Injury In PC12 Cells Research Articles

Statement of Retraction

This article refers to:RETRACTED ARTICLE: Overexpression of lncRNA ANRIL aggravated hydrogen peroxide-disposed injury in PC-12 cells via inhibiting miR-499a/PDCD4 axis-mediated PI3K/Akt/mTOR/p70S6K pathway

The Essential Oil from Acori Tatarinowii Rhizome (the Dried Rhizome of Acorus tatarinowii Schott) Prevents Hydrogen Peroxide-Induced Cell Injury in PC12 Cells: A Signaling Triggered by CREB/PGC-1α Activation.

Acori Tatarinowii Rhizome (ATR, the dried rhizome of Acorus tatarinowii Schott), a well-recognized traditional Chinese herbal medicine, is prescribed to treat neurological disorders. The essential oil is considered as the active fraction of ATR, and the neuroprotection of ATR essential oil (ATEO) is proven, including the protection against oxidative stress. However, the cellular mechanism of ATEO against oxidative stress has not been fully illustrated. In this study, to investigate the cellular mechanism of ATEO, the cytoprotective effect of ATEO against H2O2-induced injury was revealed in PC12 cells. ATEO treatment increased the viability of cells affected by H2O2-mediated injury, inhibited reactive oxygen species (ROS) accumulation, and induced the expression of several antioxidant proteins (SODs, GPx, and UCPs). The cytoprotective effect of ATEO was related to upregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, which was counteracted by PGC-1α specific knockdown. Using inhibitor of protein kinase A (PKA), we found that cAMP-response element binding protein (CREB) activation was involved in ATEO-induced PGC-1α expression. Taken together, we suggest that ATEO effectively prevents H2O2-induced cell injury possibly through the activation of CREB/PGC-1α signaling in PC12 cells. The results provide a molecular insight into the effect of ATEO on cytoprotection against oxidative stress.

Silencing MEG3 protects PC12 cells from hypoxic injury by targeting miR-21

Increasing number of literatures highlighted lncRNA maternally expressed gene 3 (MEG3) as an emerging target for hypoxic-ischaemic brain damage (HIBD). This study attempted to assess the role of MEG3 in a cell model of HIBD. Expression of MEG3 in PC12 cells was suppressed by siRNA-mediated transfection, after which the cells were subjected to hypoxia. Cell viability, apoptosis, migration and the expression of related proteins were assessed. Furthermore, the downstream gene of MEG3 and its downstream signalling pathways were explored. We found that, down-regulation of MEG3 prevented hypoxic injury in PC12 cells, as hypoxia induced viability loss, apoptosis and migration repression were attenuated by transfection with MEG3 siRNA. Meanwhile, MEG3 acted as a miR-21 sponge. The neuroprotective functions of MEG3 silence were flattened when miR-21 was suppressed. Moreover, the deactivation of PI3K/AKT pathway and the activation of NF-κB pathway induced by hypoxia were attenuated by MEG3 silence. As expected, the effects of MEG3 silence on these two signalling were via miR-21. In conclusion, the neuroprotective effects of MEG3 silence on PC12 cells injured by hypoxia were observed in this study. Mechanistically, the neuroprotective effects of MEG3 silence on PC12 cells were via sponging miR-21 and thus regulating PI3K/AKT and NF-κB pathways. HIGHLIGHTS MEG3 is highly expressed in PC12 cells following hypoxic injury; Silence of MEG3 prevents hypoxia-induced cell damage in PC12 cells; MEG3 acts as a miR-21 sponge; MEG3 sponges miR-21 to regulate PI3K/AKT and NF-κB pathways.

