Survival Of PC12 Cells Research Articles

Poliumoside inhibits apoptosis, oxidative stress and neuro-inflammation to prevent intracerebroventricular Streptozotocin-induced cognitive dysfunction in Sprague-Dawley Rats: in in-vivo, in-vitro and in-silico study.

Alzheimer's disease (AD) is a severe neurological illness causes cognitive decline and mortality if not treated early. However, the current therapeutic modalities are inefficient to manage the cognitive dysfunction of AD. Therefore, in the present manuscript, we have enumerated the pharmacological benefit of Poliumoside in the Streptozotocin-induced cognitive dysfunction in Sprague-Dawley (SD) rats. Initially, the cognitive dysfunction in rats was induced by the intracerebroventricular administration of Streptozotocin, then rats received PMD (5 mg and 10 mg/kg body weight) was given. Various behavioural analysis, such as Morris water maze (MWM), and object recognition tests (ORT), and locomotor analysis was conducted in PMD treated group. Various biochemical analysis was conducted to analyze the effect of PMD on hippocampus oxidative-nitrosative stress and pro-inflammatory cytokines. MTT assay and annexin V/PI staining was performed to analyse the effect of PMD on the cell viability and neuronal toxicity of PC12 cells, respectively. Molecular docking analysis was also conducted with crystal structure of human AChE. PMD treatment improved cognitive capacity in rats in MWM and ORT. Compared to STZ rats, PMD-treated rats had significantly higher locomotor activity and lower AChE activity. PMD also restores dopamine, 5-HT, and NE levels and reduces metabolic their deactivation as evidenced by increased levels of DOPAC, HVA, 5-HIAA. Nitrite, MDA, SOD, CAT, and GSH levels were restored near normal in PMD-treated rats, reducing hippocampus oxidative-nitrosative stress. Pro-inflammatory cytokines were similarly lowered in PMD-treated rats. In in-vitro studies, PMD did not affect PC12 cell survival at the maximal dose of 10 µM. In addition, PMD concentration-dependently prevents H₂O₂-induced neuronal death in PC12 cells. The in-silico docking analysis showed that the PMD fit snugly into the active site of human AChE by engaging with the anionic domain and the catalytic triad of Trp86, Tyr337, Phe338, and Gly121 residues. In conclusion, our study demonstrated that PMD have significant impact on AD by inhibiting ACheE and restoring neurotransmitter levels, which enhances Ach levels in rats and improves cognitive impairment in STZ rats.

Luteolin Mitigates Dopaminergic Neuron Degeneration and Restrains Microglial M1 Polarization by Inhibiting Toll Like Receptor 4.

Luteolin is a natural flavonoid and its neuroprotective and anti-inflammatory effects have been confirmed to mitigate neurodegeneration. Despite these findings, the underlying mechanisms responsible for these effects remain unclear. Toll-like receptor 4 (TLR4) is widely distributed in microglia and plays a pivotal role in neuroinflammation and neurodegeneration. Here studies are outlined that aimed at determining the mechanisms responsible for the anti-inflammatory and neuroprotective actions of luteolin using a rodent model of Parkinson's disease (PD) and specifically focusing on the role of TLR4 in this process. The mouse model of PD used in this experiment was established through a single injection of lipopolysaccharide (LPS). Mice were then subsequently randomly allocated to either the luteolin or vehicle-treated group, then motor performance and dopaminergic neuronal injury were evaluated. BV2 microglial cells were treated with luteolin or vehicle saline prior to LPS challenge. MRNA expression of microglial specific marker ionized calcium-binding adapter molecule 1 (IBA-1) and M1/M2 polarization markers, as well as the abundance of indicated pro-inflammatory cytokines in the mesencephalic tissue and BV2 were quantified by real time-polymerase chain reaction (RT-PCR) and Enzyme-linked Immunosorbent Assay (ELISA), respectively. Cell viability and apoptosis of neuron-like PC12 cell line co-cultured with BV2 were detected. TLR4 RNA transcript and protein abundance in mesencephalic tissue and BV2 cells were detected. Nuclear factor kappa-gene binding (NF-κB) p65 subunit phosphorylation both in vitro and in vivo was evaluated by immunoblotting. Luteolin treatment induced functional improvements and alleviated dopaminergic neuronal loss in the PD model. Luteolin inhibited apoptosis and promoted cell survival in PC12 cells. Luteolin treatment shifted microglial M1/M2 polarization towards an anti-inflammatory M2 phenotype both in vitro and in vivo. Finally, it was found that luteolin treatment significantly downregulated both TLR4 mRNA and protein expression as well as restraining NF-κB p65 subunit phosphorylation. Luteolin restrained dopaminergic degeneration in vitro and in vivo by blocking TLR4-mediated neuroinflammation.

