Apoptosis In PC12 Cells Research Articles

Neuroprotective schinortriterpenoids from Schisandra neglecta collected in Medog County, Tibet, China

Schisdilactones K–U (1–11), a series of previously unreported 16,17-secopreschisanartane-type schinortriterpenoids (SNTs), were isolated from the leaves and stems of Schisandra neglecta A. C. Smith. Their structures were mainly established through analysis of their spectroscopic data. Besides, schisdilactones K (1), O (5) and R (8) were confirmed by single-crystal X-ray crystallographic analysis, and the configurations of schisdilactones T and U (10 and 11) were elucidated via quantum chemical calculation of their NMR chemical shifts and electronic circular dichroism (ECD) spectra. Schisdilactones L–S (2–8) and U (11) were found to exhibit moderate protective activities against corticosterone-induced apoptosis of PC12 cells at 20 μM, with cell viability in the range of 62.95–66.97%.

Stilbene glycoside upregulates SIRT3/AMPK to promotes neuronal mitochondrial autophagy and inhibit apoptosis in ischemic stroke.

Ischemic stroke, also known as cerebrovascular accident or cerebral stroke, occupies the first place in the world's top 10 causes of death, with high incidence, mortality and disability rates. To investigate the effect of stilbene glycoside upregulated SIRT3/AMPK expression on neuronal mitochondrial autophagy and neuronal apoptosis in ischemic stroke. The PC12 cells were cultured without serum to construct an ischemic neuron model. The cells were divided into 6 groups: normal group (untreated cells), model group (ischemic treated cells), TSG group (stilbene glycoside treatment), NC group (SIRT3 and AMPK negative control treatment), si-SIRT3 group (SIRT3 silencing treatment), TSG+si-SIRT3 group (joint treatment), and TSG+si-SIRT3+oe-AMPK group (joint treatment). Cell survival and the expression of related molecules were detected. Compared with normal group, the model group had significantly decreased cell survival rate, mitochondrial membrane potential, as well as the expression of Bcl-2, LC3II/I, P62, PINK1, Parkin, SIRT3, AMPK, and p-AMPK, while showing significantly increased proportion of apoptosis and the expression of caspase 3 and Bax. Compared with the model group, TSG treatment promoted cell survival rate and mitochondrial autophagy, and inhibited apoptosis, while SIRT3 silencing treatment reduced cell survival rate and mitochondrial autophagy, and increased apoptosis. The SIRT3 silencing could block the inhibitory effect of TSG on the apoptosis of ischemic PC12 cells and promote mitochondrial autophagy, and AMPK overexpression could save the apoptosis of ischemic PC12 cells caused by SIRT3 silencing, and promote mitochondrial autophagy. By promoting the expression of SIRT3/AMPK, TSG promotes mitochondrial autophagy in ischemic neurons and inhibits their apoptosis.

Protective Effects of Crataegus pinnatifida Extracts and Crataegolic Acid Against Neurotoxicity of Paraquat in PC12 Cells

The neuroprotective mechanisms of Crataegus pinnatifida extracts and crataegolic acid were studied using paraquat induced cytotoxicity in PC12 cells. C. pinnatifida extracts were prepared using hexane, ethyl acetate, and 95% ethanol. Additionally, crataegolic acid (also known as maslinic acid) was found in C. pinnatifida extracts. Assessment methods included the examinations of cytotoxicity, intracellular reactive oxygen species and calcium changes, activity of caspase-3 and α-synuclein, apoptotic cell death, and the expression levels of the B-cell lymphoma 2 (Bcl-2) and BCL2-associated X (Bax) proteins to investigate the neuroprotective mechanisms of C. pinnatifida extracts and its active component, crataegolic acid. The three extracts and crataegolic acid exhibited potent neuroprotective actions against paraquat induced PC12 cell apoptosis at 5–20µg/mL and 80–100µM concentrations, respectively. The key protective mechanisms included decreasing cell apoptosis, upregulating Bcl-2 protein levels, and downregulating Bax protein levels. The 95% ethanol extract also decreased paraquat induced reactive oxygen species production, calcium overloading, and caspase-3 and α-synuclein activities. The beneficial effects of these extracts could be explained by the active component, crataegolic acid that also inhibited paraquat-induced apoptosis through the suppression of reactive oxygen species generation and the caspase-3 signaling pathway.

