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Background: Methamphetamine use is associated with several negative consequences, including neurotoxicity and greater probability of exhibiting a substance use disorder. Sigma1 receptor is involved in the neurobiological basis of several drug use disorders. Cannabidiol has received attention in the treatment of drug use disorders and neurotoxicity.Objectives: To investigate the effects of cannabidiol on methamphetamine-induced conditioned place preference (CPP) and the viability of PC12 cells.Methods: Adult male rats (n = 70) underwent methamphetamine (2 mg/kg, IP) induced CPP, and were administered cannabidiol (10, 20, 40, or 80 mg/kg, IP) during the methamphetamine withdrawal period for five consecutive days. Methamphetamine (0.5 mg/kg) was then injected to reactivate CPP. Four brain regions (ventral tegmental area, nucleus accumbens, prefrontal cortex, and hippocampus) were extracted after the last test. PC12 cells were treated with cannabidiol, Sigma1R-siRNA, or BD1047 before methamphetamine exposure.Results: Administration of 20, 40, or 80 mg/kg cannabidiol facilitated CPP extinction (80 mg/kg, p < .001) and prevented CPP development (80 mg/kg, p < .0001). This was associated with changes in the expression of Sigma1R (ventral tegmental area, 80 mg/kg, p < .0001) in the four brain regions. Cannabidiol protected the PC12 cell's viability (10 µM, p = .0008) and inhibited the methamphetamine-induced activation of the AKT/GSK3ß/CREB signaling pathway by mediating Sigma1R (10 µM, p < .0001).Conclusions: Cannabidiol seems to inhibit the rewarding effects of methamphetamine and the effects of this drug on cell viability. Sigma1R should be given further consideration as a potential target for cannabidiol.
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Canabidiol , Metanfetamina , Animais , Canabidiol/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Metanfetamina/efeitos adversos , Células PC12 , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/fisiologiaRESUMO
Purpose: Gait variability analysis has been clinically adopted to characterize the presentation of various neurological diseases. However, literature and practice lack a comprehensive murine model assessment of the gait deficits that result from transient focal ischemic stroke. Further, correlations between gait parameters and the gene expression profiles associated with brain ischemia have yet to be identified. This study quantitatively assesses gait deficits through a murine model of transient focal cerebral ischemia on day 7 to determine associations between gait deficits and ischemia-related gene expressions.Methods: A total of 182 dynamic and static gait parameters from the transient middle cerebral artery occlusion (MCAO) murine model for simulating human transient focal ischemic stroke on day 7 were measured using the CatWalk system. Pearson's correlation analysis and genes associated with ischemia were identified from the existing literature to aid the investigation of the relationship between gait variability and gene expression profiles.Results: Thirty-nine gait parameters and the mRNA expression levels of four of the eight ischemia-associated genes exhibited more significant change in the MCAO models (p < 0.005) on day 7. Twenty-six gait parameters exhibited strong correlations with four ischemia-associated genes.Conclusion: This examination of gait variability and the strong correlation to the gene expression profiles associated with transient focal brain ischemia on day 7 provides a quantitative and reliable assessment of the MCAO model's motor performance. This research provides valuable insights into the study of disease progression and offers novel therapeutic interventions in the murine modeling of ischemic stroke.
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Marcha/genética , Marcha/fisiologia , Expressão Gênica/genética , Expressão Gênica/fisiologia , Ataque Isquêmico Transitório/genética , Acidente Vascular Cerebral/genética , Animais , Correlação de Dados , Infarto da Artéria Cerebral Média , Ataque Isquêmico Transitório/complicações , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Camundongos , Córtex Motor/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologiaRESUMO
BACKGROUND: Stroke represents the second leading cause of death and the primary cause of long-term disability in humans. The transplantation of mesenchymal stem cells (MSC) reportedly improves functional outcomes in animal models of cerebral ischemia. Here, we evaluate the neuroprotective potential of extracellular vesicles secreted from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-EV) using preclinical cell-based and animal-based models of ischemic strokes. METHODS: hiPS-MSC-EV were isolated using an ultrafiltration method. HT22 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) injury for 2 h, followed by treatment with hiPS-MSC-EV (100 µg/mL). Male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by an intravenous injection of hiPS-MSC-EV (100 µg) at three distinct time points. RESULTS: Our experimental approach revealed hiPS-MSC-EV promoted HT22 cell proliferation, reduced apoptosis, and altered cellular morphology following OGD/R. In addition, hiPS-MSC-EV reduced the volume of infarcts, improved spontaneous movement abilities, and enhanced angiogenesis by expressing the VEGF and CXCR4 proteins in the infarcted hemisphere of the MCAO-treated mouse model. CONCLUSION: Our findings provide evidence of the potential neuroprotective effects of hiPS-MSC-derived extracellular vesicles (hiPS-MSC-EVs) in both in vitro and in vivo mouse models of ischemic stroke. These results suggest that hiPS-MSC-EVs may play a role in neurorestoration and offer insights into potential cell-free strategies for addressing cerebral ischemia.