Superhydrophobic synergistic antibacterial ZrO2-based surfaces with tertiary hierarchical structures fabricated by femtosecond laser texturing and surface modification

Zirconia (ZrO2) is a hard and brittle dental implant material without anti-infective property. This work reports the preparation of a tertiary micro-nano hierarchical structure on a ZrO2 surface via femtosecond laser texturing combined with magnetron sputtering deposition of an Ag layer (Ag/ZrO2), aiming to achieve a superhydrophobic synergistic antibacterial effect. Laser-scanning pitch and Ag layer thickness were systematically optimized for obtaining the tertiary hierarchical structure. The wettabilities, antibacterial properties and durabilities of various ZrO2 and stearic acid modified Ag/ZrO2 (S-Ag/ZrO2) surfaces were further tested and comparatively analyzed. The results revealed that the optimal process parameters were a laser-scanning pitch of 30 μm and an Ag layer thickness of 100 nm. The as-obtained S-Ag/ZrO2 sample displayed a tertiary micro-nano hierarchical structure, which was featured by a periodic regular conical micro-convex structure covered with ZrO2 nano-protrusions and Ag nanoparticles. This structure could not only greatly reduce the solid-liquid contact area to achieve outstanding superhydrophobicity and durability, it could also sustainably release bacteria-killing Ag+ to obtain an antibacterial ratio of 95.1 % and ensure a long-term antibacterial effect. In addition, the optimal S-Ag/ZrO2 sample having this structure showed a PC12 cell survival rate of up to 81.75 %, demonstrating its good biocompatibility. This work may be of great inspiration and reference for the expansion of the use of ZrO2 in dentistry.

Exploring the neuroprotection of the combination of astragaloside A, chlorogenic acid and scutellarin in treating chronic cerebral ischemia via network analysis and experimental validation

Chronic cerebral ischemia (CCI) primarily causes cognitive dysfunction and other neurological impairments, yet there remains a lack of ideal therapeutic medications. The preparation combination of Astragalus membranaceus (Fisch.) Bunge and Erigeron breviscapus (Vant.) Hand.-Mazz have been utilized to ameliorate neurological dysfunction following cerebral ischemia, but material basis of its synergy remains unclear. The principal active ingredients and their optimal proportions in this combination have been identified through the oxygen and glucose deprivation (OGD) cell model, including astragaloside A, chlorogenic acid and scutellarin (ACS), and its efficacy in enhancing the survival of OGD PC12 cells surpasses that of the combination preparation. Nevertheless, mechanism of ACS against CCI remains elusive. In this study, 63 potential targets of ACS against CCI injury were obtained by network pharmacology, among which AKT1, CASP3 and TNF are the core targets. Subsequent analysis utilizing KEGG and GO suggested that PI3K/AKT pathway may play a crucial role for ACS in ameliorating CCI injury. Then, a right unilateral common carotid artery occlusion (rUCCAO) mouse model and an OGD PC12 cell model were established to replicate the pathological processes of CCI in vivo and in vitro. These models were utilized to explore the anti-CCI effects of ACS and its regulatory mechanisms, particularly focusing on PI3K/AKT pathway. The results showed that ACS facilitated the restoration of cerebral blood flow in CCI mice, enhanced the function of the central cholinergic nervous system, protected against ischemic nerve cell and mitochondrial damage, and improved cognitive function and other neurological impairments. Additionally, ACS upregulated the expression of p-PI3K, p-AKT, p-GSK3β and Bcl-2, and diminished the expression of Cyto-c, cleaved Caspase-3 and Bax significantly. However, the PI3K inhibitor (LY294002) partially reversed the downregulation of Bax, Cyto-c and cleaved Caspase-3 expression as well as the upregulation of p-AKT/AKT, p-GSK3β/GSK3β, and Bcl-2/Bax ratios. These findings suggest that ACS against neuronal damage in cerebral ischemia may be closely related to the activation of PI3K/AKT pathway. These results declared first time ACS may become an ideal candidate drug against CCI due to its neuroprotective effects, which are mediated by the activated PI3K/AKT pathway mitigates mitochondrial damage and prevents cell apoptosis.