MiR-495 reduces neuronal cell apoptosis and relieves acute spinal cord injury through inhibiting PRDM5.

This study aims to investigate the role of miR-495 in neuronal cell apoptosis after acute spinal cord injury (ASCI). The ASCI rat model was established and the Basso, Beattie, and Bresnahan (BBB) score was assessed. miR-495, PR domain containing 5 (PRDM5), and Bcl-2 expressions were measured by qRT-PCR or western blotting. Neuronal cell line PC-12 was subjected to hypoxia condition to simulate the in vitro ASCI model. PC-12 cell apoptosis was measured by flow cytometry, and the interaction between miR-495 and PRDM5 was confirmed by dual luciferase reporter assay. Results showed that BBB score was significantly decreased in ASCI rats compared with sham rats. miR-495 expression was down-regulated in spinal cord tissue of ASCI rats and hypoxia-induced PC-12 cells, and PRDM5 protein level was up-regulated in spinal cord tissue of ASCI rats and hypoxia-induced PC-12 cells. miR-495 overexpression could reduce apoptosis of PC-12 cells, and up-regulated anti-apoptosis protein Bcl-2 protein level. Moreover, PRDM5 was a target of miR-495, and mRNA and protein levels of PRDM5 were negatively regulated by miR-495. miR-495 overexpression could reduce the hypoxia-induced PC-12 cell apoptosis, while PRDM5 overexpression abolished this inhibiting effect. The agomir-495 was injected into ASCI rats, and Bcl-2 protein level and BBB score were increased, but the PRDM5 overexpression reversed these results. Overall, we concluded that miR-495 could inhibit neuronal cell apoptosis and relieve acute spinal cord injury through inhibiting PRDM5.

Icariin promotes the repair of PC12 cells by inhibiting endoplasmic reticulum stress

BackgroundEndoplasmic reticulum stress (ERS) is one of the main mechanisms of spinal cord injury (SCI) pathology and can affect the physiological state of neurons. Icariin (ICA), the main pharmacological component of Epimedium, can relieve the symptoms of patients with SCI and has obvious protective effects on neurons through ERS.MethodsPC12 cells were induced to differentiate into neurons by nerve growth factor and identified by flow cytometry. Cell proliferation was detected by CCK8 method, cell viability was detected by SRB assay, apoptosis was detected by flow cytometry and microstructure of ER was observed by transmission electron microscope. Western blot was used to detect the protein expression of CHOP and Grp78, and qPCR was used to detect the mRNA expression of CHOP and Grp78.ResultsThe results of CCK8, SRB and flow cytometry showed that ICA could relieve ERS and reduce apoptosis of PC12 cells. The results of transmission microscope showed that ICA could reduce apoptosis of PC12 cells caused by ERS. The results of Western blot and q-PCR showed that ICA could inhibit ERS by down-regulating the expression of CHOP and Grp78.ConclusionsICA can inhibit ERS and promote the repair of PC12 cells by down-regulating the expression of CHOP and Grp78. ICA has the potential to promote the recovery of spinal cord injury.

Endoplasmic Reticulum Stress Is Involved in Glucocorticoid-Induced Apoptosis in PC12 Cells.