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Methamphetamine (METH) and HIV-1 lead to oxidative stress and their combined effect increases the risk of HIV-associated neurocognitive disorder (HAND), which may be related to the synergistic ferroptotic impairment in microglia. Ferroptosis is a redox imbalance cell damage associated with iron overload that is linked to the pathogenic processes of METH and HIV-1. NRF2 is an antioxidant transcription factor that plays a protective role in METH and HIV-1-induced neurotoxicity, but its mechanism has not been fully elucidated. To explore the role of ferroptosis in METH abuse and HIV-1 infection and the potential role of NRF2 in this process, we conducted METH and HIV-1 Tat exposure models using the BV2 microglia cells. We found that METH and HIV-1 Tat reduced the expression of ferroptotic protein GPX4 and the cell viability and enhanced the expression of P53 and the level of ferrous iron, while the above indices were significantly improved with pretreatment of ferrostatin-1. In addition, NRF2 knockdown accelerated METH and HIV-1 Tat-induced BV2 cell ferroptosis accompanied by decreased expression of SLC7A11. On the contrary, NRF2 stimulation significantly increased the expression of SLC7A11 and attenuated ferroptosis in cells. In summary, our study indicates that METH and HIV-1 Tat synergistically cause BV2 cell ferroptosis, while NRF2 antagonizes BV2 cell ferroptotic damage induced by METH and HIV-1 Tat through regulation of SLC7A11. Overall, this study provides potential therapeutic strategies for the treatment of neurotoxicity caused by METH and HIV-1 Tat, providing a theoretical basis and new targets for the treatment of HIV-infected drug abusers.
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Ferroptose , Infecções por HIV , HIV-1 , Metanfetamina , Humanos , Metanfetamina/toxicidade , HIV-1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sistema y+ de Transporte de AminoácidosRESUMO
Amanitin poisoning is one of the most life-threatening mushroom poisonings. α-Amanitin plays a key role in Amanita phalloides intoxication. α-Amanitin shows toxic effects on the liver. However, the mechanism by which α-amanitin induces liver injury has not been elucidated. Autophagy plays a crucial role in maintaining cellular homeostasis and is closely related to the occurrence of a variety of diseases. Studies have shown that autophagy may play an important role in the process of α-amanitin-induced liver injury. However, the mechanism of α-amanitin-induced autophagy remains unclear. Thus, this study aimed to explore the mechanisms of α-amanitin in inducing hepatotoxicity in Sprague Dawley (SD) rats and the normal human liver cell line L02 cells. The SD rats and L02 cells exposed to α-amanitin were observed to determine whether α-amanitin could induce the autophagy of rat liver and L02 cells. The regulatory relationship between autophagy and the AMPK-mTOR-ULK pathway by exposing the autophagy agonist (rapamycin (RAPA)), autophagy inhibitor (3-methylademine (3-MA)), and AMPK inhibitor (compound C) was also explored. Autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins were detected using Western blot. The results of the study indicated that exposure to different concentrations of α-amanitin led to morphological changes in liver cells and significantly elevated levels of ALT and AST in the serum of SD rats. Additionally, the expression levels of LC3-II, Beclin-1, ATG5, ATG7, AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 were significantly increased in the rat liver. And we found that L02 cells exposed to 0.5 µM α-amanitin for 6 h significantly induced autophagy and activated the AMPK-mTOR-ULK1 pathway. Pretreated with RAPA, 3-MA, and compound C for 1 h, the expression levels of autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins significantly changed. Our results indicates that autophagy and the AMPK-mTOR-ULK pathway are involved in the process of α-amanitin-induced liver injury. This study may foster the identification of actionable therapeutic targets for A. phalloides intoxication.