Erxian decoction, a famous Chinese medicine formula, antagonizes corticosterone-induced injury in PC12 cells, and improves depression-like behaviours in mice

Context In folk medicine, erxian decoction (EXD) is used to treat perimenopausal syndrome in women. It is also used clinically to treat depression, but the mechanism remains unknown. Objectives To investigate the neuroprotective effect of EXD, and its antidepressant potential. Materials and methods ICR mice were treated with EXD (0.5, 1.5 and 4.5 g/kg i.g.) and fluoxetine (6.0 mg/kg i.g.) for 10 days. On day 10 of the treatment, depression-like behaviour was induced by reserpine (2.5 mg/kg injected i.p.), and after 24 h of reserpine administration, it was assessed using the tail suspension and forced swimming tests. MTT assay, lactate dehydrogenase test, flow cytometry analysis, Hoechst staining and western blotting were used to assess the apoptosis of PC12 cells. Apoptosis proteins and neurotransmitter were tested in vitro and in vivo, respectively. Results MTT assay results showed corticosterone prevented cell growth, but EXD at concentrations of 100, 200 and 400 μg/mL restored cell viability (EC50: 204.016 μg/mL). EXD decreased lactate dehydrogenase leakage from 63.48 to 43.60 U/L, and upregulated expression of Bcl-2 while the expression of Bax, caspase-3 and caspase-8 were decreased in vivo and in vitro. Moreover, EXD improved depression-like behaviour in mice, and 4.5 g/kg EXD treatment increased the secretion of serotonin, dopamine and norepinephrine by 67.44, 28.12 and 42.12 pg/mg, respectively, in hypothalamus compared to that of reserpine group. Discussion and conclusions EXD demonstrated neuroprotective effects and improved depression-like behaviour in mice. Further research should be focussed on the mechanism of the active components in EXD.

Expression of Concern: Scutellaria barbata D. Don (SBD) protects oxygen glucose deprivation/reperfusion-induced injuries of PC12 cells by up-regulating Nrf2

Expression of Concern: Scutellaria barbata D. Don (SBD) protects oxygen glucose deprivation/reperfusion-induced injuries of PC12 cells by up-regulating Nrf2

Knocking down TRPM2 expression reduces cell injury and NLRP3 inflammasome activation in PC12 cells subjected to oxygen-glucose deprivation.

Transient receptor potential melastatin 2 (TRPM2) is an important ion channel that represents a potential target for treating injury caused by cerebral ischemia. However, it is unclear whether reducing TRPM2 expression can help repair cerebral injury, and if so what the mechanism underlying this process involves. This study investigated the protective effect of reducing TRPM2 expression on pheochromocytoma (PC12) cells injured by oxygen-glucose deprivation (OGD). PC12 cells were transfected with plasmid encoding TRPM2 shRNAS, then subjected to OGD by incubation in glucose-free medium under hypoxic conditions for 8 hours, after which the cells were allowed to reoxygenate for 24 hours. Apoptotic cells, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels were detected using flow cytometry. The relative expression of C-X-C motif chemokine ligand 2 (CXCL2), NACHT, LRR, and PYD domain-containing protein 3 (NALP3), and caspase-1 were detected using fluorescence-based quantitative reverse transcription-polymerase chain reaction and western blotting. The rates of apoptosis, mitochondrial membrane potentials, reactive oxygen species levels, and cellular calcium levels in the TRPM2-shRNA + OGD group were lower than those observed in the OGD group. Taken together, these results suggest that TRPM2 knockdown reduces OGD-induced neuronal injury, potentially by inhibiting apoptosis and reducing oxidative stress levels, mitochondrial membrane potentials, intracellular calcium concentrations, and NLRP3 inflammasome activation.

Typhae pollen polysaccharides protect hypoxia-induced PC12 cell injury via regulation of miR-34a/SIRT1.