Evaluating the Neuroprotective Potential of Caffeinated Coffee in the Context of Aluminum-Induced Neurotoxicity: Insights from a PC12 Cell Culture Model.

Exposure to aluminum (Al) and its compounds is an environmental factor that induces neurotoxicity, partially through oxidative stress, potentially leading to the development of neurodegenerative diseases. Components of the diet, such as caffeinated coffee, may play a significant role in preventing these diseases. In the present study, an experimental model of PC12 cells (rat pheochromocytoma tumor cells) was developed to investigate the influence of caffeine and caffeinated coffee on neurotoxicity induced by Al compounds and/or oxidative stress. For the induction of neurotoxicity, aluminum maltolate (Almal) and H2O2 were used. The present study demonstrates that 100 μM Almal reduced cell survival, while caffeinated coffee with caffeine concentrations of 5 μg/mL and 80 μg/mL reversed this effect, resulting in a higher than fivefold increase in PC12 cell survival. However, despite the observed antioxidant properties typical for caffeine and caffeinated coffee, it is unlikely that they are the key factors contributing to cell protection against neurotoxicity induced by both oxidative stress and Al exposure. Moreover, the present study reveals that for coffee to exert its effects, it is possible that Al must first activate certain mechanisms within the cell. Therefore, various signaling pathways are discussed, and modifications of these pathways might significantly decrease the risk of Al-induced neurotoxicity.

BMSC-Exosomes attenuate ALP dysfunction by restoring lysosomal function via the mTOR/TFEB Axis to reduce cerebral ischemia-reperfusion injury

BackgroundThe complex pathophysiological changes following cerebral ischemia-reperfusion injury (CIRI) include the accumulation of defective proteins and damaged organelles, which cause massive neuron demise. To preserve cellular homeostasis, the autophagy-lysosomal pathway (ALP) is crucial for neurons to dispose of these substances. Many studies have shown that bone mesenchymal stem cell exosomes (BMSC-Exos) can reduce CIRI. However, the specific mechanisms have not been well elucidated, a fact that limits its widespread clinical use. This study aimed to clarify whether BMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI via inhibiting mTOR and then activating TFEB nucleus translocation. MethodsIn this study, Flow cytometry, Nanoparticle tracking analysis (NTA), Transmission electron microscope (TEM), and Western blot were used to identify the BMSCs and BMSC-Exos used in this experiment as conforming to the requirements. In vivo experiments, SD rats were modeled with temporary middle cerebral artery occlusion (tMCAO), and BMSC-Exos was injected into the tail vein 2 h after modeling. Triphenyl tetrazolium chloride (TTC) staining, modified neurological severity scores (mNSS), corner turn test, and rotating rod test were used to detect neurological deficits in rats after BMSC-Exos intervention. Western blot and Immunofluorescence were used to detect ALP, transcription factor EB(TFEB) nucleus translocation, and mammalian target of rapamycin (mTOR) change at different time points after modeling and after BMSC-Exos intervention. In vitro experiments, pheochromocytoma cells (PC12) cells were subjected to oxygen-glucose deprivation and reperfusion (OGD/R) modeling to mimic CIRI, and were respectively intervened with BMSC-Exos, BMSC-Exos + MHY 1485 (the mTOR agonist), Rapamycin (the mTOR inhibitor). CCK8, Western blot, and Immunofluorescence were used to detect PC12 cell survival, TFEB nucleus translocation, and cathepsin B(CTSB) Immunofluorescence intensity. ResultsWe found that ALP dysfunction occurred 72 h after tMCAO, and BMSC-Exos can attenuate ALP dysfunction by restoring lysosomal function. Next, we examined TFEB nucleus translocation and the expression of mTOR, a key regulator of translocation. We found that BMSC-Exos could inhibit mTOR and activate TFEB nucleus translocation. Additional in vitro tests revealed that BMSC-Exos could increase PC12 cell survival after OGD/R, activating TFEB nucleus translocation and enhancing the fluorescence intensity of CTSB, which in turn could be reversed by the mTOR agonist, MHY1485. This effect was similar to another mTOR inhibitor, Rapamycin. ConclusionBMSC-Exos could attenuate ALP dysfunction by restoring lysosomal function after CIRI by inhibiting mTOR and then promoting TFEB nucleus translocation.