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Proteínas Quinases Ativadas por AMP , Doença Hepática Crônica Induzida por Substâncias e Drogas , Ratos , Animais , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Alfa-Amanitina , Ratos Sprague-Dawley , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais , Proteínas Relacionadas à Autofagia , Autofagia , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Methamphetamine (METH) is a psychostimulant that is abused throughout the world. METH is a highly addictive drug commonly used by persons living with HIV, and its use can result in cognitive impairment and memory deficits. METH and human immunodeficiency virus-1 transactivator of transcription (HIV-1Tat) have toxic and synergistic effects on the nervous system; however, the mechanism of their synergistic effects has not been clarified. We used BV2 cells, primary microglia, Nrf2-KO C57BL/6J mice, and autopsied brain tissues of METH-abusing, HIV infection, and METH-abusing individuals comorbid with HIV to explore the regulatory role of Nrf2/NQO1/HO-1 signal pathway on microglia autophagy. Our results showed that microglia were significantly activated by METH and HIV-1Tat protein. METH and HIV-1Tat protein combination significantly increase the autophagy-related proteins (LC3-II, Beclin-1, ATG5, and ATG7) expression in microglia and striatum of C57BL/6J mice. After silencing or knocking out the Nrf2 gene, the expression levels of autophagy-related proteins were significantly increased. In human brain tissue, microglia were activated, Nrf2, LC3-II, and Beclin-1 expression levels were raised, and the p62 expression level was decreased. Our results suggested that METH and HIV or HIV-1Tat synergistically affect autophagy. And the Nrf2 pathway plays a vital role in regulating the synergistic induction of microglial autophagy by METH and HIV-1Tat protein. This study may provide a theoretical basis and new ideas for effective targets for pharmacological intervention in HIV-infected patients with drug abuse.
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Estimulantes do Sistema Nervoso Central , Infecções por HIV , HIV-1 , Metanfetamina , Animais , Autofagia , Proteína Beclina-1/metabolismo , Estimulantes do Sistema Nervoso Central/farmacologia , Produtos do Gene tat/farmacologia , Humanos , Metanfetamina/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Microglia , NAD(P)H Desidrogenase (Quinona) , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de SinaisRESUMO
Many individuals infected with human immunodeficiency virus (HIV) are also afflicted with HIV-associated neurocognitive disorders (HANDs). Methamphetamine (METH) abuse puts HIV-1 patients at risk for HANDs because METH and HIV-1 proteins, such as trans-activator of transcription (Tat), can synergistically damage the blood-brain barrier (BBB). However, the underlying mechanism of METH- and HIV-Tat-induced BBB damage remains unclear. In this study, male adult tree shrews and human brain capillary endothelial cells were treated with HIV-Tat, METH, and gastrodin. We used western blotting to examine the expressions of glucose transporters (GLUT1 and GLUT3), tight junctions, and junctional adhesion molecule A (JAMA) and to evaluate the damage and detect Evans blue (EB) and fluorescein sodium in the brain to assess BBB permeability to study the effect of METH and the HIV-1 Tat protein on BBB function in vitro and in vivo. The results showed that the group treated with Tat and METH experienced a significant change at the ultrastructural level of the tree shrew cerebral cortex, decreased protein levels of occluding, claudin-5, Zonula occludens 1 (ZO1), and JAMA in vitro and in vivo, and increased levels of EB and fluorescein sodium in the tree shrew cerebral cortex. The protein levels of GLUT1 and GLUT3 was downregulated in patients with Tat- and METH-induced BBB damage. Pretreatment with gastrodin significantly increased the levels of EB and fluorescein sodium in the tree shrew cerebral cortex and increased the expressions of occluding, ZO1, JAMA, and GLUT1 and GLUT. These results indicate that gastrodin may offer a potential therapeutic option for patients with HANDs.
