Neuronal PC12 Cells Research Articles

Size, Surface Properties, and Ion Release of Zinc Oxide Nanoparticles: Effects on Cytotoxicity, Dopaminergic Gene Expression, and Acetylcholinesterase Inhibition in Neuronal PC-12 Cells.

The extensive applications of zinc oxidenanoparticles (ZnO NPs) have resulted in a substantial risk of human exposure. However, the knowledge of the toxicity of these NPs in the nervous system is still limited. A comparative analysis of ZnO NPs of various sizes and NPs of the same size, with and without surface coating, and the potential role of released zinc ions is yet to be thoroughly explored. As a result, we have studied the cellular toxicity of two different-sized ZnO NPs, ZnO-22 (22nm) and ZnO-43 (43nm), and NPs with similar size but with polyvinylpyrrolidone coating (ZnO-P, 45nm). The findings from our study suggested a time-, size-, and surface coating-dependent cytotoxicity in PC-12 cells at a concentration ≥ 10μg/ml. ZnO NP treatment significantly elevated reactive oxygen and reactive nitrogen species, thereby increasing oxidative stress. The exposure of ZnO-22 and ZnO-43 significantly upregulated the expression of monoamine oxidase-A and downregulated the α-synuclein gene expression associated with the dopaminergic system. The interaction of NPs enzymes in the nervous system is also hazardous. Therefore, the inhibition activity of acetylcholinesterase enzyme was also studied for its interaction with these NPs, and the results indicated a dose-dependent inhibition of enzyme activity. Particle size, coating, and cellular interactions modulate ZnO NP's cytotoxicity; smaller sizes enhance cellular uptake and reactivity, while coating reduces cytotoxicity by limiting direct cell contact and potentially mitigating oxidative stress. Furthermore, the study of released zinc ions from the NPs suggested no significant contribution to the observed cytotoxicity compared to the NPs.

Design and synthesis of 9-phenanthranilamide derivatives and the study of anti-inflammatory, antioxidant and neuroprotective activities

Oxidative stress and a series of excessive inflammatory responses are major obstacles to neurological functional recovery after ischemic stroke. In this study, we synthesized several novel 9-phenanthranilamide derivatives and evaluated their anti-inflammatory and antioxidant activities. Among the initially screened compounds, most could strongly inhibi lipopolysaccharide (LPS)-stimulated production of IL-1β, IL-6 and TNF-α in microglial cells. Additionally, compounds 8b, 8q, 8r and 8s significantly inhibited the production of NO, and they also had dose-dependent protective effects on PC12 neuronal cells induced by H2O2. The antineuroinflammatory effects of 8r and 8s were associated with the downregulation of LPS-induced inflammatory mediators of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and both compounds inhibited the NF-κB signaling pathway. Further examinations showed that 8s had a significant neuroprotective effect on rats with middle cerebral artery occlusion (MCAO). It decreased the infarct volume and the neurological deficit score. Overall, our results suggested that compound 8s might be a promising agent for stroke treatment.

Based on spinal central sensitization creating analgesic screening approach to excavate anti-neuropathic pain ingredients of Corydalis yanhusuo W.T.Wang