Tetrahydropiperine, a natural alkaloid with neuroprotective effects in ischemic stroke

BackgroundIschemic stroke (IS) is a life-threatening neurological disease with various pathological mechanisms. Tetrahydropiperine (THP) is a natural alkaloid with protective effects against multiple diseases, such as seizure, and pain. This study was to examine the impact of THP on IS and investigate its potential mechanism. Material and methodsWe employed network pharmacology and molecular docking techniques to identify the target proteins of THP for intervention in IS. Adult male Sprague-Dawley rats were used to create a permanent middle cerebral artery occlusion model. PC-12 cells were chosen to establish an oxygen–glucose deprivation (OGD) cell model. Disease modeling followed by nimodipine (NIMO); 3-methyladenine (3-MA) and rapamycin (RAP) interventions. Open field test, Longa score, balance beam test, and forelimb grip test were used to measure motor and neurological functions. The degree of neurological damage recovery was assessed through behavioral analysis, and cerebral infarction volume was determined using TTC staining. Morphological changes were examined through HE and Nissl staining, and ultrastructural changes in neurons were observed using transmission electron microscopy. The protein expression of autophagy and related pathways was analyzed through Western blot (WB). The appropriate hypoxia time and drug concentration were determined using CCK-8 assay, which also measured cell survival rate. ResultsThe network pharmacology findings indicated that the impact of THP on IS was enhanced in the PI3K/Akt signaling pathway. THP demonstrated robust docking capability with proteins associated with the autophagy and PI3K/Akt/mTOR, as indicated by the molecular docking outcomes. THP significantly improved behavioral damage, reduced the area of cerebral infarction, ameliorated histopathological damage from ischemia, increase neuronal survival, and alleviated ultrastructural damage in neurons (P < 0.05). THP enhanced the survival of PC-12 cells induced by OGD and ameliorated the morphological harm to the cells (P < 0.05). THP was found to elevate the quantities of P62, LC3-Ⅰ, PI3K, P-AKt/Akt, and P-mTOR/mTOR proteins while reducing the levels of Atg7 and Beclin1 proteins. The results of transmission electron microscopy showed no autophagosomes in the THP, 3-MA, and 3-MA + THP groups. ConclusionThe activation of the PI3K/Akt/mTOR signaling pathway by THP inhibits autophagy and provides relief from neurological damage in IS.

Metabotropic Glutamate Receptor 8 Suppresses M1 Polarization in Microglia by Alleviating Endoplasmic Reticulum Stress and Mitochondrial Dysfunction.

Microglia-mediated neuroinflammation is a hallmark of neurodegeneration. Metabotropic glutamate receptor 8 (GRM8) has been reported to promote neuronal survival in neurodegenerative diseases, yet the effect of GRM8 on neuroinflammation is still unclear. Calcium overload-induced endoplasmic reticulum (ER)-mitochondrial miscommunication has been reported to trigger neuroinflammation in the brain. The aim of this study was to investigate putative anti-inflammatory effects of GRM8 in microglia, specifically focusing on its role in calcium overload-induced ER stress and mitochondrial dysfunction. BV2 microglial cells were pretreated with GRM8 agonist prior to lipopolysaccharide administration. Pro-inflammatory cytokine levels and the microglial polarization state in BV2 cells were then quantified. Cellular apoptosis and the viability of neuron-like PC12 cells co-cultured with BV2 cells were examined using flow cytometry and a Cell Counting Kit-8, respectively. The concentration of cAMP, inositol-1,4,5-triphosphate receptor (IP3R)-dependent calcium release, ER Ca2+ concentration, mitochondrial function as reflected by reactive oxygen species levels, ATP production, mitochondrial membrane potential, expression of ER stress-sensing protein, and phosphorylation of the nuclear factor kappa B (NF-κB) p65 subunit were also quantified in BV2 cells. GRM8 activation inhibited pro-inflammatory cytokine release and shifted microglia polarization towards an anti-inflammatory-like phenotype in BV2 cells, as well as promoting neuron-like PC12 cell survival when co-cultured with BV2 cells. Mechanistically, microglial GRM8 activation significantly inhibited cAMP production, thereby desensitizing the IP3R located within the ER. This process markedly limited IP3R-dependent calcium release, thus restoring mitochondrial function while inhibiting ER stress and subsequently deactivating NF-κB signaling. Our results indicate that GRM8 activation can protect against microglia-mediated neuroinflammation by attenuating ER stress and mitochondrial dysfunction, and that IP3R-mediated calcium signaling may play a vital role in this process. GRM8 may thus be a potential target for limiting neuroinflammation.