Objective The present study selected PC12 cells to construct a neuronal injury model induced by glucocorticoids (GC) in vitro, aiming to explore whether the endoplasmic reticulum stress (ERS) PKR-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP-homologous protein (CHOP) and inositol requirement 1 (IRE1)-apoptosis signal regulating kinase 1 (ASK1)-C-Jun amino-terminal kinase (JNK) signaling pathways are associated with the neuronal injury process induced by GC and provide morphological evidence. Methods Cell models with different doses and different durations of GC exposure were established. The viability of PC12 cells was detected by the CCK-8 assay, and the apoptosis rate of PC12 cells was detected by the flow cytometry assay. The expression of microtubule-associated protein 2 (Map2); glucocorticoids receptor (GR); cellular oncogene fos (C-fos); and ERS-related proteins, glucose-regulated protein 78 (GRP78), p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP, was observed by immunofluorescence staining. Results The results of immunofluorescence staining showed that PC12 cells abundantly expressed Map2 and GR. The CCK-8 assay revealed that high-concentration GC exposure significantly inhibited the cell viability of PC12 cells. The flow cytometry assay indicated that high-concentration GC exposure significantly increased the apoptosis rate of PC12 cells. Immunofluorescence staining showed that GC exposure significantly increased the expression of C-fos, GRP78, p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP. Treatment with ERS inhibitor 4-phenylbutyric acid (4-PBA) and GR inhibitor RU38486 attenuated related damage and downregulated the expression of the abovementioned proteins. Conclusion High-concentration GC exposure can significantly inhibit the viability of PC12 cells and induce apoptosis. PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways are involved in the above damage process.

The orphan nuclear receptor Nur77 plays a vital role in BPA-induced PC12 cell apoptosis

Bisphenol A (BPA) is a typical environmental endocrine disruptor that can migrate into organisms through skin contact, breathing, diet and various other approaches. The reproductive toxicity and neurotoxicity of BPA has been confirmed by several toxicological studies. However, the neurotoxicity of BPA is still controversial. In the present study, we used PC12 cells as a model to investigate the mechanism of BPA-induced neuronal apoptosis. BPA exposure reduced cell viability, altered cell morphology and aggravated intracellular Lactate dehydrogenase (LDH) release, intracellular Ca2+ concentration, Reactive oxygen species (ROS) levels, apoptosis and the reduction in the mitochondrial transmembrane potential (ΔΨm). Moreover, the results of the Western blot (WB) and Real-time quantitative polymerase chain reaction (RT-qPCR) assays indicated that the expression levels of Nur77 in the BPA group were down-regulated and accompanied by the downregulation of the NF-κb/Bcl-2 proteins and the upregulation of cleaved-caspase 3, which is a marker of apoptosis. However, these changes were significantly reversed with the upregulation of the Nur77 protein by introducing plasmids carrying the nur77 gene. These results indicated that BPA-induced apoptosis was closely related to Nur77-mediated inhibition of the NF-κb/Bcl-2 pathway.

Longxuetongluo Capsule protects against cerebral ischemia/reperfusion injury through endoplasmic reticulum stress and MAPK-mediated mechanisms

IntroductionLongxuetongluo Capsule (LTC) is wildly applied to treat ischemic stroke in clinical practice in China. However, the pharmacological mechanism of LTC on ischemic stroke is still unstated. ObjectiveOur research was designed to study the protective effect of LTC against cerebral ischemia–reperfusion (I/R) injury and reveal the underlying mechanism both in vivo and in vitro. MethodsPC12 cells treated with glucose deprivation/reperfusion (OGD/R) were used to simulate in vitro ischemia/reperfusion (I/R) injury. The cell viability, apoptosis rate, and protein expressions of PC12 cells were evaluated. In vivo validation of the protective effect of LTC was carried out by middle cerebral artery occlusion (MCAO)/reperfusion treatment, and the underlying mechanism of its anti-apoptosis ability was further revealed by immunohistochemistry staining and Western blotting. ResultsIn the current study, we observed that LTC effectively inhibited oxygen-glucose deprivation/reperfusion (OGD/R) induced apoptosis of PC12 cells through suppressing the cleavage of poly ADP-ribose polymerase (PARP), caspase-3, and caspase-9. Further investigation revealed that OGD/R insult remarkably triggered the endoplasmic reticulum stress responses (ER stress) to induce PC12 cell apoptosis. LTC treatment alleviated OGD/R induced ER stress by inhibiting the activation of protein kinase RNA (PKR)-like ER kinase (PERK)/eukaryotic translation initiation factor 2 (eIF2α) and inositol requiring enzyme 1 (IRE1)/tumor necrosis factor receptor-associated factor 2 (TRAF2) pathways. Additionally, LTC also restrained the OGD/R-induced PC12 cell apoptosis by reversing the activated mitogen-activated protein kinase (MAPK) through IRE1/TRAF2 pathway. Animal studies demonstrated LTC significantly restricted the infarct region induced by middle cerebral artery occlusion (MCAO)/reperfusion, the activation of ER stress and apoptosis of neuronal cells had also been suppressed by LTC in the penumbra region. ConclusionLTC protects the cerebral neuronal cell against ischemia/reperfusion injury through ER stress and MAPK-mediated mechanisms.