Ethnopharmacological relevanceCorydalis Rhizome (RC) as a traditional analgesic Chinese medicine is the dried tuber of Corydalis yanhusuo W.T.Wang. Many efforts have revealed that RC could effectively alleviate neuropathic pain, while its active ingredients in neuropathic pain are still not clear. Aim of the studySpinal central sensitization contributes greatly to neuropathic pain, and neuron, astrocyte and microglia play important roles in spinal central sensitization. The aim of the present study is to excavate active compounds in RC regulating spinal central sensitization to inhibit neuropathic pain. Materials and methodsImmunofluorescence and western blotting were used to determine protein expression levels. Gene expression levels were detected by RT-PCR. PC12 neuronal cells, C6 astrocyte cells, and BV2 microglia cells were cultured for in vitro studies. Targeting multi types of cells extraction combined with HPLC-Q-TOF-MS/MS was established to identify components binding to above cells. Animal studies were used to verify the analgesic activities of components. ResultsTotal alkaloids of RC (RC-TA) significantly relieved neuropathic pain in chronic constriction injury (CCI) rats and repressed spinal central sensitization. Eight components of RC-TA were found to bind to PC12, C6, or BV2 cells. They could respectively suppress the activation of cells in vitro and alleviate CCI-induced neuropathic pain, among which glaucine and dehydrocorydaline induced antinociception was stronger than l-THP. Meanwhile, glaucine had no effect on acute or chronic inflammatory pain, and its antinociception in neuropathic pain could be abolished by dopamine D1 receptor agonist. ConclusionsEmploying multi types of cells based on spinal central sensitization rather than single cell may allow for more thorough excavation of active substances. Glaucine was firstly found could attenuate neuropathic pain but not other types of pain which indicated that different alkaloids in RC exert distinct analgesic effects on different pain models, and gluacine has the potential to be developed as an analgesic drug specifically for neuropathic pain relieving.

BRAP silencing protects against neuronal inflammation, oxidative stress and apoptosis in cerebral ischemia-reperfusion injury by promoting PON1 expression.

BRCA1 associated protein (BRAP) participates in the regulation of myocardial infarction and atherosclerosis. But the function of BRAP in cerebral ischemia-reperfusion (CIR) injury has not been elucidated yet. BRAP expression in PC12 cells in response to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment was examined with Western blot assay. PC12 cells underwent OGD/R-treatment and were subsequently transfected with pcDNA-BRAP or sh-BRAP, followed by determination of viability, lactate dehydrogenase (LDH) production, apoptosis, inflammatory cytokine secretion, and oxidative stress marker protein levels. Paraoxonase 1 (PON1) promoter methylation was evaluated with methylation-specific PCR assay. the effect of BRAP/PON1 axis on CIR injury was investigated by rescue experiments. Additionally, sh-BRAP was injected into a middle cerebral artery occlusion (MCAO) rat model, and the changes of neurological damage were evaluated. BRAP overexpression exacerbated OGD/R-induced viability reduction, LDH production, apoptosis, inflammatory cytokine secretion and oxidative stress in PC12 neuronal cells. In contrast, BRAP silencing showed the opposite results. Mechanistically, BRAP reduced PON1 expression by promoting DNA methyl transferase1 (DNMT1)-mediated PON1 promoter methylation. PON1 silencing reversed BRAP-mediated neuroprotection. Additionally, BRAP silencing alleviated CIR-induced neurological damage in MCAO rats. BRAP silencing suppressed OGD/R-induced neuronal apoptosis, inflammation, and oxidative stress, and alleviated CIR-induced neurological damage in MCAO rats through facilitating PON1 expression.

3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) is protective against Aβ42-induced toxicity in vitro and in an Alzheimer's Disease Drosophila.

Alzheimer's disease (AD) is the most prevalent type of dementia globally. The accumulation of amyloid-beta (Aβ) extracellular senile plaques in the brain is one of the hallmark mechanisms found in AD. Aβ42 is the most damaging and aggressively aggregating Aβ isomer produced in the brain. Although Aβ42 has been extensively researched as a crucial peptide connected to the development of the characteristic amyloid fibrils in AD, the specifics of its pathophysiology are still unknown. Therefore, the main objective was to identify novel compounds that could potentially mitigate the negative effects of Aβ42. 3-[[(3S)-1,2,3,4-Tetrahydroisoquinoline-3-carbonyl]amino]propanoic acid (THICAPA) was identified as a ligand for Aβ42 and for reducing fibrillary Aβ42 aggregation. THICAPA also improved cell viability when administered to PC12 neuronal cells that were exposed to Aβ42. Additionally, this compound diminished Aβ42 toxicity in the current AD Drosophila model by rescuing the rough eye phenotype, prolonging the lifespan and enhancing motor functions. Through Next-generation RNA-sequencing, immune response pathways were downregulated in response to THICAPA treatment. Thus, this study suggests THICAPA as a possible disease-modifying treatment for AD.

