The present study may provide new insights into the role of RKIP in ischemic stroke.

4-Hydroxybenzyl-substituted glutathione derivatives from Gastrodia elata

Seven new 4-hydroxybenzyl-substituted glutathione derivatives (2–8), together with a known analogue (1), were isolated from the aqueous extract of Gastrodia elata Blume rhizomes. Their structures were determined by using spectroscopic and chemical methods. The absolute configurations of 1–8 were assigned by using Marfey's method, combined with comparing the NMR and CD spectroscopic data of sulfoxide moieties in 3–6 with those of S-(4-hydroxybenzyl)cysteine sulfoxide stereoisomers (9–12) synthesized as authentic samples. The configurations of 9–12 were confirmed by electronic CD calculations based on the quantum-mechanical time-dependent density functional theory. Furthermore, the structures of 1, 3, 5, 7, and 8 were verified by synthesis. Compound 3 was active against serum deprivation-induced PC12 cell damage and synthetic 9–14 exhibited activity against Fe2+-cysteine induced rat liver microsomal lipid peroxidation.

Protective effect of Hibiscus sabdariffa against serum/glucose deprivation-induced PC12 cells injury.

Findings natural products with antioxidant and antiapoptotic properties has been one of the interesting challenges in the search for the treatment of neurodegenerative diseases including ischemic stroke. Serum/glucose deprivation (SGD) has been used as a model for the understanding of the molecular mechanisms of neuronal damage during ischemia in vitro and for the expansion of neuroprotective drugs against ischemia-induced brain injury. Recent studies showed that Hibiscus sabdariffa exert pharmacological actions such as potent antioxidant. Therefore, in this study we investigated the protective effect of extract of H. sabdariffa against SGD-induced PC12 cells injury. Cells were pretreated with different concentrations of H. sabdariffa extract (HSE) for 2 hr, and then exposed to SGD condition for 6, 12 and 18 hr. SGD caused a major reduction in cell viability after 6, 12, and 18 hr as compared with control cells (p< 0.001). Pretreatment with HSE (30-500 𝜇g/mL) significantly increased cell viability following SGD insult for 6, 12 and 18 hr. A significant increase in cell apoptosis was seen in cells under SGD condition after 12hr as compared with control cells (p< 0.001). Pretreatment with HSE significantly decreased cell apoptosis subsequent SGD conditionafter12hr at concentration of 60, 125 and 250. These data showed that HSE had a protective property under SGD condition in PC12 cells, suggesting that H. sabdariffa has the potential to be used as a new therapeutic approach for neurodegenerative disorders.

Anti-apoptotic effect of taxodione on serum/glucose deprivation-induced PC12 cells death.

Taxodione, a diterpenoid from the roots of Salvia chorassanica Bunge, possesses cytotoxic, apoptotic, and antimicrobial activity. This study was designed to investigate the protective effects of taxodione on serum/glucose deprivation-induced ischemic injury in PC12 cells and related mechanisms. In an in vitro model of ischemia, PC12 cells were exposed to serum and glucose deprivation for 6 and 18h. The protective effects of the methanol extract of S. chorassanica and taxodione were assessed using alamarBlue(®) assay. Intracellular ROS production was measured by fluorimetry using 2',7'-dichlorofluorescin diacetate (DCFH-DA). The levels of PARP, Bcl-2, and Bax proteins were detected after western blot analysis. It was shown that taxodione (0.2-1.5μM) significantly increased cell viability in a dose-dependent manner after ischemic insult. Taxodione has antioxidant activity and protects PC12 cells against oxidative stress-induced apoptotic cell death. Meanwhile, pretreatment with taxodione significantly induced an increase in Bcl-2 and a decrease in Bax protein level. The results of this study confirmed the protective effect of taxodione in serum/glucose deprivation-induced ischemic injury and the putative role of apoptosis as a underling mechanisms. Thus, it would be fair to consider taxodione as a promising ingredient of S. chorassanica for the expansion on novel class of anti-ischemic agents.

The expression changes of vacuolar protein sorting 4B (VPS4B) following middle cerebral artery occlusion (MCAO) in adult rats brain hippocampus.

Vacuolar protein sorting 4 (VPS4), is a member of ATPases associated with diverse cellular activities protein family. VPS4 is composed of VPS4A and VPS4B, VPS4B plays an important role in the lysosomal degradation pathway, intracellular protein trafficking, virus budding and abscission of cytokinesis. However, information regarding its distribution and possible function in the central nervous system is limited. Therefore, we performed a middle cerebral artery occlusion (MCAO) in adult rats and detected the dynamic changes of VPS4B in hippocampus CA1 subregion. We found that the VPS4B expression was increased strongly after MCAO and reached the peak after 3 days. VPS4B mainly located in the cytoplasm of neurons, but not astrocytes and microglia. Moreover, there was a concomitant up-regulation of active caspase-3. In vitro studies indicated that the up-regulation of VPS4B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knock-down of VPS4B in cultured differentiated PC12 cells by siRNA showed that VPS4B promoted the expression of active caspase-3. Collectively, all these results and MTT assay suggested that the up-regulation of VPS4B played an important role in the pathophysiology after MCAO, and further research is needed to have a good understanding of its function and mechanism.

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.