Integrated network pharmacology, molecular docking and pharmacodynamic study reveals protective effects and mechanisms of corilagin against cerebral ischemia-induced injury

BackgroundStroke is one of the leading causes of death and long-term disability worldwide. Previous studies have found that corilagin has antioxidant, anti-inflammatory, anti-atherosclerotic and other pharmacological activities and has a protective effect against cardiac and cerebrovascular injury. ObjectivesThe aim of this study was to investigate the protective effects of corilagin against ischemic stroke and to elucidate the underlying molecular mechanisms using network pharmacology, molecular docking, and animal and cell experiments. MethodsWe investigated the potential of corilagin to ameliorate cerebral ischemia–reperfusion injury using in vivo rat middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro oxygen–glucose deprivation/reoxygenation (OGD/R) models. ResultsOur results suggest that corilagin may exert its anti-ischemic stroke effect by interacting with 92 key targets, including apoptosis-associated proteins (Bcl-2, Bax, caspase-3) and PI3K/Akt signaling pathway-related proteins. In vivo and in vitro experiments showed that corilagin treatment improved neurological deficits, attenuated cerebral infarct volume, and mitigated neuronal damage in MCAO/R rats. Corilagin treatment also enhanced the survival of PC12 cells exposed to OGD/R, reduced the rate of LDH leakage, inhibited cell apoptosis, and activated the PI3K/Akt signaling pathway. Importantly, the effects of corilagin on the PI3K/Akt signaling pathway and apoptosis-associated proteins were reversed by the PI3K-specific inhibitor LY294002. ConclusionsThese results indicate that the molecular mechanism of the anti-ischemic effect of corilagin involves inhibiting neuronal apoptosis and activating the PI3K/Akt signaling pathway. These findings provide a theoretical and experimental basis for the further development and application of corilagin as a potential anti-ischemic stroke agent.

Exosomes from ectoderm mesenchymal stem cells inhibits lipopolysaccharide-induced microglial M1 polarization and promotes survival of H2O2-exposed PC12 cells by suppressing inflammatory response and oxidative stress

To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells (EMSCs-exo) for repairing secondary spinal cord injury. EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa, identified by transmission electron microscope, nanoparticle tracking analysis (NTA), and Western blotting, and quantified using the BCA method. Neonatal rat microglia purified by differential attachment were induced with 100 μg/L lipopolysaccharide (LPS) and treated with 37.5 or 75 mg/L EMSCs-exo. PC12 cells were exposed to 400 μmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L. The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT- PCR, and the concentrations of IL- 6, IL-10, and IGF-1 in the supernatants were measured with ELISA. The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry. The isolated rat EMSCs showed high expressions of nestin, CD44, CD105, and vimentin. The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63, CD81, and TSG101 but not vimentin. In LPS-treated microglia, EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression (P < 0.05). EMSCs-exo treatment at 75 mg/L, as compared with the lower concentration at 37.5 mg/L, more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia, and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells (P < 0.05). EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival, suggesting its potential in controlling secondary spinal cord injury.

Parkin-mediated mitophagy is a potential treatment for oxaliplatin-induced peripheral neuropathy.