P-Coumaric acid attenuates the effects of Aβ42 in vitro and in a Drosophila Alzheimer’s disease model

Alzheimer's disease (AD) is the most common neurodegenerative condition in civilizations worldwide. The distinctive occurrence of amyloid-beta (Aβ) accumulation into insoluble fibrils is part of the disease pathophysiology with Aβ42 being the most toxic and aggressive Aβ species. The polyphenol, p-Coumaric acid (pCA), has been known to boost a number of therapeutic benefits. Here, pCA's potential to counteract the negative effects of Aβ42 was investigated. First, pCA was confirmed to reduce Aβ42 fibrillation using an in vitro activity assay. The compound was next examined on Aβ42-exposed PC12 neuronal cells and was found to significantly decrease Aβ42-induced cell mortality. pCA was then examined using an AD Drosophila melanogaster model. Feeding of pCA partially reversed the rough eye phenotype, significantly lengthened AD Drosophila's lifespan, and significantly enhanced the majority of the AD Drosophila’s mobility in a sex-dependent manner. The findings of this study suggest that pCA may have therapeutic benefits for AD.

Peptide fraction from B. jararaca snake venom protects against oxidative stress-induced changes in neuronal PC12 cell but not in astrocyte-like C6 cell

Venom-derived proteins and peptides have prevented neuronal cell loss, damage, and death in the study of neurodegenerative disorders. The cytoprotective effects of the peptide fraction (PF) from Bothrops jararaca snake venom were evaluated against oxidative stress changes in neuronal PC12 cells and astrocyte-like C6 cells. PC12 and C6 cells were pre-treated for 4 h with different concentrations of PF, and then H2O2 was added (0.5 mM in PC12 cells; 0.4 mM in C6 cells) and incubated for 20 h more. In PC12 cells, PF at 0.78 μg mL−1 increased viability (113.6 ± 6.3%) and metabolism (96.3 ± 10.3%) cell against H2O2-induced neurotoxicity (75.6 ± 5.8%; 66.5 ± 3.3%, respectively), reducing oxidative stress markers such as ROS generation, NO production, and arginase indirect activity through urea synthesis. Despite that, PF showed no cytoprotective effects in C6 cells, but potentiated the H2O2-induced damage at a concentration lower than 0.07 μg mL−1. Furthermore, the role of metabolites derived from L-arginine metabolism was verified in PF-mediated neuroprotection in PC12 cells, using specific inhibitors of two of the key enzymes in the L-arginine metabolic pathway: the α-Methyl-DL-aspartic acid (MDLA) to argininosuccinate synthetase (AsS), responsible for the recycling of L-citrulline to L-arginine; and, L-NΩ-Nitroarginine methyl ester (L-Name) to nitric oxide synthase (NOS), which catalyzes the synthesis of NO from L-arginine. The inhibition of AsS and NOS suppressed PF-mediated cytoprotection against oxidative stress, indicating that its mechanism is dependent on the production pathway of L-arginine metabolites such as NO and, more importantly, polyamines from ornithine metabolism, which are involved in the neuroprotection mechanism described in the literature. Overall, this work provides novel opportunities for evaluating whether the neuroprotective properties of PF shown in particular neuronal cells are sustained and for exploring potential drug development pathways for the treatment of neurodegenerative diseases.