This current research was performed to investigate the role of typhae pollen polysaccharides (TPP) in hypoxia-treated PC12 cell which was an in vitro cell model of cerebral ischemia. Hypoxia-treated cells were treated with TPP for 12 h. Cell viability and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2 5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. Cell apoptotic proteins and PI3K/AKT and Ras/Raf/MEK/ERK signal pathway–associated proteins were also examined by western blot. Furthermore, abnormal expression of miR-34a and silent information regulator 1 (SIRT1) was achieved by transfection. Besides, the expression of miR-34a and SIRT1 was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of SIRT1 was detected by qRT-PCR and western blot. The relationship between miR-34a and SIRT1 was verified by luciferase assay. We found that TPP enhanced cell viability and inhibited apoptosis in hypoxia-treated PC12 cells. Moreover, TPP increased the accumulated levels of Bcl-2 while decreased expression of Bax, cleaved Caspase-3, and cleaved PARP. TPP downregulated miR-34a expression while induced by hypoxia. Further results showed that miR-34a overexpression reversed the results led by TPP in cell viability, apoptosis, and its related proteins. In addition, SIRT1 was upregulated by TPP and was verified to be a target of miR-34a. Silence of SIRT1 led to the opposite results led by TPP. In the end, TPP activated PI3K/AKT and Ras/Raf/MEK/ERK signal pathways. In conclusion, TPP plays important roles in regulating cell viability and apoptosis in hypoxia-treated PC12 cells via modulating miR-34a/SIRT1, as well as activating PI3K/AKT and Ras/Raf/MEK/ERK signal pathways.

Overexpression of miR-146b-5p Ameliorates Neonatal Hypoxic Ischemic Encephalopathy by Inhibiting IRAK1/TRAF6/TAK1/NF-αB Signaling.

PurposeNeonatal hypoxic ischemic encephalopathy (HIE) is an essential factor underlying neonatal death and disability. This study sought to explore the role of miR-146b-5p in regulating neonatal HIE.Materials and MethodsIn vitro and in vivo HIE models were established in PC12 cells and 10-day neonatal Sprague Dawley rats, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to assess miR-146b-5p expression and inflammatory factors [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] in brain lesions and PC12 cells, while enzyme-linked immunosorbent assay was employed to detect the expression of oxidative stress factors (SOD and GSH-Px). Gain- and loss-assays of miR-146b-5p were conducted to verify its role in modulating the viability and apoptosis of PC12 cells under oxygen-glucose deprivation (OGD) treatment. Expression of TLR4, IRAK1, TRAF6, TAK1, and NF-κB were examined by qRT-PCR and/or Western blot. Dual luciferase activity assay was conducted to identify relationships between miR-146b-5p and IRAK1.ResultsIn the HIE models, significant oxidative stress and inflammatory responses emerged upon upregulation of TLR4/IRAK1/TRAF6/TAK1/NF-κB signaling. Overexpression of miR-146b-5p greatly inhibited OGD-induced PC12 cell injury, inflammatory responses, and oxidative stress. Inhibiting miR-146b-5p, however, had the opposite effects. IRAK1 was found to be a target of miR-146b-5p, and miR-146b-5p overexpression suppressed the activation of IRAK1/TRAF6/TAK1/NF-κB signaling.ConclusionThis study demonstrated that miR-146b-5p overexpression alleviates HIE-induced neuron injury by inhibiting the IRAK1/TRAF6/TAK1/NF-κB pathway.

1,8-Cineol Attenuated AΒ25-35-Induced PC12 Cell Injury through Reducing Caspase 3 Expression and NO Production

Objective To investigate the effect of 1,8-cineol on caspase 3 expression and NO production induced by Aβ25-35 in PC12 cells. Methods PC12 cells were cultured in vitro, and cell injury was induced by Aβ25-35 with a concentration of 20 μM. 1,8-cineol (1, 3, 10 μM) was pretreated before Aβ25-35 treatment. PC12 cell viability was evaluated by MTT detection assay. Caspase 3 protein expression was detected by Western blotting. The level of NO production in PC12 cells was measured using ELISA detection assay kit. Results In cultured PC12 cells in vitro, MTT results showed that 20 μM of Aβ25-35 reduced cell viability significantly compared with control group. The cell viability was increased by pretreatment with 1,8-cineol with concentrations of 3 and 10 μM compared with Aβ25-35 only group. Western blotting results showed compared with control group, caspase 3 expression was increased significantly in 20 μM Aβ25-35 group. Compared with Aβ25-35 group, 1,8-cineol of 3 and 10 μM group reduced caspase 3 protein expression significantly. The level of NO production in PC12 cells was increased significantly, which was decreased by pretreatment with 3 and 10 μM of 1,8-cineol. Conclusions: Our results revealed a protective effect of 1,8-cineol on Aβ25-35 induced PC12 cell injury through inhibition of caspase 3 expression and NO production.