Oxaliplatin-induced peripheral nerve pain (OIPNP) is a common chemotherapy-related complication, but the mechanism is complex. Mitochondria are vital for cellular homeostasis and regulating oxidative stress. Parkin-mediated mitophagy is a cellular process that removes damaged mitochondria, exhibiting a protective effect in various diseases; however, its role in OIPNP remains unclear. In this study, we found that Parkin-mediated mitophagy was decreased, and reactive oxygen species (ROS) was upregulated in OIPNP rat dorsal root ganglion (DRG) in vivo and in PC12 cells stimulated with oxaliplatin (OXA) in vitro. Overexpression of Parkin indicated that OXA might cause mitochondrial and cell damage by inhibiting mitophagy. We also showed that salidroside (SAL) upregulated Parkin-mediated mitophagy to eliminate damaged mitochondria and promote PC12 cell survival. Knockdown of Parkin indicated that mitophagy is crucial for apoptosis and mitochondrial homeostasis in PC12 cells. In vivo study also demonstrated that SAL enhances Parkin-mediated mitophagy in the DRG and alleviates peripheral nerve injury and pain. These results suggest that Parkin-mediated mitophagy is involved in the pathogenesis of OIPNP and may be a potential therapeutic target for OIPNP.NEW & NOTEWORTHY This article discusses the effects and mechanisms of Parkin-mediated mitophagy in oxaliplatin-induced peripheral nerve pain (OIPNP) from both in vivo and in vitro. We believe that our study makes a significant contribution to the literature because OIPNP has always been the focus of clinical medicine, and mitochondrial quality regulation mechanisms especially Parkin-mediated mitophagy, have been deeply studied in recent years. We use a variety of molecular biological techniques and animal experiments to support our argument.

Low beauvericin concentrations promote PC-12 cell survival under oxidative stress by regulating lipid metabolism and PI3K/AKT/mTOR signaling

Beauvericin (BEA), a naturally occurring cyclic peptide with good pharmacological activity, has been widely explored in anticancer research. Although BEA is toxic, studies have demonstrated its antioxidant activity. However, to date, the antioxidant mechanisms of BEA remain unclear. Herein, we conducted a comprehensive and detailed study of the antioxidant mechanism of BEA using an untargeted metabolomics approach, subsequently validating the results. BEA concentrations of 0.5 and 1 μM significantly inhibited H2O2-induced oxidative stress (OS), decreased reactive oxygen species levels in PC-12 cells, and restored the mitochondrial membrane potential. Untargeted metabolomics indicated that BEA was primarily involved in lipid-related metabolism, suggesting its role in resisting OS in PC-12 cells by participating in lipid metabolism. BEA combated OS damage by increasing phosphatidylcholine, phosphatidylethanolamine, and sphingolipid levels. In the current study, BEA upregulated proteins related to the PI3K/AKT/mTOR pathway, thereby promoting cell survival. These findings support the antioxidant activity of BEA at low concentrations, warranting further research into its pharmacological effects.

Production of a Bioink Containing Decellularized Spinal Cord Tissue for 3D Bioprinting.

For the past few years, three-dimensional (3D) bioprinting has emerged as a promising approach in the field of regenerative medicine. This technique allows for the production of 3D scaffolds to support cell transplantation due to its ability to mimic the extracellular environment. One alternative to enhancing cell adhesion, survival, and proliferation is the use of decellularized extracellular matrix as a bioink component. The aim of this study was to produce a bioink using lyophilized rat decellularized spinal cord tissue (DSCT) for 3D bioprinting of nervous tissue. DNA quantification, hematoxylin and eosin and DAPI staining indicated that 1% sodium dodecyl sulfate and 9 h processing were effective in removing the cells from the spinal cord samples. The cell viability assay showed that the decellularized matrix is not cytotoxic for PC12 cells. The hydrogel containing DSCT, alginate, and gelatine used as the base for the bioink has a shear thinning behavior and low G″/G' ratio, allowing for good printability without compromising cell viability after 3D bioprinting. The bioink supported long-term PC12 cell survival, with 93% of live cells 4 weeks after printing, and stimulated the production of laminin-1 and neurofilament-M. This bioink, therefore, represents an easily available biomaterial for central nervous system tissue engineering.

