Treated SH-SY5Y Cells Research Articles

LncRNA BDNF-AS promotes autophagy and apoptosis in MPTP-induced Parkinson’s disease via ablating microRNA-125b-5p

BackgroundsRecently, extensive evidence has indicated that the biological role of long non-coding RNAs (lncRNAs) in neurodegenerative diseases is becoming increasingly evident. The lncRNA brain-derived neurotrophic factor anti-sense (BDNF-AS) has been found to be dysregulated in Huntington’s Disease. However, the function of BDNF-AS in Parkinson's disease (PD) remains unknown. The purpose of this present study was to explore the effect of BDNF-AS on PD and its underlying molecular mechanisms. MethodsThe MPTP-induced mouse model of PD and MPP+-induced SH-SY5Y cell model were established. Immunofluorescence was performed to determine the number of TH + positive cells. Mice behavioral changes were detected by pole and rota-rod test. SH-SY5Y cells viability, apoptosis was detected by MTT assay and flow cytometry. The number of autophagosome was measured by transmission electron microscopy. Dopamine content was tested by high performance liquid chromatography. Dual-luciferase reporter gene assay was utilized to verify the correlation between BDNF-AS and miR-125b-5p. qRT-PCR and western blot were used to detect gene expression levels. ResultsOur results showed that BDNF-AS was up-regulated in MPTP-induced PD model and dopamine neurons, and MPP + treated SH-SY5Y cells, while miR-125b-5p was down-regulated. The expression of BDNF-AS was positively related with the MPP + concentration. BDNF-AS knockdown could significantly promote cell proliferation, while inhibit apoptosis and autophagy in SH-SY5Y cells treated by MPP + . Silencing BDNF-AS could also increase TH positive neurons and significantly suppress the autophagy of PD mice. Additionally, miR-125b-5p, a putative target gene of BDNF-AS, was involved in the effects of BDNF-AS on SH-SY5Y cell apoptosis and autophagy. ConclusionsOur study demonstrated that knockdown of BDNF-AS could elevate SH-SY5Y cell viability, inhibit autophagy and apoptosis in MPTP-induced PD models through regulating miR-125b-5p, suggesting that BDNF-AS might act as a potential therapeutic target for PD.

Knockdown of long non-coding RNA AL049437 mitigates MPP+ -induced neuronal injury in SH-SY5Y cells via the microRNA-205-5p/MAPK1 axis

BackgroundLong noncoding RNAs (lncRNAs) have been defined as critical regulators of various human diseases. However, the functions of lncRNAs in Parkinson's disease (PD) have not yet been elucidated. In this study, we investigated the role of lncRNA AL049437 in PD and its underlying mechanism. MethodsAn in vivo model of PD was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), while an in vitro model was created using N-methyl-4-phenylpyridinium (MPP+). Gene expression was evaluated using quantitative reverse transcriptase polymerase chain reaction and western blotting. The effects and mechanism of AL049437 in PD were explored using Cell Counting Kit-8 assay, flow cytometry, enzyme-linked immunosorbent assay, and 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate fluorescence assay. The interaction between AL049437, miR-205-5p, and mitogen-activated protein kinase 1 (MAPK1) was evaluated using luciferase reporter and RNA pull-down assays. ResultsThe expression of AL049437 was upregulated, while that of miR-205-5p was downregulated in MPTP-induced PD mouse model and MPP+-treated SH-SY5Y cells. Silencing of AL049437 mitigated MPP+-induced neurotoxicity in SH-SY5Y cells, as demonstrated by increased cell viability and reduced cell apoptosis. Furthermore, silencing of AL049437 alleviated MPP+-induced neuroinflammation and oxidative stress, as indicated by the reduction in tumor necrosis factor-α and interleukin-6 levels and reactive oxygen species production. In addition, AL049437 was predominantly localized in the cytoplasm of SH-SY5Y cells and functioned as an miR-205-5p sponge. Moreover, MAPK1 was identified as a downstream target of miR-205-5p. Remarkably, the impact of AL049437 silencing on MPP+-induced neuronal damage could be blocked by miR-205-5p inhibition or MAPK1 overexpression. ConclusionKnockdown of lncRNA AL049437 mitigates MPP+ -induced neuronal injury in SH-SY5Y cells by regulating the miR-205-5p/MAPK1 axis. Our research reveals a novel regulatory mechanism of AL049437 in PD progression.