Polycyclic polyprenylated acylphloroglucinol derivatives with neuroprotective effects from Hypericum monogynum

A new polycyclic polyprenylated acylphloroglucinol (PPAP), hypermonin C (1), along with nine known PPAPs (2–10) were obtained from the leaves and twigs of Hypericum monogynum. The structures of the isolates were determined on the basis of extensive spectroscopic analysis. The neuroprotective effects of the isolates against several chemical-induced injuries in SH-SY5Y and PC12 cells were assessed, and most of the compounds exhibited significant protective effects at 10 μg/ml. Especially, three compounds (1, 3, and 7) showed excellent neuroprotective activity with a cell viability of 92.4% ∼ 95.8% in KCl-induced SH-SY5Y cell injury. Their preliminary structure-activity relationship was also discussed and the configuration of substituent in furohyperforin may be critical for the neuroprotective activity of PPAP derivatives.

Phenylethanoid glycosides from Paraboea martinii protect rat pheochromocytoma (PC12) cells from hydrogen peroxide-induced cell injury.

In this study, we isolated eight phenylethanoid glycosides from Paraboea martinii for the first time, and evaluated the mechanism underlying their neuroprotective effects against H2O2-induced injury in PC12 cells. The MTS method was utilized to screen the phenylethanoid glycosides for protective ability. Next, qRT-PCR and western blotting analysis were used to detect the transcription levels of HO-1 and GCLC, which are regulated by Nrf2. The inhibitor ZnPP was used to analyze the involvement of Nrf2 in HO-1 expression. Analyses showed that caleolarioside B, paraboside B, and paraboside II also upregulated the expression of HO-1, but showed no obvious effect on GCLC. Pretreatment with ZnPP significantly reduced the neuroprotective effects. Thus, phenylethanoid glycosides isolated from P. martinii protected PC12 cells from H2O2-induced damage by upregulating HO-1. The results provided evidence that P. martinii might be a potential therapeutic agent for the treatment of neurodegenerative diseases.

Upregulation of miR-376c-3p alleviates oxygen\u2013glucose deprivation-induced cell injury by targeting ING5

BackgroundThe expression level of miR-376c-3p is significantly lower in infants with neonatal hypoxic-ischemic encephalopathy (HIE) than in healthy infants. However, the biological function of this microRNA remains largely elusive.MethodsWe used PC-12 and SH-SY5Y cells to establish an oxygen–glucose deprivation (OGD) cell injury model to mimic HIE in vitro. The miR-376c-3p expression levels were measured using quantitative reverse transcription PCR. The CCK-8 assay and flow cytometry were utilized to evaluate OGD-induced cell injury. The association between miR-376c-3p and inhibitor of growth 5 (ING5) was validated using the luciferase reporter assay. Western blotting was conducted to determine the protein expression of CDK4, cyclin D1, Bcl-2 and Bax.ResultsMiR-376c-3p was significantly downregulated in the OGD-induced cell injury model. Its overexpression elevated cell viability and impaired cell cycle G0/G1 phase arrest and apoptosis in PC-12 and SH-SY5Y cells after OGD. Downregulation of miR-376c-3p gave the opposite results. We further demonstrated that ING5 was a negatively regulated target gene of miR-376c-3p. Importantly, ING5 knockdown had a similar effect to miR-376c-3p-mediated protective effects against cell injury induced by OGD. Its overexpression abolished these protective effects.ConclusionOur data suggest that miR-376c-3p downregulated ING5 to exert protective effects against OGD-induced cell injury in PC-12 and SH-SY5Y cells. This might represent a novel therapeutic approach for neonatal HIE treatment.

Neuroprotective Effect of Cyperi rhizome against Corticosterone-Induced PC12 Cell Injury via Suppression of Ca2+ Overloading.