3D culture of fibroblasts and neuronal cells on microfabricated free-floating carriers

3D cell culture is a relatively recent trend in biomedical research for artificially mimicking in vivo environment and providing three dimensions for the cells to grow in vitro, particularly with regard to surface-adherent mammalian cells. Different cells and research objectives require different culture conditions which has led to an increase in the diversity of 3D cell culture models. In this study, we show two independent on-carrier 3D cell culture models aimed at two different potential applications. Firstly, micron-scale porous spherical structures fabricated from poly (lactic-co-glycolic acid) or PLGA are used as 3D cell carriers so that the cells do not lose their physiologically relevant spherical shape. Secondly, millimetre-scale silk fibroin structures fabricated by 3D inkjet bioprinting are used as 3D cell carriers to demonstrate cell growth patterning in 3D for use in applications which require directed cell growth. The L929 fibroblasts demonstrated excellent adherence, cell-division and proliferation on the PLGA carriers, while the PC12 neuronal cells showed excellent adherence, proliferation and spread on the fibroin carriers without any evidence of cytotoxicity from the carriers. The present study thus proposes two models for 3D cell culture and demonstrates, firstly, that easily fabricable porous PLGA structures can act as excellent cell carriers for aiding cells easily retain their physiologically relevant 3D spherical shape in vitro, and secondly, that 3D inkjet printed silk fibroin structures can act as geometrically-shaped carriers for 3D cell patterning or directed cell growth in vitro. While the ‘fibroblasts on PLGA carriers’ model will help achieve more accurate results than the conventional 2D culture in cell research, such as drug discovery, and cell proliferation for adoptive cell transfer, such as stem cell therapy, the ‘neuronal cells on silk fibroin carriers’ model will help in research requiring patterned cell growth, such as treatment of neuropathies.

The Flavonoid Agathisflavone Directs Brain Microglia/Macrophages to a Neuroprotective Anti-Inflammatory and Antioxidant State via Regulation of NLRP3 Inflammasome.

Agathisflavone, purified from Cenostigma pyramidale (Tul.) has been shown to be neuroprotective in in vitro models of glutamate-induced excitotoxicity and inflammatory damage. However, the potential role of microglial regulation by agathisflavone in these neuroprotective effects is unclear. Here we investigated the effects of agathisflavone in microglia submitted to inflammatory stimulus in view of elucidating mechanisms of neuroprotection. Microglia isolated from cortices of newborn Wistar rats were exposed to Escherichia coli lipopolysaccharide (LPS, 1 µg/mL) and treated or not with agathisflavone (1 µM). Neuronal PC12 cells were exposed to a conditioned medium from microglia (MCM) treated or not with agathisflavone. We observed that LPS induced microglia to assume an activated inflammatory state (increased CD68, more rounded/amoeboid phenotype). However, most microglia exposed to LPS and agathisflavone, presented an anti-inflammatory profile (increased CD206 and branched-phenotype), associated with the reduction in NO, GSH mRNA for NRLP3 inflammasome, IL1-β, IL-6, IL-18, TNF, CCL5, and CCL2. Molecular docking also showed that agathisflavone bound at the NLRP3 NACTH inhibitory domain. Moreover, in PC12 cell cultures exposed to the MCM previously treated with the flavonoid most cells preserved neurites and increased expression of β-tubulin III. Thus, these data reinforce the anti-inflammatory activity and the neuroprotective effect of agathisflavone, effects associated with the control of NLRP3 inflammasome, standing out it as a promising molecule for the treatment or prevention of neurodegenerative diseases.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Abstract 3042: BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy

Abstract Background: Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating side effect associated with many antineoplastic chemotherapeutic agents including cytotoxic and targeted agents. It significantly impacts cancer patients’ quality of life during treatment, and may cause lasting neuropathy and shorten treatment regimen, potentially impacting clinical benefit. The pathology of CIPN is complex and still not completely understood. Damages to nerve cells are believed to be directly resulting from the antineoplastic agents’ cytotoxicity, and inflammation and the immune system are believed to be involved as well. Evidence suggests that intervention of certain GPCRs (e.g., cannabinoid receptors and others) may be useful as potential treatments. Altered neuronal sphingolipid metabolism has been linked to neuropathic pain and elevated plasma levels of sphingosine-1-phosphate (S1P) have also been associated with patients receiving chemotherapy and developing CIPN; therefore, modulating S1P levels may also be a potential treatment. Method: BXQ-350 is a nanovesicle of Saposin C, an allosteric activator of sphingolipid metabolism, that lowers systemic S1P; Saposin C has been reported to activate certain GPCRs, including GPR37. BXQ-350 was investigated in an adult Phase 1 dose-escalation safety study in heavily pretreated all-comer cancer patients with advanced solid malignancies (NCT02859857). The cytoprotective properties of BXQ-350 against multiple agents known to induce CIPN were investigated in vitro in neuronal PC12 cells and BXQ-350’s CIPN mitigation properties were investigated in vivo in a CIPN preclinical model. Results: A pancreatic cancer patient with chronic CIPN at time of enrollment in the Phase 1 study spontaneously reported a significant improvement of her neuropathic symptoms shortly after receiving BXQ-350; several patients had similar improvements. Results from preclinical experiments in PC12 cells revealed that BXQ-350 at concentration as low as 50 nM protected cells from oxaliplatin, paclitaxel, bortezomib or MMAE and promoted neurite growth. BXQ-350 was subsequently investigated in a murine oxaliplatin-induced CIPN preclinical model, showing a dose-dependent reduction of thermal and mechanical allodynia correlating with decreasing systemic S1P levels. Conclusions: Preclinical results demonstrated that BXQ-350 was highly effective at protecting neuronal cells from antineoplastic agents known to induce CIPN and prevent CIPN in a preclinical model. These results appeared to support the clinical observation that BXQ-350 alleviated CIPN symptoms in several patients soon after receiving BXQ-350. Additional studies are on-going. Citation Format: Nikhil Wilkins, Tim Stephens, Robin Furnish, Charlie Cruze, Darren Wolfe, Gilles H. Tapolsky, Ray Takigiku. BXQ-350 may protect peripheral nerves from the direct cytotoxicity of chemotherapeutic agents leading to chemotherapy induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3042.

Abstract 2686: The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells

Abstract Glioblastoma is a lethal brain tumor. Despite aggressive treatment, the median survival time is 6 to 12 months. These tumors acquire resistance against many therapeutic regimens. There is a need to screen for potential new drugs and drug combinations. Cucurbitacin B (CuB), a compound obtained from plants, and evodiamine (Evo), a supplement, have shown promising results in various neoplasms but have not been well studied in glioblastoma and especially, as a combination treatment. This study investigated the combined effects of CuB and Evo on U-87MG human glioma cell line. Results demonstrated that CuB and Evo individually decreased the U-87MG cell viability in time and dose dependent manners. The effect was significantly pronounced with the combined treatment. The combination treatment of CuB and Evo induced cellular apoptosis most likely by interfering with the PI3K/AKT and JNK/ERK pathways. In addition, the flow cytometry analysis showed that the combination treatment arrested the cell cycle at the G2M phase and inhibited cell propagation. Moreover, CuB and Evo combined effect successfully inhibited cellular migration. The cytotoxic effect of combination treatment of CuB and Evo was three times lower in neuronal PC-12 cells compared to U-87MG cells. Overall, a combination of cucurbitacin B and evodiamine proved to be a superior potential anti-neoplastic treatment in U-87MG cells as compared to individual treatment, and may be considered as a potential adjuvant therapy for GBM. Citation Format: Vrutti V. Mehta, Kenza E. Benzeroual. The combination of Cucurbitacin B and Evodiamine inhibits cell migration and propagation, and induces apoptosis in in vitro glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2686.

Curcumin-dependent phenotypic transformation of microglia mediates resistance to pseudorabies-induced encephalitis