Astrocyte-Derived Exosomal microRNA miR-200a-3p Prevents MPP+-Induced Apoptotic Cell Death Through Down-Regulation of MKK4.

Astrocytes release exosomes that regulate neuronal cell function. 1-methyl-4-phenylpyridinium (MPP+) is a well-known neurotoxin used to induce cell death in in vitro Parkinson's disease models, and microRNA (miRNA) transferred by released exosomes can regulate its mechanisms. Here, we demonstrated that exosomes released from normal astrocytes (ADEXs), but not exosomes derived from MPP+-stimulated astrocytes (MPP+-ADEXs), significantly attenuate MPP+-induced cell death in SH-SY5Y cells and primary mesencephalic dopaminergic neuron cultures, and reduce expression of mitogen-activated protein kinase kinase 4 (MKK4), an important upstream kinase in the c-Jun N-terminal kinase cell death pathway. Similar neuroprotective results were obtained from primary hippocampal neuron cultures, an in vitro glutamate excitotoxicity model. Through small-RNA sequencing of exosomal miRNA, we identified miR-200a-3p as the most down-regulated miRNA expressed in MPP+-ADEXs. miRNA target analysis and reporter assay confirmed that miR-200a-3p targets MKK4 through binding to two independent sites on the 3'-UTR of Map2k4/MKK4 mRNA. Treatment with miR-200a-3p mimic suppressed both MKK4 mRNA and protein expressions, and attenuated cell death in MPP+-treated SH-SY5Y cells and glutamate-treated hippocampal neuron cultures. Our results suggest that normal astrocytes release miR-200a-3p which exhibits a neuroprotective effect through down-regulation of MKK4.

Melatonin reduces OGD/R-induced neuron injury by regulating redox/inflammation/apoptosis signaling.

To investigate the roles and underlying mechanisms of melatonin in oxygen-glucose deprivation/reoxygenation (OGD/R)-insulted SH SY5Y cells. SH SY5Y cells were cultured for OGD/R stimulation. Cell viability and cytotoxicity were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and Hoechst 33258/propidium iodide (PI) staining assays. The mRNA levels of high mobility group box-1 (HMGB1), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) were analyzed by quantitative Real Time-PCR assays. Nitric oxide (NO) production was assessed by Griess reagent. Reactive oxygen species (ROS) production was detected by fluorescent probe. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) were examined by commercial kits. Cell apoptosis was analyzed by flow cytometry and caspase-3 activity. The protein levels were detected by Western blot. Melatonin enhanced the viability and reduced the death and LDH release of OGD/R exposed SH SY5Y cells. Melatonin repressed the HMGB1, TNF-α, and iNOS mRNA expression, NO production, and nuclear factor κB (NF-κB) activation in OGD/R challenged SH SY5Y cells. Melatonin reduced the ROS, MDA, 4-HNE, and 8-OHdG contents but further enhanced the levels of the nuclear factor E2-related factor-2 (Nrf2) and heme oxygenase (HO-1). Melatonin-increased viability and melatonin-decreased LDH release were also mediated by the blockage of NF-κB or reversed by Nrf2 or HO-1 knockdown. Melatonin exerted antiapoptotic effect on OGD/R treated SH SY5Y cells partly by activating Akt signaling. OGD/R challenged SH SY5Y cell autophagy was also repressed by melatonin, as evidenced by the decreased levels of LC-II and beclin-1 and the increased phosphorylation of mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP-1). Melatonin protected SH SY5Y cells from OGD/R induced oxidative stress, inflammation, apoptosis, and autophagy by blocking NF-κB signaling and activating Nrf2/HO-1, Akt, and mTOR/p70S6K/4E-BP-1 pathways, thereby indicating that melatonin is a potential and novel therapeutic drug for ischemic stroke.

FGF21 Protects Dopaminergic Neurons in Parkinson's Disease Models Via Repression of Neuroinflammation.