Cyperi Rhizoma (CR) is a well-known functional food and traditional herbal medicine in Asian countries for the treatment of menstrual or emotional disturbances in women. Recent studies have shown the pharmacological effects of CR on neuronal diseases, such as Parkinson’s disease (PD) and depression. Thus, the neuroprotective effect of CR might play a vital role in exerting its effect. Here, corticosterone-induced PC12 cells were applied to screen the active fraction of CR and evaluate its neuroprotective effect. The results indicated that the fraction containing medium-polarity chemical constituents (CR-50E) displayed the best protection effect. CR-50E could increase the cell viability and reduce cell apoptosis through inhibiting oxidative stress and decreasing the lactate dehydrogenase LDH release induced by corticosterone. Further, the mechanism of action was explored by cell metabolomics. The result showed CR-50E mediated the sphingolipids metabolism of corticosterone-induced PC12 cells, which suggested inhibition of Ca2+ overloading may involve the protection of CR-50E against cell damage. The expression levels of three key proteins in calcium transport, including phospholipase A2 (PLA2), calcium/calmodulin independent protein kinase II (CaMK II), and caspase-3, confirmed the above result by Western blot. The findings suggest that CR-50E can suppress the disequilibrium of calcium homeostasis-mediated apoptosis by improving the abnormal sphingolipids metabolism as well as remedying the damage of the cell membrane.

Protective functions of salvianolic acid B in PC-12 cells against hydrogen peroxide-triggered damage by mediation of microRNA-26a

Spinal cord injury (SCI) can lead to varying degrees of sensory and motor dysfunction. Salvianolic acid B (Sal-B) is the dominating bioactive constituent of Danshen, which has been reported to alleviate liver fibrosis and exert neuroprotective effects. But, the influence of Sal-B in SCI remains mysterious. The research planned to delve the protective function of Sal-B in hydrogen peroxide (H2O2)-caused PC-12 cell injury. H2O2-caused PC-12 cells injury model was built, CCK-8, Transwell and flow cytometry experiments were enforced to assess cell proliferation, migration and apoptosis. The microRNA (miR)-26a plasmid and the matching control were transfected into PC-12 cells, subsequently, the influence of miR-26a inhibition in H2O2-corrupted PC-12 cells was evaluated. The cell growth-correlated factors and PI3K/AKT and MEK/ERK pathways were assayed through western blot assay. Results corroborated that Sal-B eased H2O2-evoked injury in PC-12 cells. Ascended miR-26a was monitored in Sal-B and H2O2-exposed cells. MiR-26a inhibition annulled the protective action of Sal-B in H2O2-corrupted cells. The protective function of Sal-B was enabled through activating PI3K/AKT and MEK/ERK pathways. These findings delineated that Sal-B protected PC-12 cells against H2O2-caused injury through ascending miR-26a via initiating PI3K/AKT and MEK/ERK pathways. Highlights H2O2 causes PC-12 cell injury; Sal-B eases H2O2-caused PC-12 cell injury; Sal-B protects PC-12 cells against H2O2-caused injury via elevating miR-26a; Sal-B activates AKT and MEK/ERK pathways via modulating miR-26a.

Schizandrin Protects against OGD/R-Induced Neuronal Injury by Suppressing Autophagy: Involvement of the AMPK/mTOR Pathway.

The neuroprotective role of schizandrin (SA) in cerebral ischemia-reperfusion (I/R) was recently highlighted. However, whether SA plays a regulatory role on autophagy in cerebral I/R injury is still unclear. This study aimed to explore whether the neuroprotective mechanisms of SA were linked to its regulation of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/autophagy pathway in vivo and in vitro. The present study confirmed that SA significantly improved oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced PC12 cells injury. The results of immunoblotting and confocal microscope showed that SA decreased autophagy in OGD/R-injured PC12 cells, which was reflected by the decreased Beclin-1 and LC3-II expression, autophagy flux level, and LC3 puncta formation. In addition, the autophagy inducer rapamycin partially prevented the effects of SA on cell viability and autophagy after OGD/R, whereas the autophagy inhibitor 3-methyladenine (3-MA) exerted the opposite effect. The results of Western blotting showed that SA markedly decreased the phosphorylation of AMPK (p-AMPK), whereas the phosphor-mTOR (p-mTOR) levels increased in the presence of OGD/R insult. Furthermore, pretreatment with the AMPK inducer AICAR partially reversed the protective effects and autophagy inhibition of SA. However, AMPK inhibitor Compound C pretreatment further promoted the inhibition of SA on autophagy induction and cell damage induced by OGD/R. Taken together, these findings demonstrate that SA protects against OGD/R insult by inhibiting autophagy through the regulation of the AMPK-mTOR pathway and that SA may have therapeutic value for protecting neurons from cerebral ischemia.