Pseudorabies virus (PRV) causes viral encephalitis, a devastating disease with high mortality worldwide. Curcumin (CUR) can reduce inflammatory damage by altering the phenotype of microglia; however, whether and how these changes mediate resistance to PRV-induced encephalitis is still unclear. In this study, BV2 cells were infected with/without PRV for 24 h and further treated with/without CUR for 24 h. The results indicated that CUR promoted the polarization of PRV-infected BV2 cells from the M1 phenotype to the M2 phenotype and reversed PRV-induced mitochondrial dysfunction. Furthermore, M1 BV2 cell secretions induced signalling pathways leading to apoptosis in PC-12 neuronal cells, and this effect was abrogated by the secretions of M2 BV2 cells. RNA sequencing and bioinformatics analysis predicted that this phenotypic shift may be due to changes in energy metabolism. Furthermore, Western blot analysis showed that CUR inhibited the increase in AMP-activated protein kinase (AMPK) phosphorylation, glycolysis, and triacylglycerol synthesis and the reduction in oxidative phosphorylation induced by PRV infection. Moreover, the ATP levels in M2 BV2 cells were higher than those in M1 cells. Furthermore, CUR prevented the increase in mortality, elevated body temperature, slowed growth, nervous system excitation, brain tissue congestion, vascular cuffing, and other symptoms of PRV-induced encephalitis in vivo. Thus, this study demonstrated that CUR protected against PRV-induced viral encephalitis by switching the phenotype of BV2 cells, thereby protecting neurons from inflammatory injury, and this effect was mediated by improving mitochondrial function and the AMPK/NF-κB p65-energy metabolism-related pathway.

3D Printed Conductive Multiscale Nerve Guidance Conduit with Hierarchical Fibers for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have become a promising alternative for peripheral nerve regeneration; however, the outcome of nerve regeneration and functional recovery is greatly affected by the physical, chemical, and electrical properties of NGCs. In this study, a conductive multiscale filled NGC (MF-NGC) consisting of electrospun poly(lactide-co-caprolactone) (PCL)/collagen nanofibers as the sheath, reduced graphene oxide /PCL microfibers as the backbone, and PCL microfibers as the internal structure for peripheral nerve regeneration is developed. The printed MF-NGCs presented good permeability, mechanical stability, and electrical conductivity, which further promoted the elongation and growth of Schwann cells and neurite outgrowth of PC12 neuronal cells. Animal studies using a rat sciatic nerve injury model reveal that the MF-NGCs promote neovascularization and M2 transition through the rapid recruitment of vascular cells and macrophages. Histological and functional assessments of the regenerated nerves confirm that the conductive MF-NGCs significantly enhance peripheral nerve regeneration, as indicated by improved axon myelination, muscle weight increase, and sciatic nerve function index. This study demonstrates the feasibility of using 3D-printed conductive MF-NGCs with hierarchically oriented fibers as functional conduits that can significantly enhance peripheral nerve regeneration.

Silencing of the circ-Arhgap5 RNA protects neuronal PC12 cells against injury and depends on the miR-29a-3p/Rock1 axis.

Circular RNAs (circRNAs) are abundantly expressed in human central nervous system. Here, we explored the role of circ_Arhgap5 in cerebral ischemia/reperfusion (I/R)-induced nerve injury in PC12 cells and its associated mechanism. Cell proliferation ability was assessed by 5-Ethynyl-2'-deoxyuridine (Edu) assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCM) was applied to assess cell apoptotic rate. Cell death was analyzed by lactate dehydrogenase (LDH) assay. Oxygen-glucose deprivation and reoxygenation (OGD/R) up-regulates the expression of circRNA Rho GTPase activating protein 5 (circ_Arhgap5; mmu_circ_0000377) in PC12 cells. OGD/R exposure inhibited the proliferation and induced the apoptosis of PC12 cells, and the silence of circ_Arhgap5 attenuated OGD/R-induced injury in PC12 cells. miR-29a-3p was identified as a target of circ_Arhgap5 in PC12 cells. Circ_Arhgap5 knockdown-mediated protective effects in OGD/R-induced PC12 cells were reversed by the interference of miR-29a-3p. miR-29a-3p interacted with the 3' untranslated region (3'UTR) of Rho-associated coiled-coil-containing protein kinase 1 (Rock1), and circ_Arhgap5 can positively regulate Rock1 expression by sponging miR-29a-3p in PC12 cells. miR-29a-3p overexpression protected PC12 cells against OGD/R-induced damage by down-regulating Rock1. In conclusion, circ_Arhgap5 silencing protected PC12 cells from OGD/R-induced injury through mediating miR-29a-3p/Rock1 axis.