Fibroblast growth factor21 (FGF21), a member of the FGF family, plays multiple biological functions including anti-inflammation, anti-oxidative stress, and anti-apoptosis. It has been shown that FGF21 protects cells from acute injury in several kinds of cells such as islet β-cells, endothelial cells, cardiomyocytes, and dopaminergic neurons. However, whether FGF21 plays neuroprotective roles against Parkinsonian syndrome in vivo has not been elucidated. Our results showed that FGF21 markedly improves cell survival in MPP+-treated SH-SY5Y cells and primary dopaminergic neurons. Furthermore, we treated MPTP-induced Parkinson's disease (PD) model mice with the recombinant FGF21 via intranasal pathway. The results showed that FGF21 treatment significantly improves behavioral performances and prevents tyrosine hydroxylase (TH) loss in the substantia nigra par compacta (SNpc) and striatum. Mechanistically, FGF21 stimulates the AMPK/PGC-1α axis to promote mitochondrial functions. Moreover, FGF21 attenuates microglia and astrocyte activation induced by MPTP, leading to a low level of inflammation in the brain. Our data indicate that FGF21 prevents dopaminergic neuron loss and shows beneficial effects against MPTP-induced PD syndrome in mice, indicating it might be a potent candidate for developing novel drugs to deal with PD.

MiR-652 protects rats from cerebral ischemia/reperfusion oxidative stress injury by directly targeting NOX2

Ischemic stroke is a devastating central nervous disease associated with oxidative stress and NOX2 is the main source of ROS responsible for brain tissue. miRNAs are a class of negative regulator of genes in mammals and involves the pathogenesis of ischemic stroke. This study aims to observe the role of target miRNA(miR-652) of NOX2 in ischemic stroke. A rat cerebral ischemia/reperfusion (CI/R) injury model and an SH-SY5Y cell hypoxia/reoxygenation(H/R) model were used to simulate ischemic stroke, and corresponding gene expression, biochemical indicators and pathophysiological indicators were measured to observe the role of miR-652. NOX2 significantly increased in brain tissues subjected to I/R or in SH-SY5Y cells subjected to H/R, while the expression level of miR-652(potential target of NOX2) significantly decreased in both brain tissues and plasma. Overexpression of miR-652 significantly suppressed NOX2 expression and ROS generation in H/R treated SH-SY5Y cells and reduced the relative luciferase activity of cells transfected with plasmid NOX2-WT (reporter gene plasmid). MiR-652 agomir significantly decreased the expression of NOX2 and ROS generation in brain tissues of CIR rats, as well as tissue injury. These data indicated that miR-652 protected rats from cerebral ischemia reperfusion injury by directly targeting NOX2, is a novel target for ischemic stroke therapy.

Pro-survival and anti-inflammatory roles of NF-κB c-Rel in the Parkinson's disease models

The pathological hallmarks of Parkinson's disease (PD) are the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the presence of overactivated glial cells and neuroinflammation. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) c-Rel subunit is closely related in the pathological progress of PD, however the roles and mechanisms of c-Rel in PD development remain unclear. Here, in neurotoxins-induced PD models, the dynamic changes of NF-κB c-Rel and its functions were evaluated. We found that c-Rel was rapidly activated in the nigrostriatal pathway, which mainly occurred in dopaminergic neurons and microglia. c-Rel could maintain neuronal survival by initiating the anti-apoptotic gene expression in MPP+-treated SH-SY5Y cells and it could inhibit microglial overactivation by suppressing the inflammatory gene expression in LPS-challenged BV2 cells. c-Rel inhibitor IT901 aggravated the damage of MPTP on dopaminergic neurons and promoted the activation of microglia in the nigrostriatal pathway of mice. Moreover, the expression of c-Rel in blood samples of PD patients decreased dramatically. Our results indicate that the NF-κB/c-Rel subunit plays an important role in neuroprotection and neuroinflammation inhibition during PD progression.

Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) protein levels. ACH caused reduction in ERβ protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERβ protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERβ expression, increase in ERα, may contribute to reduction in ERβ levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERβ localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH–induced increase in ERβ mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

SNHG1 promotes MPP+-induced cytotoxicity by regulating PTEN/AKT/mTOR signaling pathway in SH-SY5Y cells via sponging miR-153-3p