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Synergistic impairment of the blood-brain barrier (BBB) induced by methamphetamine (METH) and HIV-Tat protein increases the risk of HIV-associated neurocognitive disorders (HAND) in HIV-positive METH abusers. Studies have shown that oxidative stress plays a vital role in METH- and HIV-Tat-induced damage to the BBB but have not clarified the mechanism. This study uses the human brain microvascular endothelial cell line hCMEC/D3 and tree shrews to investigate whether the transient receptor potential melastatin 2 (TRPM2) channel, a cellular effector of the oxidative stress, might regulate synergistic damage to the BBB caused by METH and HIV-Tat. We showed that METH and HIV-Tat damaged the BBB in vitro, producing abnormal cell morphology, increased apoptosis, reduced protein expression of the tight junctions (TJ) including Junctional adhesion molecule A (JAMA) and Occludin, and a junctional associated protein Zonula occludens 1 (ZO1), and increased the flux of sodium fluorescein (NaF) across the hCMEC/D3 cells monolayer. METH and HIV-Tat co-induced the oxidative stress response, reducing catalase (CAT), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) activity, as well as increased reactive oxygen species (ROS) and malonaldehyde (MDA) level. Pretreatment with n-acetylcysteine amide (NACA) alleviated the oxidative stress response and BBB damage characterized by improving cell morphology, viability, apoptosis levels, TJ protein expression levels, and NaF flux. METH and HIV-Tat co-induced the activation and high protein expression of the TRPM2 channel, however, early intervention using 8-Bromoadenosine-5'-O-diphosphoribose (8-Br-ADPR), an inhibitor of TPRM2 channel, or TRPM2 gene knockdown attenuated the BBB damage. Oxidative stress inhibition reduced the activation and high protein expression of the TRPM2 channel in the in vitro model, which in turn reduced the oxidative stress response. Further, 8-Br-ADPR attenuated the effects of METH and HIV-Tat on the BBB in tree shrews-namely, down-regulated TJ protein expression and increased BBB permeability to Evans blue (EB) and NaF. In summary, the TRPM2 channel can regulate METH- and HIV-Tat-induced oxidative stress and BBB injury, giving the channel potential for developing drug interventions to reduce BBB injury and neuropsychiatric symptoms in HIV-infected METH abusers.
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Gait analysis has been widely used to examine the behavioral presentation of numerous neurological disorders. Thorough murine model evaluation of the subarachnoid hemorrhage (SAH)-associated gait deficits is missing. This study measures gait deficits using a clinically relevant murine model of SAH to examine associations between gait variability and SAH-associated gene expressions. A total of 159 dynamic and static gait parameters from the endovascular perforation murine model for simulating clinical human SAH were determined using the CatWalk system. Eighty gait parameters and the mRNA expression levels of 35 of the 88 SAH-associated genes were differentially regulated in the diseased models. Totals of 42 and 38 gait parameters correlated with the 35 SAH-associated genes positively and negatively with Pearson's correlation coefficients of >0.7 and <-0.7, respectively. p-SP1453 expression in the motor cortex in SAH animal models displays a significant correlation with a subset of gait parameters associated with muscular strength and coordination of limb movements. Our data highlights a strong correlation between gait variability and SAH-associated gene expression. p-SP1453 expression could act as a biomarker to monitor SAH pathological development and a therapeutic target for SAH.
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Análise da Marcha , Hemorragia Subaracnóidea/genética , Transcriptoma , Animais , Encéfalo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Força Muscular , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/fisiopatologiaRESUMO
[This corrects the article DOI: 10.1093/toxres/tfaa021.].