LncRNA SNHG15 Knockdown Protects Against OGD/R-Induced Neuron Injury by Downregulating TP53INP1 Expression via Binding to miR-455-3p.

Cerebral ischemia-reperfusion (I/R) injury is the common symptom of ischemic stroke, which poses a heavy burden to human health. Long non-coding RNA (lncRNA) is indicated to be a critical regulator in cerebral ischemia. This study aims to reveal the effects of lncRNA small nucleolar RNA host gene 15 (SNHG15) on oxygen-glucose deprivation and reoxygenation (OGD/R)-induced neuron injury and underlying mechanism. The expression levels of SNHG15, microRNA-455-3p (miR-455-3p) and tumour protein p53 inducible nuclear protein 1 (TP53INP1) mRNA were determined by quantitative real time polymerase chain reaction in P12 cells. The protein levels of TP53INP1, cleaved caspase-3, caspase-3, B-cell lymphoma-2 and BCL2-associated x protein (Bax) were detected by western blot in P12 cells. Cell viability and apoptosis were revealed by cell counting kit-8 assay and flow cytometry analysis, respectively, in P12 cells. Caspase-3 activity, the levels of tumor necrosis factor-α and interleukin-1β and the production of reactive oxygen species (ROS) were severally determined by caspase-3 activity assay, Enzyme-linked immunosorbent assay and ROS detection assay in P12 cells. The binding relationship between miR-455-3p and SNHG15 or TP53INP1 was predicted by starbase online database, and identified by dual-luciferase reporter, RNA pull-down or RNA immunoprecipitation assay. SNHG15 expression and the mRNA and protein levels of TP53INP1 were dramatically upregulated, while miR-455-3p expression was apparently downregulated in OGD/R-induced PC12 cells. SNHG15 silencing hindered the effects of OGD/R treatment on cell viability, apoptosis, inflammation and oxidative in PC12 cells; however, these impacts were restored after miR-455-3p inhibitor transfection. Additionally, SNHG15 acted as a sponge of miR-455-3p and miR-455-3p bound to TP53INP1. SNHG15 contributed to OGD/R-induced neuron injury by regulating miR-455-3p/TP53INP1 axis, which provided a novel insight to study lncRNA-directed therapy in ischemia stoke.

Mechanism of Action of Xiaoyao San in Treatment of Ischemic Stroke is Related to Anti-Apoptosis and Activation of PI3K/Akt Pathway.

ObjectiveThe traditional Chinese medicine (TCM) formulation Xiaoyao San (XYS) has a good clinical effect in treating ischemic stroke (IS). We explored the mechanism and material basis of XYS in IS treatment.MethodsNetwork pharmacology was used to construct a network of XYS components and IS targets. R software was used to analyze the biological process and pathway analysis of the targets of XYS in IS treatment. In vitro, a model of apoptosis of PC12 cells induced by oxygen-glucose deprivation/reperfusion (OGD/R) was established to evaluate the neuroprotective effect of XYS and its influence on the expression of apoptotic protein-related genes. The affinity between the potentially active compounds in XYS and apoptotic proteins was evaluated by molecular docking.ResultsXYS was shown to have 136 chemical components that exert potential anti-IS activity by acting on 175 proteins. Bioinformatics analysis showed that apoptosis and the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) signaling pathway were the main signaling pathways of XYS. In vitro experiments showed that XYS could improve the effect of OGD/R on PC12-cell activity (EC50 = 0.43 mg/mL) and inhibit apoptosis. The main mechanisms were related to the improvement of oxidative stress and regulation of apoptosis-related gene expression. Molecular docking showed that C22, C102 and other components in XYS had a strong affinity with apoptosis-related proteins.ConclusionNetwork pharmacology, in vitro experiments, and molecular docking were used, for the first time, to study the material basis and molecular mechanism of XYS in IS treatment from the perspective of multiple targets and multiple pathways. We provided a new approach for the future study of TCM formulations in the treatment of complex diseases.