In Vitro Neurotrophic Properties and Structural Characterization of a New Polysaccharide LTC-1 from Pyrola corbieri Levl (Luticao).

Pyrola corbieri Levl has been used to strengthen bones and nourish the kidney (the kidney governs the bone and is beneficial to the brain) by the local Miao people in China. However, the functional components and neurotrophic activity have not been reported. A new acidic homogeneous heteropolysaccharide named LTC-1 was obtained and characterized by periodate oxidation, Smith degradation, partial acid hydrolysis, GC-MS spectrometry, methylation analysis, and Fourier transform infrared spectroscopy, and its molecular weight was 3239 Da. The content of mannuronic acid (Man A) in LTC-1 was 46%, and the neutral sugar was composed of L-rhamnose (L-Rha), L-arabinose (L-Ara), D-xylose (D-Xyl), D-mannose (D-Man), D-glucose (D-Glc) and D-galactose (D-Gal) with a molar ratio of 1.00:3.63:0.86:1.30:6.97:1.30. The main chain of LTC-1 was composed of Glc, Gal, Man, Man A and the branched chain Ara, Glc, Gal. The terminal residues were composed of Glc and Gal. The main chain and branched chains were linked by (1→5)-linked-Ara, (1→3)-linked-Glc, (1→4)-linked-Glc, (1→6)-linked-Glc, (1→3)-linked-Gal, (1→6)-linked-Gal, (1→3, 6)-linked-Man and ManA. Meanwhile, neurotrophic activity was evaluated through PC12 and primary hippocampal neuronal cell models. LTC-1 exhibited neurotrophic activity in a concentration-dependent manner, which significantly induced the differentiation of PC12 cells, promoted the neurite outgrowth of PC12 cells, enhanced the formation of the web architecture of dendrites, and increased the density of dendritic spines in hippocampal neurons and the expression of PSD-95. These results displayed significant neurotrophic factor-like activity of LTC-1, which suggests that LTC-1 is a potential treatment option for neurodegenerative diseases.

Peptide-based inhibitors of amyloid beta aggregation and toxicity.

Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease. Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Previous work has shown that seven overlapping fragments of Aβ1-42 can disrupt the process of fibril formation, possibly through intercalation into the growing aggregate. The potential neuroprotective properties of these fragments were not directly examined. In this project, Aβ1-42 was allowed to aggregate in aqueous buffer for 2, 4, or 24 h in the absence and presence of an Aβ fragment, at 1:2 Aβ1-42/fragment molar ratio. Neuronal PC12 cells were then incubated with the samples for 50 h before conducting a cell viability assay. Thioflavin-T fluorescence assays to detect the fibrillar form of Aβ1-42 were also conducted. There was an inverse correlation between the time span of Aβ1-42 aggregation and cellular toxicity, with the 2 h sample exhibiting the greatest level of toxicity. Aβ fragments displayed varying effects on the inhibition of Aβ1-42 fibrillogenesis and suppression of its toxicity. Three fragments completely neutralized the toxicity of the 2 h aggregate, while two others conferred weak cellular protection against Aβ1-42 toxicity. One fragment enhanced Aβ1-42 toxicity at all stages of aggregation. Every fragment disrupted fibril formation to some extent, but there was no obvious correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. This suggests that fragment intercalation into the growing fibril may block further aggregation but not the toxicity of oligomeric Aβ1-42. Fragments that block both Aβ1-42 aggregation and toxicity represent potential targets for therapeutic development.

Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury.