Protective Effect ofPunica granatumL. against Serum/Glucose Deprivation-Induced PC12 Cells Injury

The discovery and development of natural products with potent antioxidant, anti-inflammatory, and antiapoptotic properties have been one of the most interesting and promising approaches in the search for the treatment of many neurodegenerative diseases including ischemic stroke. Serum/glucose deprivation (SGD) has served as an excellent in vitro model for the understanding of the molecular mechanisms of neuronal damage during ischemia and for the development of neuroprotective drugs against ischemia-induced brain injury. Recent studies suggested that pomegranate (Punica granatum L.) or its active constituents exert pharmacological actions such as antioxidant, anti-inflammatory, and neuroprotective properties. Therefore, in this study we investigated the possible protective effects of different extracts of pomegranate against SGD-induced PC12 cells injury. Initially, the cells were pretreated with different concentrations of pulp hydroalcoholic extract (PHE), pulp aqueous extract (PAE) and pomegranate juice (PJ) for 2 h and then deprived of serum/glucose (SGD) for 6 and 12 h. SGD caused a significant reduction in cell viability (measured by the MTT assay) after 6 and 12 h, as compared with control cells (P < 0.001). Pretreatment with PHE, PAE, and PJ significantly and concentration-dependently increased cell viability following SGD insult for 6 and 12 h. A significant increase in DNA damage (measured by the comet assay) was seen in nuclei of cells following SGD for 12 h (P < 0.001). In control groups, no significant difference was seen in DNA damage between PHE, PAE, and PJ-pretreated and vehicle-pretreated PC12 cells (P > 0.05). PHE, PAE, and PJ pretreatment resulted in a significant decrease in DNA damage following ischemic insult (P < 0.001). This suppression of DNA damage by PHE, PAE and PJ was found to be concentration dependent. These data indicate that there is a cytoprotective property in PHE, PAE, and PJ under SGD condition in PC12 cells, suggesting that pomegranate has the potential to be used as a new therapeutic strategy for neurodegenerative disorders.

Minocycline reduces oxygen–glucose deprivation-induced PC12 cell cytotoxicity via matrix metalloproteinase-9, integrin β1 and phosphorylated Akt modulation

Minocycline has shown anti-inflammatory, anti-apoptotic, and antioxidative activities in many models of cerebral ischemia and human acute ischemic stroke. However, the cellular and molecular bases for its neuroprotective effects have not been fully elucidated. In this study, we investigated whether pre-treatment with minocycline could attenuate oxygen-glucose deprivation-induced PC12 cytotoxicity. The activity of matrix metalloproteinase-9 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. And the expressions of integrin β1, Akt and phosphorylated Akt were analyzed by Western blot. Our results showed that minocycline could ameliorate oxygen-glucose deprivation-induced PC12 cell cytotoxicity at concentrations of 20 nM-20 μM, down-regulate the production and activity of matrix metalloproteinase-9, inhibit the degradation of integrin β1, and up-regulate Akt phosphorylation at optimal concentration of 200 nM. The results may provide a new area for minocycline's therapeutic intervention for improving the outcomes of cerebral ischemia.

Coumarin Glycosides and Iridoid Glucosides with Neuroprotective Effects from Hydrangea paniculata

Five new coumarin glycosides, umbelliferone 7-O-sophoroside (1), umbelliferone 7-O-β-D-glucopyranosyl(1 → 3)-β-D-glucopyranoside (2), umbelliferone 7-O-β-D-glucopyranosyl(1 → 3)-[β-D-apiofuranosyl(1 → 6)]-β-D-glucopyranoside (3), umbelliferone 7-O-β-D-glucopyranosyl(1 → 2)-β-D-apiofuranosyl(1 → 6)-β-D-glucopyranoside (4), and umbelliferone 7-O-β-D-glucopyranosyl(1 → 5)-β-D-apiofuranosyl(1 → 6)-β-D-glucopyranoside (5), and two new iridoid glucosides, 7-O-E-isoferuloyl loganic acid (6) and 7-O-β-D-glucopyranosyl loganin (7), together with eight known compounds (8-15) were isolated from the stems of Hydrangea paniculata. Their structures were established by spectroscopic analysis and chemical methods. At 10 µM, compounds 1, 3, 5, 6, 8, 9, and 13 showed neuroprotective effects against serum deprivation-induced PC12 cell damage.

The neuroprotective effects of an extract of Gastrodia elata

Ethnopharmacological relevanceGastrodia elata (GE) Blume (family Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy, indicating neuronal protective functions. Aim of the studyTo evaluate the neuroprotection of GE and its molecular mechanism in preventing serum deprivation-induced PC12 cell apoptosis. Materials and methodsAn MTT assay and Hoechst staining were used to respectively validate serum deprivation-induced cell death and apoptosis. Cyclic (c)AMP formation and protein kinase (PK)A activity were also measured after GE treatment. Western blotting was used to detect the phosphorylation of the cAMP response element-binding (CREB) protein. Transient transfection of a dominant negative CREB was used to validate the importance of CREB. ResultsGE targeted the adenosine A2A receptor (A2A-R). GE increased cAMP formation, PKA activity, and phosphorylation of the CREB protein. GE-induced CREB protein phosphorylation and protection was blocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively. ConclusionsThese results support the neuroprotective effects of GE. The protective mechanism might be mediated through an A2A-R/cAMP/PKA/CREB-dependent pathway.

Protective effects of pulp aqueous and hydro-alcoholic extracts of Punica granatum, on serum/glucose deprivation-induced PC12 cells injury

Protective effects of pulp aqueous and hydro-alcoholic extracts of Punica granatum, on serum/glucose deprivation-induced PC12 cells injury