Knockdown of SNHG1 alleviates autophagy and apoptosis by regulating miR-362-3p/Jak2/stat3 pathway in LPS-injured PC12 cells.

Spinal cord injury (SCI) is a serious neurological disease. Long non-coding RNA (lncRNA) small nucleolar RNA host gene (SNHG1) and microRNA-362-3p (miR-362-3p) were confirmed to be related to neurological disorders. However, it is unclear whether SNHG1 was involved in the development of SCI via regulating miR-362-3p. PC12 cells were treated with lipopolysaccharide (LPS) to imitate the in vitro cell model of SCI. Cell ciability and apoptosis rate were detected by cell counting kit-8 (CCK-8) assay and flow cytometry assay. The levels of SNHG1, miR-362-3p, and Janus kinase-2 (Jak2) were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between miR-362-3p and SNHG1 or Jak2. Besides, the levels of apoptosis- and autophagy- related proteins were detected by western blot assay. In present research, LPS suppressed cell viability, and induced apoptosis and autophagy in PC12 cells. SNHG1 knockdown could affect cell viability, and suppress cell apoptosis and autophagy in LPS-treated PC12 cells. Moreover, miR-362-3p was a target of SNHG1, miR-362-3p targeted Jak2 and negatively regulated Jak2/stat3 pathway. Our data also demonstrated that SNHG1 depletion inactivated Jak2/stat3 pathway to affect cell viability and confine apoptosis, autophagy in LPS-treated PC12 cells. Taken together, SNHG1 regulated cell viability, apoptosis and autophagy in LPS-treated PC12 cells by activating Jak2/stat3 pathway via sponging miR-362-3p.

MicroRNA-137-mediated lysine demethylase 4A regulates the recovery of spinal cord injury via the SFRP4-Wnt/β-Catenin axis

Objective Spinal cord injury (SCI) causes great harm to the normal life of patients. Histone demethylase is involved in many biological processes, including SCI. Hence, this study explored the role and mechanism of histone lysine demethylase 4A (KDM4A) in SCI. Methods The acute SCI (ASCI) rat model was established after spinal compression and the SCI neuronal model was induced via treating PC12 cells with lipopolysaccharide (LPS). KDM4A expression during SCI was detected. The microRNA (miRNA) targeting KDM4A was predicted and verified. The miRNA and KDM4A expression patterns were intervened in LPS-stimulated PC12 cells to evaluate their combined effects on neuronal cells in SCI. The downstream pathways of KDM4A were predicted, and SFRP4 and H3K9me3 expressions were determined. After the intervention of SFRP4 in LPS-treated cells, β-Catenin expression and the effect of SFRP4 on neuronal cells in SCI were detected. Finally, the effectiveness of the miR-137/KDM4A/SFRP4/Wnt/β-Catenin axis was verified in vivo. Results KDM4A was abnormally elevated in SCI. miR-137 targeted KDM4A. miR-137 effectively inhibited the apoptosis of LPS-challenged PC12 cells, which could be reversed after overexpressing KDM4A. KDM4A promoted SFRP4 expression through demethylation of H3K9me3. Overexpression of SFRP4 blocked the Wnt/β-Catenin pathway and promoted apoptosis of LPS-stimulated cells. In vivo, miR-137 overexpression remarkably improved SCI symptoms, accompanied by obviously increased β-Catenin expression and notably decreased KDM4A and SFRP4 expressions, while overexpressed KDM4A treatment showed the opposite trend in the presence of miR-137. Conclusion We demonstrated that miR-137 targeted KDM4A and then downregulated SFRP4 to ameliorate SCI in a Wnt/β-Catenin-dependent manner.

Lycibarbarines A-C, Three Tetrahydroquinoline Alkaloids Possessing a Spiro-Heterocycle Moiety from the Fruits of Lycium barbarum.