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

Exosomes from magnetic particles-primed mesenchymal stem cells enhance neural differentiation of PC12 cells

This study aimed to investigate the effects of mesenchymal stem cell exosomes loaded with Fe3O4 magnetic particles (Fe3O4@ MSC-exo) on the survival and neural differentiation of PC12 cells. Exosomes were separated from Fe3O4 magnetic nanoparticles-primed umbilical cord mesenchymal stem cells condition medium by ultracentrifugation and characterized by transmission electron microscopy, flow nano analysis, and western blotting. PC12 cells were treated with culture medium containing exosomes. The effects of Fe3O4@ MSC-exo on PC12 cell proliferation, migration, and neural differentiation were analyzed using CCK-8 assay, transwell migration assay, RT-qPCR, and immunofluorescence, respectively. Additionally, miRNA sequencing was performed on Fe3O4@ MSC-exo, followed by bioinformatic analysis of the results. We found that Fe3O4@ MSC-exo can promote PC12 cell proliferation, migration, and neural differentiation. According to the sequencing results, there were a total of 43 differentially expressed miRNAs. The present study indicated that Fe3O4@ MSC-exo might enhance nerve cell function, laying the foundation for targeted therapy of nerve injury.

Molecular docking and In-Ovo analysis of human amniotic fluid extracellular vesicles loaded with sulforaphane: A potential therapy for neurological disorders

Sulforaphane (SFN) is a bioactive component found in broccoli that has neuroprotective and anti-cancer effects. SFN has been encapsulated in various drug carriers to improve its stability and effectiveness. Specifically, this study presents an SFN delivery system based on human Amniotic Fluid-EVs (hAFEVs) for the treatment of neurological disorders. The binding affinity between hAFEVs (<200 nm) and SFN was investigated by spectroscopic analysis, revealing a temperature-dependent binding mechanism. The binding constants (ksv, kq, and kb) were also assessed, indicating a static quenching mechanism. Additionally, the docking studies of SFN and CD9, CD81, HSP70, and TSG101 were performed using AutoDock 4.2.6 software's Lamarckian genetics algorithm to investigate the type of interaction and their effects on the targetability of hAFEVs. The obtained results showed that SFN-loaded hAFEVs had a favorable effect on increasing the survival and proliferation of human PC12 cells (137%). The results of gene expression studies showed that this release system can upregulate the expression of Nrf2 and decrease the expression of IL-6, which shows the support of nerve cells against oxidative stress. In the In-Ovo study, treatment with SFN-hAFEVs resulted in improved grey and white matter cohesion and the improved orientation of bipolar neurons. The results indicate a significant increase in synaptophysin expression and the number of doublecortin-positive cells improved neuronal development and synapse formation. The findings of this study suggest that SFN-hAFEVs have great potential as a treatment for neurological disorders.

Comparison of Biocompatibility and Morphology of PC12 Cell Line on a Polycaprolactane/Silymarin Scaffold and a Polycaprolactane/Tragacanth Scaffold

Background: The goal of tissue engineering is to create biological solutions that restore, maintain, and improve the function of damaged tissue. Scaffolds are structures based on extracellular matrix materials that have undergone various treatments. Objectives: This study aimed to prepare polycaprolactone/silymarin and polycaprolactane/tragacanth scaffolds and compare the morphology and viability of PC12 cell lines. Methods: A 7% polycaprolactane solution (dissolved in acetic acid) was mixed with a 0.9% silymarin solution to prepare the polycaprolactane scaffold and silymarin loading, and a 7% polycaprolactane solution was mixed to prepare the polycaprolactane and tragacanth scaffold. Tragacanth solution was mixed at a concentration of 0.7% by weight, and then two scaffolds were prepared by electrospinning. The morphology of the scaffolds was studied by scanning electron microscopy (SEM), and the chemical structure of the scaffolds was studied by ATR-FTIR spectroscopy. The biocompatibility of the scaffolds and cell survival of PC12 cells was investigated by MTT assay. Results: The morphology of the scaffolds and their chemical structure showed good porosity and successful loading of silymarin onto PCL and a suitable combination of tragacanth with PCL. The biocompatibility of the scaffolds was evaluated at 24, 48, and 72 hours after PC12 cell culture. Cell survival was found to increase on polycaprolactane/silymarin scaffolds compared to polycaprolactane/tragacanth. Conclusions: From the results of this study, loading polycaprolactane scaffold with silymarin increases the proliferation and survivability of PC12 cells compared to polycaprolactane/supporting scaffold, which may be a good candidate for neural tissue engineering.

A candidate protective factor in amyotrophic lateral sclerosis: heterogenous nuclear ribonucleoprotein G.