The study of neuroprotective effect of ferulic acid based on cell metabolomics

Ferulic acid (FA), a naturally derived phenolic compound, has antioxidant and antidepressant-like effects. It is still a challenge to study its mechanism due to the complexity of the pathophysiology of depression. In this study, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to perform metabolomics studies based on biochemical changes in differentiated rat pheochromocytoma (PC12) cells treated with corticosterone-induced neurological damage after FA treatment. A total of 31 metabolites were identified as potential biomarkers for corticosterone-induced PC12 cells injury. Among them, 24 metabolites were regulated after FA treatment. Pathway analysis revealed that these metabolites were mainly involved in the amino acid metabolism, energy metabolism and glycerophospholipid metabolism. In addition, based on the results of metabolomics, three cell signaling pathways related to glutamate were discovered. To further study the interactions between FA and major targets in three signaling pathways, a molecular docking method was employed. The results showed that FA had the strongest binding power with protein kinase B (AKT). Furthermore, the result of mRNA changes analyzed by quantitative real time RT-PCR indicated that AKT and protein kinase A (PKA) in the signaling pathway were up regulated after treatment with FA compared with model group. This study shows that strategies based on cell metabolomics associated with molecular docking and molecular biology is a helpful tool to elucidate the neuroprotective mechanism of FA.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells.

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury. Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology, the mechanisms are not fully understood. To address this issue, we first co-cultured 1.5 × 105 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1, and then intervened with cobalt chloride (CoCl2) for 24 hours. Reactive oxygen species in PC12 cells was measured by Mito-sox. Mitochondrial membrane potential (?Ψm) in PC12 cells was determined by JC-1 staining. Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining. Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy. Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry. Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria. Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells. CoCl2-induced PC12 cell damage was dose-dependent. Co-culture with mesenchymal stem cells significantly reduced apoptosis and restored ?Ψm in the injured PC12 cells under CoCl2 challenge. Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling, the disappearance of cristae, and chromatin margination in the injured PC12 cells. After direct co-culture, mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells. The transfer efficiency was greatly enhanced in the presence of CoCl2. More importantly, inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury. Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Damage and Phenotype Change in PC12 Cells Induced by Lipopolysaccharide Can Be Inhibited by Antioxidants Through Reduced Cytoskeleton Protein Synthesis.

The present study investigated changes in cellular phenotype and oxidative stress during the inflammatory response in PC12 cells stimulated by lipopolysaccharide (LPS) and assessed the effects of minocycline, astragalus (AST), and baicalin on inflammation. PC12 cells were exposed to LPS with or without minocycline, AST, or baicalin. Cell viability was measured by a thiazolyl blue tetrazolium bromide (MTT) assay. Contrast and laser confocal microscopy were used to analyze changes in cellular phenotype and cytoskeleton synthesis. Western blotting tested the expression of α7nAChR and vimentin. Inhibitory ratio of superoxide dismutase (SOD) activity and leakage of lactate dehydrogenase (LDH) were detected to evaluate cellular oxidative stress. Results showed that LPS could attenuate PC12 cell viability in a time- and dose-dependent manner, which could be rescued by minocycline. In addition, minocycline could reverse PC12 cell phenotypic change and the synthesis of the mesenchymal cytoskeleton protein vimentin, both induced by LPS. During LPS-initiated inflammation, α7nAChR and vimentin expression were obviously inhibited by minocycline, AST, or baicalin. The inhibitory rate of SOD activity and LDH leakage in PC12 cells were increased by LPS and attenuated significantly when exposed to minocycline, AST, or baicalin. These findings suggest phenotype change, altered cytoskeleton protein synthesis, and oxidative stress are all involved in the inflammatory response in PC12 cells during which α7 nicotinic acetylcholine receptor (α7nAChR) is induced by LPS stimulation. Minocycline, AST, and baicalin have a protective effect against PC12 cell injury, acting as antioxidants and inhibitors of mesenchymal proteins.