Three tetrahydroquinoline alkaloids, lycibarbarines A-C (1-3), possessing a unique tetracyclic tetrahydroquinoline-oxazine-ketohexoside fused motif, were isolated from the fruits of Lycium barbarum. Their structures were determined by spectroscopic analysis and quantum-chemical calculations. Compounds 1 and 3 exhibited neuroprotective activity when evaluated for corticosterone-induced injury by reducing the apoptosis of PC12 cells through the inhibition of caspase-3 and caspase-9.

Neuroprotective effects of maize tetrapeptide-anchored gold nanoparticles in Alzheimer’s disease

Nanopeptide assembled from peptide-anchored nanoparticles possess an enormous research potential in the field of cellular medicine and disease treatment. The aim of this study was to explore the neuroprotective effects of maize tetrapeptide anchored gold nanoparticles against l-glutamic acid-induced PC12 cell apoptosis and a murine Alzheimer’s disease model induced by aluminum chloride and d-galactose. The results revealed that the nanopeptide antioxidant inhibited intracellular ROS accumulation and promoted cell differentiation than that of maize bioactive tetrapeptide. Compared with untreated Alzheimer’s disease model mice, nanopeptide administration shortened the escape latency time in a water maze test and improved the movements in the autonomic activity test. After 16 days of nanopeptide administration, the central cholinergic system function of acetylcholine and cholineacetyltransferase were enhanced, and the level of acetylcholinesterase was reduced. It also increased superoxide dismutase and glutathione peroxidase activity in sera and hypothalami. Moreover, nanopeptide treatment upregulated cerebral nuclear factor erythroid 2-related factor 2 and heme-oxygenase-1 and downregulated kelch-like ECH-associated protein 1 relative to untreated Alzheimer’s disease model mice. Thus, the novel nanopeptide is expected to be used as the neuroprotective agent to prevent Alzheimer's disease.

NF-κB 1-induced LINC00665 regulates inflammation and apoptosis of neurons caused by spinal cord injury by targeting miR-34a-5p

ABSTRACT Background: Spinal cord injury (SCI) has high disability rate and low cure rate, which frustrates the patients and brings a heavy burden to their families. This study aimed to explore whether NF-κB1 could induce the expression of LINC00665 and form a feedback loop with miR-34a-5p to regulate inflammation and apoptosis of neurons. Results: Basso, Beattie, and Bresnahan (BBB) scoring was decreased, damage for spinal cord tissue was aggravated and neuron number was decreased in SCI rats. The levels of TNF-α, IL-1β and IL-6 in serum and the expression of LINC00665 and NF-κB1 in spinal cord tissues were all increased in SCI rats. After LPS induction, PC12 cell viability was decreased. The expression of LINC00665 and NF-κB1 in LPS-induced PC12 cells was increased, which was partially reversed by BAY11-7082 (NF-κB inhibitor). Inhibition of LINC00665 improved cell viability, suppressed apoptosis and inflammation and down-regulated the NF-κB1 expression in LPS-induced PC12 cells. Furthermore, miR-34a-5p expression was decreased in LPS-induced PC12 cells, which could be promoted by inhibition of LINC00665. miR-34a-5p inhibitor restrained the effect of inhibition of LINC00665 on NF-κB1 expression in LPS-induced PC12 cells. Conclusion: inhibition of LINC00665 improved cell viability, suppressed apoptosis and inflammation in LPS-induced PC12 cells, and the NF-κB1/LINC00665/miR-34a-5ploop might be a useful therapeutic target in SCI treatment.

Protective effects of ajwain (Trachyspermum ammi L.) extract against cadmium-induced cytotoxicity and apoptosis in PC12 cells