Heterogenous nuclear ribonucleoprotein G is down-regulated in the spinal cord of the Tg(SOD1*G93A)1Gur (TG) amyotrophic lateral sclerosis mouse model. However, most studies have only examined heterogenous nuclear ribonucleoprotein G expression in the amyotrophic lateral sclerosis model and heterogenous nuclear ribonucleoprotein G effects in amyotrophic lateral sclerosis pathogenesis such as in apoptosis are unknown. In this study, we studied the potential mechanism of heterogenous nuclear ribonucleoprotein G in neuronal death in the spinal cord of TG and wild-type mice and examined the mechanism by which heterogenous nuclear ribonucleoprotein G induces apoptosis. Heterogenous nuclear ribonucleoprotein G in spinal cord was analyzed using immunohistochemistry and western blotting, and cell proliferation and proteins (TAR DNA binding protein 43, superoxide dismutase 1, and Bax) were detected by the Cell Counting Kit-8 and western blot analysis in heterogenous nuclear ribonucleoprotein G siRNA-transfected PC12 cells. We analyzed heterogenous nuclear ribonucleoprotein G distribution in spinal cord in the amyotrophic lateral sclerosis model at various time points and the expressions of apoptosis and proliferation-related proteins. Heterogenous nuclear ribonucleoprotein G was mainly localized in neurons. Amyotrophic lateral sclerosis mice were examined at three stages: preonset (60-70 days), onset (90-100 days) and progression (120-130 days). The number of heterogenous nuclear ribonucleoprotein G-positive cells was significantly higher in the anterior horn of the lumbar spinal cord segment of TG mice at the preonset stage than that of control group but lower than that of the control group at the onset stage. The number of heterogenous nuclear ribonucleoprotein G-positive cells in both central canal and surrounding gray matter of the whole spinal cord of TG mice at the onset stage was significantly lower than that in the control group, whereas that of the lumbar spinal cord segment of TG mice was significantly higher than that in the control group at preonset stage and significantly lower than that in the control group at the progression stage. The numbers of heterogenous nuclear ribonucleoprotein G-positive cells in the posterior horn of cervical and thoracic segments of TG mice at preonset and progression stages were significantly lower than those in the control group. The expression of heterogenous nuclear ribonucleoprotein G in the cervical spinal cord segment of TG mice was significantly higher than that in the control group at the preonset stage but significantly lower at the progression stage. The expression of heterogenous nuclear ribonucleoprotein G in the thoracic spinal cord segment of TG mice was significantly increased at the preonset stage, significantly decreased at the onset stage, and significantly increased at the progression stage compared with the control group. heterogenous nuclear ribonucleoprotein G expression in the lumbar spinal cord segment of TG mice was significantly lower than that of the control group at the progression stage. After heterogenous nuclear ribonucleoprotein G gene silencing, PC12 cell survival was lower than that of control cells. Both TAR DNA binding protein 43 and Bax expressions were significantly increased in heterogenous nuclear ribonucleoprotein G-silenced cells compared with control cells. Our study suggests that abnormal distribution and expression of heterogenous nuclear ribonucleoprotein G might play a protective effect in amyotrophic lateral sclerosis development via preventing neuronal death by reducing abnormal TAR DNA binding protein 43 generation in the spinal cord.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Relationship between Neuroprotective Effects and Structure of Procyanidins.

This study evaluated the relationship between the neuroprotective effects of procyanidins and their structural characteristics. In vitro, a rat pheochromocytoma cell line (PC12) was exposed to the grape seed-derived procyanidin monomers: catechin (C), epicatechin (EC), and epicatechin gallate (ECG); the procyanidin dimers: procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), and procyanidin B2-3-O-gallate (B2-G); and the procyanidin trimers: procyanidin C1 (C1) and N-acetyl-l-cysteine (NAC) for 24 h. Cells were then incubated with 200 μM H2O2 for 24 h. In vivo, zebrafish larvae (AB strain) 3 days post-fertilization were incubated with NAC or procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) in 300 µM H2O2 for 4 days. Different grape seed procyanidins increased the survival of PC12 cells challenged with H2O2, improved the movement behavior disorder of zebrafish caused by H2O2, inhibited the increase of ROS and MDA and the decrease of GSH-Px, CAT, and SOD activities, and up-regulated the Nrf2/ARE pathway. The neuroprotective effects of the procyanidin trimer C1 treatment group were greater than the other treatment groups. These results suggest that the neuroprotective effect of procyanidins is positively correlated with their degree of polymerization.