Corticosterone induces neurotoxicity in PC12 cells via disrupting autophagy flux mediated by AMPK/mTOR signaling.

AimsOur previous study indicated that chronic stress caused autophagy impairment and subsequent neuron apoptosis in hippocampus. However, the mechanism underlying the stress‐induced damage to neurons is unclear. In present work, we investigated whether stress‐level glucocorticoids (GCs) GCs promoted PC12 cell damage via AMPK/mTOR signaling‐mediated autophagy.MethodsChronic stress‐induced PC12 cell injury model was built by treatment with high level corticosterone (CORT). Cell injury was evaluated by flow cytometry assay and transmission electron microscopy observation.ResultsAutophagy flux was measured based on the changes in LC3‐II and P62 protein expressions, and the color alteration of mCherry‐GFP‐LC3‐II transfection. Our results showed that CORT not only increased cell injury and apoptosis, but also dysregulated AMPK/mTOR signaling‐mediated autophagy flux, as indicated by the upregulated expression of LC3‐II and P62 proteins, and the lowered ration of autolysosomes to autophagosomes. Mechanistically, our results demonstrated that autophagy activation by AMPK activator metformin or mTOR inhibitor rapamycin obviously promotes cell survival and autophagy flux, improved mitochondrial ultrastructure, and reduced expression of Cyt‐C and caspase‐3 in CORT‐induced PC12 cells.ConclusionThese results indicate that high CORT triggers PC12 cell damage through disrupting AMPK/mTOR‐mediated autophagy flux. Targeting this signaling may be a promising approach to protect against high CORT and chronic stress‐induced neuronal impairment.

Triclosan induces PC12 cells injury is accompanied by inhibition of AKT/mTOR and activation of p38 pathway

Triclosan (TCS) has been widely used as a disinfectant and antiseptic in multiple consumer and healthcare products due to its clinical effectiveness against various bacteria, fungi and protozoa. Recently, several studies have reported the adverse effects of TCS on various nerve cells, arousing concerns about its potential neurotoxicity. The present study aimed to investigate the neurotoxicity of TCS in rat pheochromocytoma PC12 cells. After differentiation, the stabilized PC12 cells were treated with 1, 10, 50 μM TCS for 12 h. At the end of the treatment, the generation of reactive oxygen species (ROS), protein expression of apoptotic-related genes, AMPK-AKT/mTOR, as well as p38 in PC12 cells were determined. The concentrations were chosen based on the results of cell viability and lactic dehydrogenase (LDH) assays in response to TCS treatment (ranging from 0.001 to 100 μM) for varied time periods. The results showed that TCS is cytotoxic to PC12 cells, causing decreased cell viability accompanied by increased LDH release. TCS treatment at 10 and 50 μM for 12 h increased the mRNA and protein expression of the pro-apoptotic gene Bax, while Bcl-2 levels remained unchanged. Moreover, an increase in the generation of reactive oxygen species (ROS) was found in TCS-treated PC12 cells at the concentrations of 1 and 10 μM. Pretreatment with 100 μM N-acetyl cysteine (NAC- ROS scavenger) for 1 h normalized the ROS generations in TCS-treated PC12 cells. Additionally, the suppression of the phosphorylation of Akt and mTOR was observed in TCS-treated PC12 cells at 10 and 50 μM for 12 h, concomitant with the activation of p38 MAPK pathway at 50 μM TCS. However, there were no effects of TCS on the phosphorylation of AMPK in these cells. Taken together, these results suggest that TCS may cause adverse effects and oxidative stress in PC12 cells accompanied by inhibition of Akt/mTOR and activation of p38.