BackgroundLong non-coding RNA small molecule RNA host gene 1 (SNHG1) was previously identified to be relevant with Parkinson’s disease (PD) pathogenesis. This work aims to further elucidate the regulatory networks of SNHG1 involved in PD.Methods1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-hydrochloride (MPTP)-induced mice and 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells were respectively constructed as the in vivo and in vitro PD models. Expression levels of SNHG1 and miR-153-3p were detected by qRT-PCR. Protein expression levels of phosphate and tension homology deleted on chromosome ten (PTEN) were measured by western blotting assay. Cell viability and apoptosis were determined by MTT and flow cytometry assays. The interactions among SNHG1, miR-153-3p and PTEN were identified by luciferase reporter assay, RNA immunoprecipitation, and/or RNA pull-down analysis.ResultsIncreased SNHG1 expression was found in midbrain of MPTP-induced PD mice and MPP+-treated SH-SY5Y cells. Overexpression of SNHG1 lowered viability and enhanced apoptosis in MPP+-treated SH-SY5Y cells. Moreover, SNHG1 acted as a molecular sponge to inhibit the expression of miR-153-3p. Furthermore, miR-153-3p-mediated suppression of MPP+-induced cytotoxicity was abated following SNHG1 up-regulation. Additionally, PTEN was identified as a direct target of miR-153-3p, and SNHG1 could serve as a competing endogenous RNA (ceRNA) of miR-153-3p to improve the expression of PTEN. Besides, enforced expression of PTEN displayed the similar functions as SNHG1 overexpression in regulating the viability and apoptosis of MPP+-treated SH-SY5Y cells. Finally, SNHG1 was found to activate PTEN/AKT/mTOR signaling pathway in SH-SY5Y cells by targeting miR-153-3p.ConclusionSNHG1 aggravates MPP+-induced cellular toxicity in SH-SY5Y cells by regulating PTEN/AKT/mTOR signaling via sponging miR-153-3p, indicating the potential of SNHG1 as a promising therapeutic target for PD.

Mesenchymal Stem Cell-Conditioned Medium Improves Mitochondrial Dysfunction and Suppresses Apoptosis in Okadaic Acid-Treated SH-SY5Y Cells by Extracellular Vesicle Mitochondrial Transfer.

Mesenchymal stem cells-conditioned medium (MSC-CM) provides a promising cell-free therapy for Alzheimer's disease (AD) mainly due to the paracrine of MSCs, but the precise mechanisms remain unclear. Studies suggests that mitochondrial dysfunction precedes the accumulation of amyloid-β plaques and neurofibrillary tangles, and involves in the onset and development of AD. In the present study, we evaluated the protective effects and explored the related-mitochondrial mechanisms of human umbilical cord derived MSC-CM (hucMSC-CM) in an AD model in vitro. To this end, an AD cellular model was firstly established by okadaic acid (OA)-treated SH-SY5Y cells, and then treated by hucMSC-CM to assess the oxidative stress, mitochondrial function, apoptosis, AD-related genes, and signaling pathways. hucMSC-CM significantly deceased tau phosphorylated at Thr181 (p181-tau) level, which was increased in AD. hucMSC-CM also alleviated intracellular and mitochondrial oxidative stress in OA-treated SH-SY5Y cells. In addition, hucMSC-CM suppressed apoptosis and improved mitochondrial function in OA-treated SH-SY5Y cells. Flow cytometric analysis indicated that hucMSC-CM exerted the protective effects relying on or partly extracellular vesicle (EV) mitochondrial transfer from hucMSCs to OA-treated SH-SY5Y cells. Moreover, RNA sequencing data further demonstrated that hucMSC-CM regulated many AD-related genes, signaling pathways and mitochondrial function. These results indicated that MSC-CM or MSC-EVs containing abundant mitochondria may provide a novel potential therapeutic approach for AD.

MiR-142-5p Protects Against 6-OHDA-Induced SH-SY5Y Cell Injury by Downregulating BECN1 and Autophagy.

Background:MiR-142-5p has been demonstrated to hold significant implications in neurological diseases. However, the impact and underlying regulatory mechanism of miR-142-5p in Parkinson’s disease (PD) are still ominous.Methods:To simulate the PD, 6-hydroxydopamine (6-OHDA)-treated SH-SY5Y cell model was used in this study. Levels of messenger RNA and protein were tested by quantitative real-time polymerase chain reaction and Western blot analyses, respectively. The direct interaction between miR-142-5p and Beclin 1 (BECN1) was assessed by luciferase reporter assay. Furthermore, Cell Counting Kit-8 assay was performed to assess cytotoxicity of SH-SY5Y cell.Results:In consequence, a significant decrease of miR-142-5p was observed in 6-OHDA-induced SH-SY5Y cells. Over-/Low-expressed miR-142-5p resulted in a significant enhancement/inhibition on cell vitalities of 6-OHDA-treated SH-SY5Y cells, which might be modulated by repressing cellular autophagy through inhibiting level of BECN1 and LC3 II/LC3 I and elevating P62 level. Luciferase reporter assay showed that the BECN1 was the target gene of miR-142-5p. Additionally, the loss/gain of BECN1 rescued/blocked the effects of miR-142-5p on the viability of 6-OHDA-induced SH-SY5Y cells.Conclusions:These results highlight that miR-142-5p functions as a neuroprotective regulator in 6-OHDA-induced neuronal SH-SY5Y cells simulating PD model in vitro via regulating autophagy-related protein BECN1 and autophagy to influence cell viability.