A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia-mediated neuroinflammation and neuronal apoptosis after SCI. Stimulation of microglia with lipopolysaccharide (LPS) to simulate neuroinflammation models in vitro, the effect of Alpinetin on the release of pro-inflammatory mediators in LPS-induced microglia and its mechanism were detected. In addition, a co-culture system of microglia and neuronal cells was constructed to assess the effect of Alpinetin on activating microglia-mediated neuronal apoptosis. Finally, rat spinal cord injury models were used to study the effects on inflammation, neuronal apoptosis, axonal regeneration, and motor function recovery in Alpinetin. Alpinetin inhibits microglia-mediated neuroinflammation and activity of the JAK2/STAT3 pathway. Alpinetin can also reverse activated microglia-mediated reactive oxygen species (ROS) production and decrease of mitochondrial membrane potential (MMP) in PC12 neuronal cells. In addition, in vivo Alpinetin significantly inhibits the inflammatory response and neuronal apoptosis, improves axonal regeneration, and recovery of motor function. Alpinetin can be used to treat neurodegenerative diseases and is a novel drug candidate for the treatment of microglia-mediated neuroinflammation.

Preventive Effect of 3,3′‐Dimethoxy‐4,4′‐dihydroxystilbene Triazole against Alzheimer's Disease by Inhibition of Neuronal Apoptosis

AbstractThe present study investigated 3,3′‐dimethoxy‐4,4′‐dihydroxy‐stilbene triazole (DMDHSB) in vitro for alleviation of β‐amyloid mediated damage to the PC12 neuronal cells. Exposure of PC12 cells to DMDHSB significantly (P<0.05) reversed β‐amyloid induced loss of viability of PC12 cells in dose‐dependent manner. Moreover, DMDHSB exposure significantly (P<0.05) inhibited over production of PGE2 activated by β‐amyloid in PC12 cells. The β‐amyloid promoted higher levels of COX‐2 and NO were also down‐regulated in PC12 cells on exposure to DMDHSB. The exposure to DMDHSB led to a significant (P<0.05) inhibition of β‐amyloid promoted higher expression of iNOS. The β‐amyloid promoted higher release of iNOS was effectively alleviated by DMDHSB exposure in PC12 cells. In DMDHSB exposed PC12 cells, the β‐amyloid promoted elevation of NF‐κB nuclear translocation was effectively prevented compared to the control. It was observed from the docking studies that one‐pose of DMDHSB interacted with COX‐2 protein at binding affinity of −9.6 kcal/mol at 0‐rmsd. Thus, β‐amyloid promoted loss of PC12 cell viability is prevented on DMDHSB exposure through targeting inflammatory response. Therefore, DMDHSB may be developed as therapeutic candidate for investigation in detail against Alzheimer's disease.

Protective effect of Capsosiphon fulvescens on oxidative stress-stimulated neurodegenerative dysfunction of PC12 cells and zebrafish larva models

Reactive oxygen species (ROS) at high concentrations induce oxidative stress, an imbalanced redox state that is a prevalent cause of neurodegenerative disorders. This study aimed to investigate the protective effect of Capsosiphon fulvescens (CF) extract on oxidative stress-induced impairment of cognitive function in models of neurodegenerative diseases. CF was extracted with subcritical water and several solvents and H2O2 (0.25 mM) or aluminum chloride (AlCl3; 25 μM) as an inducer of ROS was treated in PC12 neuronal cells and zebrafish larvae. All statistical analyses were performed using one-way analysis of variance and Dunnett’s test using GraphPad Prism. H2O2 and AlCl3 were found to significantly induce ROS production in PC12 neuronal cells and zebrafish larvae. In addition, they strongly affected intracellular Ca2+ levels, antioxidant enzyme activity, brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) signaling, acetylcholinesterase (AChE) activity, and hallmarks of Alzheimer’s disease. However, treatment of H2O2-induced PC12 cells or AlCl3-induced zebrafish larvae with CF subcritical water extract at 90°C and CF water extract effectively regulated excessive ROS production, intracellular Ca2+ levels, and mRNA expression of superoxide dismutase, glutathione peroxide, glycogen synthase kinase-3 beta, β-amyloid, tau, AChE, BDNF, and TrkB. Our study suggested that CF extracts can be a potential source of nutraceuticals that can improve the impairment of cognitive function and synaptic plasticity by regulating ROS generation in neurodegenerative diseases.