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Methamphetamine (METH) is a highly addictive psychostimulant. Cannabidiol (CBD) is an exogenous cannabinoid without psychostimulating activity, which has potential therapeutic effects on opioid addiction. However, it is unclear whether CBD has therapeutic effects on METH-induced motivational effects. The present study examines whether CBD has a protective effect on METH-induced conditioned place preference (CPP) in rats by regulating the Sigma1R and AKT-GSK3ß-CREB signaling pathway. Seventy rats were equally and randomly divided into seven groups. The rat CPP model was established via the intraperitoneal injection (IP) of 2 mg/kg of METH. Next, the intraperitoneal injection of 10, 20, 40, and 80 mg/kg CBD was performed 1 h prior to the injection of saline or METH. The protein expression levels of Sigma1R, AKT, p-AKT, GSK-3ß, p-GSK-3ß, CREB, and p-CREB in the rats' prefrontal cortex, nucleus accumbens, and hippocampus and ventral tegmental were detected using western blot analysis. CBD was found to inhibit METH-induced CPP in a dose-dependent fashion. The expression levels of Sigma1R, p-AKT, p-GSK3ß, and p-CREB increased significantly in the METH-induced CPP model. Treatment involving different doses of CBD caused differential inhibitory responses in the cellular protein abundance of Sigma1R, p-AKT, p-GSK3ß, and p-CREB across various brain regions. The present study found that METH can induce CPP in rats. When a pretreatment of CBD is applied, the CBD can weaken CPP in METH-induced rats by regulating the SigmaR1/AKT/GSK-3ß/CREB signaling pathway. The results of this study indicate that CBD has a potential therapeutic effect on METH-induced rewarding effects.
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AIMS: Premature ovarian failure (POF) is a phenomenon in which the ovaries fail before the age of 40 years. Prior research has used a wide range of mouse models designed to reflect different causes of POF, including genetic factors, iatrogenic factors, and immune factors. The current study employed a mouse model of POF induced by 4-vinylcyclohexene diepoxide (VCD). VCD can specifically kill primordial and primary ovarian follicles, which destroys the follicular reserve and causes POF. The current study sought to specify and extend the applications of this model by examining the effect of timing and VCD dose and by exploring the effect of the model on systems outside of the ovaries. MATERIALS AND METHODS: A VCD-induced mouse model of POF was constructed using established methods (VCD injected continuously at a concentration of 160 mg/kg for 15 days). Evidence for a graded effect of VCD was observed using a range of concentrations, and the best windows for examining VCD's effects on follicles and associated tissues were identified. KEY FINDINGS: The mouse model used here successfully simulated two common complications of POF - emotional changes and decreased bone density. The model's application was then extended to examine the links between disease and intestinal microorganisms, and evidence was found linking POF to the reproductively relevant composition of the gut microbiota. SIGNIFICANCE: These findings provide novel methodological guidance for future research, and they significantly extend the applications and scope of VCD-induced POF mouse models.
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Modelos Animais de Doenças , Microbioma Gastrointestinal/fisiologia , Folículo Ovariano/patologia , Insuficiência Ovariana Primária/fisiopatologia , Animais , Densidade Óssea/fisiologia , Cicloexenos/administração & dosagem , Cicloexenos/toxicidade , Relação Dose-Resposta a Droga , Emoções/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Ovariana Primária/complicações , Insuficiência Ovariana Primária/microbiologia , Compostos de Vinila/administração & dosagem , Compostos de Vinila/toxicidadeRESUMO
INTRODUCTION: This study aims to establish a methamphetamine (METH)-induced behavioral sensitization model using tree shrews, as well as to measure the protein expression of the dopamine D3 receptor (D3R) and dopamine transporter (DAT). METHODS: Forty tree shrews were equally and randomly divided into four experimental groups: those administered with 1, 2, and 4 mg/kg METH and a control group (treated with an equal amount of normal saline). Each experimental group was repeatedly exposed to METH for nine consecutive days to induce the development of behavioral sensitization, followed by four days of withdrawal (without the METH treatment) to induce the transfer of behavioral sensitization, then given 0.5 mg/kg of METH to undergo the expression of behavioral sensitization. Altered locomotor and stereotypic behaviors were measured daily via open-field experiments during the development and expression stages, and weight changes were also recorded. Then, the Western blot method was used to detect the expression levels of D3R and DAT in three brain regions: the nucleus accumbens, prefrontal cortex, and dorsal striatum 24 hr after the last behavioral test. RESULTS: METH administration augmented motor-stimulant responses and stereotypic behaviors in all experimental groups, and stereotypic behaviors intensified more in the groups treated with 2 and 4 mg/kg METH. Motion distance, speed, and trajectory were significantly elevated in all experimental, however, METH at 4 mg/kg induced more stereotypic behaviors, decreasing these locomotor activities as compared with the 2 mg/kg METH group. 2 and 4 mg/kg METH significantly upregulated and downregulated D3R and DAT expression levels, respectively, in three brain regions, and these changes are more pronounced in 2 mg/kg METH. CONCLUSIONS: These results indicated that this animal model may be used to study the neurobiological mechanisms that underly the development and expression of behavioral sensitization to METH. Deregulated D3R and DAT expression may be involved in the METH-induced behavioral sensitization.