Astilbin attenuates cerebral ischemia/reperfusion injury by inhibiting the TLR4/MyD88/NF-κB pathway.

Ischemic stroke is the second most common cause of death worldwide and cerebral ischemia/reperfusion (I/R) injury also leads to serious tissue damage. Astilbin, a natural bioactive flavonoid compound, has been reported to have protective effects on neurological diseases. This study aims to investigate the effects of astilbin on cerebral I/R injury and determine the mechanisms involved. The results demonstrated that, in cerebral I/R rats, astilbin could attenuate I/R injury in the hippocampal region, decreasing the activity of lactate dehydrogenase (LDH) and malondialdehyde (MDA) in the rat brain. Astilbin also inhibited the I/R-induced upregulation of pro-inflammatory mediators (TNFα, IL-1β, IL-6). Similarly, in hypoxia/reperfusion (H/R) treated human neuroblastoma cells, astilbin could increase the cell viability of SH-SY5Y, decrease the activity of LDH and MDA, and inhibit the H/R-induced upregulation of pro-inflammatory mediators. For the mechanism study, western blot results indicated that astilbin could inhibit the expression of Toll-like receptor 4 (TLR4), myeloid differential protein 88 (MYD88) and phosphorylated NF-κB p65 in H/R treated SH-SY5Y cells. The research indicated that astilbin ameliorated cerebral I/R injury partly via the TLR4/MyD88/NF-κB pathway. Astilbin may have potential therapeutic effects on cerebral ischemia.

Differentially expressed long-chain noncoding RNAs in human neuroblastoma cell line (SH-SY5Y): Alzheimer’s disease cell model

ABSTRACTA number of complex human diseases including neurological diseases is characterized by dysregulation of long-chain noncoding RNA (lncRNA). The pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder is believed to involve alterations in lncRNAs. However, the specific lncRNAs modified in AD remain to be determined. The aim of this study was to identify lncRNAs associated with AD using human neuroblastoma cell line (SH-SY5Y) treated with beta-amyloid (Aβ) as a model of this disease. The differential expressions of lncRNA were compared between beta-amyloid (Aβ) SH-SY5Y cells and normal SH-SY5Y cells utilizing Illumina X10 gene sequencing. The differential expression profiles of amyloid (Aβ)-treated SH-SY5Y cells were determined and verified by qRT-PCR method. The expression levels of lncRNA were expressed by calculating the abundance of FPKM (measure gene expression). The differential expression of log2 (multiple change) >1 or log2 (multiple change) < −1 had statistical significance (P< .05). The differential expression profiles of amyloid (Aβ)-treated SH-SY5Y cells showed 40 lncRNA were up-regulated, while 60 lncRNA were down-regulated. GO and KEGG analysis demonstrated that differentially expressed genes were predominantly involved in the mitogen-activated protein kinase (MAPK) signaling pathway, p53 signaling pathway, hepatitis B, cell cycle, post-translational protein modification, and regulation. In conclusion, approximately 100 dysregulated lncRNA transcripts were found in amyloid (Aβ)-treated SH-SY5Y cells and these lncRNAs may play an important role in the occurrence and development of AD through altered signal pathways.

A 2x folic acid treatment affects epigenetics and dendritic spine densities in SHSY5Y cells

Many diseases are now associated with aberrant epigenetics and gene expression changes. Epigenetics can be modified by factors like diet. One dietary factor, folic acid, is consumed in various forms including supplements, energy drinks, and fortified grains. It was hypothesized high levels of folic acid would affect gene expression and enzyme activity of chromatin modifying enzymes as well as dendritic spine densities in a commonly utilized neuron model, the SHSY5Y cell. Decreased MBD2 and MECP2 were discovered upon treatment of SHSY5Y cells with a 2x folic acid dose. Corresponding decreases in dendritic spines were apparent in the 2x folic acid treated cells as well. Activity of DNMTs and H3K4 HMTs was altered. Further, H3K4me1, H3K4me3, H3K9Ac, and global DNA methylation were decreased in the 2x folic acid treated cells. Further studies are warranted to determine if the effects of excess folic acid are detrimental to organismal physiology.