Cadmium (Cd2+) is a heavy metal that can induce cytotoxicity leading to many chronic diseases. Ajwain (Trachyspermum ammi L.) is a popular spice with diverse pharmacological properties. It is used for the remedy of many pathological conditions that could be manifested by Cd2+ exposure e.g. inflammatory, toxic, neurological, genital or respiratory tract disorders. To reduce the Cd2+-induced cytotoxicity and apoptosis, PC12 cells were exposed/co-exposed to Cd2+ (5 or 10 μM) with/without non-toxic dose (240 μg/mL) of ethanolic extract of ajwain (AE) for 24 h. The cytotoxicity and apoptosis were evaluated by cell viability, lactate dehydrogenase (LDH) activity, glutathione levels, genomic DNA fragmentation and expressions of Bax, Bcl-2, Bcl-xL, NF-КB, cytosolic cytochrome c and caspase-3. Cd2+ reduced the cell viability significantly 24 h after exposure; however, the co-exposure of AE with Cd2+ reduced the cell death. The co-exposure also raised up glutathione levels, and decreased LDH activity. AE lessened the DNA fragmentation caused by Cd2+. AE suppressed the Cd2+-induced increased expression of apoptotic protein Bax, and promoted suppressed expressions of anti-apoptotic proteins Bcl-2, Bcl-xL and NF-КB. Moreover, it reduced the cytosolic cytochrome c levels and the caspase-3 expressions increased by Cd2+. Thus, it was suggested that AE reduced cytotoxicity and apoptosis caused by Cd2+ in PC12 cells. It inhibited Cd2+-induced apoptosis through intrinsic pathway possibly boosting up the antioxidant defense. Therefore, AE can be a candidate for a potential agent against heavy metals and/or other toxicants. Additionally, this study validated the ethno-pharmacological therapeutic uses of ajwain in various pathological conditions.

Norwogonin attenuates hypoxia-induced oxidative stress and apoptosis in PC12 cells

BackgroundNorwogonin is a natural flavone with three phenolic hydroxyl groups in skeletal structure and has excellent antioxidant activity. However, the neuroprotective effect of norwogonin remains unclear. Here, we investigated the protective capacity of norwogonin against oxidative damage elicited by hypoxia in PC12 cells.MethodsThe cell viability and apoptosis were examined by MTT assay and Annexin V-FITC/PI staining, respectively. Reactive oxygen species (ROS) content was measured using DCFH-DA assay. Lactate dehydrogenase (LDH), malondialdehyde (MDA) and antioxidant enzyme levels were determined using commercial kits. The expression of related genes and proteins was measured by real-time quantitative PCR and Western blotting, respectively.ResultsWe found that norwogonin alleviated hypoxia-induced injury in PC12 cells by increasing the cell viability, reducing LDH release, and ameliorating the changes of cell morphology. Norwogonin also acted as an antioxidant by scavenging ROS, reducing MDA production, maintaining the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and decreasing the expression levels of HIF-1α and VEGF. In addition, norwogonin prevented cell apoptosis via inhibiting the expression levels of caspase-3, cytochrome c and Bax, while increasing the expression levels of Bcl-2 and the ratio of Bcl-2/Bax.ConclusionsNorwogonin attenuates hypoxia-induced injury in PC12 cells by quenching ROS, maintaining the activities of antioxidant enzymes, and inhibiting mitochondrial apoptosis pathway.

Propofol protects PC12 cells from cobalt chloride-induced injury by mediating miR-134.

Propofol (PRO) was reported to exert a neuroprotective effect by decreasing microRNA-134 (miR-134), a brain-specific miRNA, thus, the role of PRO against cobalt chloride (CoCl₂)-induced injury in rat pheochromocytoma cells (PC12) via mediating miR-134 was explored. CoCl₂-induced PC12 cells treated with PRO were transfected with or without miR-134 negative control (NC)/ inhibitor/mimic, and the following detections were then performed using cell counting kit-8 (CCK-8), Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) and Hoechst 33258 staining. Autophagy was observed by transmission electron microscope (TEM). Mitochondrial membrane potential (MMP) was detected by Rhodamine-123 (Rh123) staining, and reactive oxygen species (ROS) by dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. Protein and gene expressions were measured by Western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), respectively. PRO reversed the CoCl₂-induced decrease in the PC12 cell viability and increased miR-134 in a dose-dependent manner. CoCl₂ increased LC3II/I ratio and Beclin-1 expression, but decreased p62 expression, which was abolished by PRO. In addition, an increased cell apoptosis rates triggered by CoCl₂ were reduced by PRO with the down-regulations of Bax and Caspase-3 and the up-regulation of Bcl-2. Furthermore, PRO decreased methylenedioxyamphetamine (MDA), nitric oxide (NO) and ROS in CoCl₂-induced PC12 cells accompanying the increase in glutathione peroxidase (GSH-Px) and MMP. The effects of PRO on autophagy, apoptosis and oxidative stress in CoCl₂-induced PC12 cell were reversed by miR-134 mimic. PRO may mitigate CoCl₂-induced autophagy in PC12 cells with decreased apoptosis and improved oxidative stress via mediating miR-134.