Epigallocatechin-3-Gallate AttenuatesMicroglial Inflammation and Neurotoxicity by Suppressing the Activation of Canonical and Noncanonical Inflammasome via TLR4/NF-κB Pathway.

In this study, it has been investigated whether the neuroprotective efficacy of epigallocatechin-3-gallate (EGCG) is mediated by inhibition of canonical and noncanonical inflammasome activation via toll-like receptor 4 (TLR4)/NF-κB pathway both in LPS+Aβ-induced microglia in vitro and in APP/PS1 mice in vivo. In BV2 cells, EGCG inhibits the expressions of Iba-1, cleaved IL-1β, and cleaved IL-18 induced by LPS+Aβ. Then, thesupernatantsare used to treat SH-SY5Y cells, and EGCG treatment significantly recovers the neurotoxicity from LPS+Aβ-induced microglial conditioned media. Subsequently, it has been found that EGCG reduces the microglial expressions of caspase-1 p20, NLRP3, and caspase-11 p26. Furthermore, the expression levels of Toll-like receptor 4 (TLR4), p-IKK/IKK, and p-NF-κB/NF-κB were decreased after EGCG treatment. As expected, when a caspase-1 specific inhibitor Z-YVAD-FMK, and an IKK and caspase-11 inhibitor wedelolactone are used for blocking, Z-YVAD-FMK and wedelolactone exacerbate the inhibitory efficacy than using EGCG alone. Finally, consistent with the results obtained in BV2 cells, EGCG treatment reduces microglial inflammation and neurotoxicity by suppressing the activation of canonical NLRP3 and noncanonical caspase-11-dependent inflammasome via TLR4/NF-κB pathway in LPS+Aβ-induced rat primary microglia and hippocampus of APP/PS1 mice. EGCG attenuates microglial inflammation and neurotoxicity by inhibition of canonical NLRP3 and noncanonical caspase-11-dependent inflammasome activation via TLR4/NF-κB pathway.

Luteolin-7-O-glucoside protects dopaminergic neurons by activating estrogen-receptor-mediated signaling pathway in MPTP-induced mice

BackgroundParkinson's disease (PD) is a neurodegenerative disorder that is characterized by the degeneration of dopaminergic neurons in substantia nigra (SN). Accumulating evidences implicate the beneficial role of estrogen in the therapy of PD. MethodsIn the present study, the protective function of luteolin-7-O-glucoside (LUT-7G), a natural flavonoid, was investigated in 1-methyl-4-phenylpyridinium (MPP+) treated SH-SY5Y cells and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mice. ResultsPre-treatment of LUT-7G increased the viability and reduced the apoptosis of SH-SY5Y cells treated by MPP+. At molecular level, the Bcl-2/Bax ratio was increased, while the expression of cleaved caspase 3 was markedly lessened. Moreover, LUT-7G increased the expression of estrogen receptor (ER), ERα and ERβ, and enhanced the activation of ERK1/2/STAT3/c-Fos that could be abolished by ER antagonists. Furthermore, in vivo experiment indicated that pre-treatment of LUT-7G improved the bradykinesia, and enhanced the muscle strength as well as the balancing capacity of mice treated with MPTP. And LUT-7G prevented the injury of TH positive cells in substantia nigra and increased TH positive nerve fibers in striatum. In addition, pre-treatment of LUT-7G also significantly diminished the MPTP-induced gliosis in substantia nigra. ConclusionsLUT-7G effectively protected dopaminergic neurons against MPP+ or MPTP-induced toxicity, probably by activating the ER-mediated signaling pathway. Our findings explore the therapeutic potential of LUT-7G for PD therapy.

Metformin inhibits Aβ25-35 -induced apoptotic cell death in SH-SY5Y cells.

Metformin, a first-line drug for type-2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aβ25-35 -treated SH-SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl-2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aβ25-35 -treated SH-SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca2+ and ROS in Aβ25-35 -treated SH-SY5Y cells. These findings suggest that metformin inhibits Aβ25-35 -treated SH-SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca2+ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.

NEAT1 regulates MPP+-induced neuronal injury by targeting miR-124 in neuroblastoma cells

Long non-coding RNAs (lncRNAs) have been reported to play important roles in Parkinson’s disease (PD) pathogenesis. It was indicated that lncRNA nuclear enriched abundant transcript 1 (NEAT1) is involved in PD. However, the underlying mechanism of NEAT1 is still not fully explored. Human neuroblastoma cell line SH-SY5Y was treated with 1-Methyl-4-phenylpyridinium (MPP+) to mimic PD model in vitro. qRT-PCR was employed to detect the expression of NEAT1, IL-1β, IL-6 and TNF-α. Starbase database, RNA pull-down assay and RNA immunoprecipitation (RIP) assay were performed to verify the relationship between NEAT1 and miR-124. MTT assay and flow cytometry assay were used to detect cell viability and apoptosis. Elisa was introduced to measure the levels of IL-1β, IL6 and TNF-α in culture media. We found that NEAT1 expression was significantly increased in SH-SY5Y cells after MPP+ treatment in dose- and time- dependent manners. MPP+ induced the expression and secretion of IL-1β, IL-6 and TNF-α, inhibited cell viability and induced apoptosis while these effects could be rescued by NEAT1 silencing. miR-124 was a target of NEAT1. Anti-miR-124 could reverse the effects caused by NEAT1 knockdown in MPP+ treated SH-SY5Y cells. Therefore, we speculated that NEAT1 may regulate MPP+ induced neuronal injury by targeting miR-124 in SH-SY5Y cells.

KHG21834 attenuates glutamate-induced mitochondrial damage, apoptosis, and NLRP3 inflammasome activation in SH-SY5Y human neuroblastoma cells

New compounds were screened to develop effective drugs against glutamate-induced toxicity. The present study assessed the effects of the novel thiazole derivative KHG21834 against glutamate-induced toxicity in human neuroblastoma SH-SY5Y cell cultures. Treatment of SH-SY5Y cells with KHG21834 significantly protected cells against glutamate-induced toxicity in a dose-dependent manner, with an optimum concentration of 50 μM. KHG21834 protected SH-SY5Y cells against glutamate toxicity by suppressing glutamate-induced oxidative stress by 50%. KHG21834 also attenuated glutamate-induced mitochondrial membrane potential, ATP level reductions, and intracellular Ca2+ influx. Furthermore, KHG21834 efficiently reduced glutamate-induced ER stress and NLRP3 inflammasome activation (59% and 65% of glutamate group, respectively). In addition, KHG21834 effectively attenuated glutamate-induced levels of Bax, Bcl-2, cleaved caspase-3, p-p38, p-JNK proteins, and TUNEL positive cells. To our knowledge, this is the first study showing that KHG21834 can effectively protect SH-SY5Y cells against glutamate toxicity, suggesting that this compound may be a valuable therapeutic agent for the treatment of glutamate toxicity.

Attenuation of Endoplasmic Reticulum Stress, Impaired Calcium Homeostasis, and Altered Bioenergetic Functions in MPP+-Exposed SH-SY5Y Cells Pretreated with Rutin.

Parkinson's disease (PD) is a common neurodegenerative disorder that affects approximately 1% of the population over the age of 65years. While treatment options for PD are limited, reports show that plant-derived bioactive compounds such as rutin possess numerous pharmacological benefits, including antioxidant and antiapoptotic activities. This study aimed to investigate the potential role of rutin in MPP+-treated SH-SY5Y neuroblastoma cells, an established cell model of PD. Our findings reveal increased concentrations of Ca2+ and endoplasmic reticulum (ER) stress as well as impaired mitochondrial membrane potential and bioenergetic status in SH-SY5Y cells treated with MPP+ only. This is demonstrated by a significant reduction in the expression levels of BiP, significantly reduced basal respiration, maximal respiration, and spare respiratory capacity as well as a significant increase in the expression levels of CHOP; however, these effects were significantly attenuated following pretreatment with rutin. Also, rutin significantly improved basal and compensatory glycolysis as a response to an impaired oxidative phosphorylation system triggered by MPP+, characterized by deficient ATP production. In conclusion, our findings provide the first evidence on the ability of rutin to maintain Ca2+ homeostasis, inhibit ER stress, and protect the mitochondria in MPP+-treated SH-SY5Y cells.