Silenced lncRNA H19 and up-regulated microRNA-129 accelerates viability and restrains apoptosis of PC12 cells induced by Aβ25-35 in a cellular model of Alzheimer’s disease

ABSTRACT Accumulating data manifest that long non-coding RNA (lncRNAs) are involved in all kinds of neurodegenerative disorders, consisting of the onset and progression of Alzheimer’s disease (AD). The study was for the research of the mechanism of lncRNA H19 (H19) in viability and apoptosis of PC12 cells induced by Aβ25-35 in a cellular model of AD with the regulation of microRNA (miR)-129 and high mobility group box-1 protein (HMGB1). An AD cellular model of PC12 cells was established using Aβ25-35. The Aβ25-35-induced PC12 cells were transfected with si-H19 or miR-129 mimic to figure their roles in cell viability,apoptosis, mitochondrial membrane potential dysfunction and oxidative stress in AD. Luciferase reporter assay and RNA-pull down assay were employed for verification of the binding relationship between H19 and miR-129 and the targeting relationship between miR-129 and HMGB1. An AD mouse model was induced and brain tissues were collected. H19, miR-129 and HMGB1 were detected in Aβ25-35-treated cells and brain tissues of AD mice. Elevated H19, HMGB1 and decreased miR-129 were found in Aβ25-35-treated PC12 cells as well as in brain tissues of AD mice. Silenced H19 or elevated miR-129 promoted viability, inhibited apoptosis, prevented mitochondrial membrane potential dysfunction and decreased oxidative stress in Aβ25-35-treated PC12 cells. H19 could specifically bind to miR-129. MiR-129 specifically suppressed HMGB1 expression. This study suggests that silenced H19 and up-regulated miR-129 accelerates viability and represses apoptosis of PC12 cells stimulated by Aβ25-35 in AD, which is beneficial for AD treatment.

Antiepileptic Effects of Cicadae Periostracum on Mice and Its Antiapoptotic Effects in H2O2-Stimulated PC12 Cells via Regulation of PI3K/Akt/Nrf2 Signaling Pathways.

Cicadae Periostracum (CPM), a commonly used animal traditional Chinese medicine (TCM), possesses antifebrile, spasmolytic, antiasthmatic, and antiphlogistic effects. In our present paper, we aimed to systemically investigate the antiepileptic effects of CPM in epileptic mice and explore the related molecular mechanism. Pentylenetetrazole- (PTZ) and strychnine-induced convulsion mice were established, and the results showed CPM could prolong the latency of convulsion and death and improve the neuronal damage in the hippocampus of PTZ-induced mice. Furthermore, the H2O2-treated PC12 cells were prepared to explore the possible mechanisms for the antiepileptic effects of CPM. CCK-8 results showed that CPM significantly improved the cell viability of H2O2-treated PC12 cells. Results of the acridine orange- (AO-) ethidium bromide (EB) staining, cell mitochondrial membrane potential (MOMP) analysis, and flow cytometry analysis showed that CPM significantly suppressed the H2O2-induced apoptosis in PC12 cells. In addition, CPM also downregulated the proapoptosis proteins, including Bax, cleaved- (C-) caspase-3, and C-caspase-9, and upregulated Bcl-2. Furthermore, CPM reduced the reactive oxygen species (ROS) levels via increasing antioxidative enzyme activities, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Importantly, CPM could increase the phosphorylation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) in H2O2-induced PC12 cells and can promote the nuclear transfer of the nuclear factor E2-related factor 2 (Nrf2) and increase the expression of heme oxygenase-1 (HO-1) in the cytoplasm. In conclusion, our present study suggested CPM possessed antiepileptic effects through antiapoptosis of neuron cells via regulation of the PI3K/Akt/Nrf2 signaling pathway.