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Encéfalo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Metanfetamina/farmacologia , Receptores de Dopamina D3/metabolismo , Animais , Comportamento Animal , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sensibilização do Sistema Nervoso Central , Estimulantes do Sistema Nervoso Central/farmacologia , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Comportamento Estereotipado/efeitos dos fármacos , Comportamento Estereotipado/fisiologia , TupaiidaeRESUMO
[This corrects the article DOI: 10.3389/fcell.2020.00587.].
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BACKGROUND: 4-vinylcyclohexene diepoxide (VCD) has long been considered a hazardous occupational chemical that promotes ovarian failure. However, VCD is also used as a research compound to chemically induce animal models of premature ovarian insufficiency (POI), and in related work we unexpectedly found that VCD apparently exhibits both dose- and duration-dependent opposing, hormone-like effects on the maintenance of the primordial follicle pool, follicle development, and ovulation induction. RESULTS: We conducted experiments with cultured murine ovaries and performed transplantation experiments using postnatal day (PD) 2 and PD12 mice and found that low-dose, short-term exposure to VCD (VCDlow) actually protects the primordial/primary follicle pool and improves the functional ovarian reserve (FOR) by disrupting follicular atresia. VCDlow inhibits follicular apoptosis and regulates the Pten-PI3K-Foxo3a pathway. Short-term VCD exposure in vivo (80 mg/kg, 5 days) significantly increases the number of superovulated metaphase II oocytes, preovulatory follicles, and corpus luteum in middle-aged mice with diminished ovarian reserve (DOR). We demonstrate that low-dose but not high-dose VCD promotes aromatase levels in granulosa cells (GCs), thereby enhancing the levels of estradiol secretion. CONCLUSION: Our study illustrates a previously unappreciated, hormone-like action for the occupational "ovotoxin" molecule VCD and strongly suggests that VCDlow should be explored for its potential utility for treating human ovarian follicular development disorders, including subfertility in perimenopausal women.
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Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency-naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten-/- mouse ESCs with the CRISPR-Cas9 system and verified that Pten-/- ESCs maintained naïve pluripotency by blocking Gsk3ß activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3ß modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.
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Diferenciação Celular , Linhagem da Célula , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Embrionárias Murinas/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Linhagem Celular , Corpos Embrioides/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/genética , Camundongos , Camundongos Nus , Mutação , PTEN Fosfo-Hidrolase/genética , Fenótipo , Transdução de Sinais , Teratoma/enzimologia , Teratoma/genética , Teratoma/patologiaRESUMO
Methamphetamine (METH) has been shown to induce neuropathological dysfunction and irreversible brain cell damage. Prior studies indicated the involvement of autophagy in METH-induced neurotoxicity. However, the underlying mechanism by which autophagy contributes to METH-induced neurotoxicity remains elusive. Gastrodin, a primary bioactive constituent of Gastrodia elata-an orchid used in traditional Chinese medicine-is used widely to treat stroke, dementia, and headache. This study investigates whether METH induces autophagy in the human dopaminergic neuroblastoma cell line SH-SY5Y, then examines the neuroprotective effects of gastrodin against autophagy in METH-treated SH-SY5Y cells. The effects of METH on the protein expressions of autophagy-related genes (LC3B and Beclin-1) were evaluated with and without gastrodin. The presence of autophagosomes in the METH-induced treatment with and without gastrodin is revealed through transmission electron microscopy. Pharmacological intervention was employed to study the role of the AKT/mTOR signaling pathway in the gastrodin-mediated neuroprotection against METH-induced autophagy. The present results indicate that METH exposure elevates the protein expression levels of LC3B and Beclin-1 in a dose- and time-dependent manner. Gastrodin is observed to block the METH-induced upregulation of LC3B and Beclin-1 protein expression significantly. Gastrodin is found to exhibit an anti-autophagic effect on the inhibition of the METH-induced Beclin-1 protein expression, partly via the AKT/mTOR These findings may aid the development of a gastrodin-based therapeutic strategy for treating METH-induced neurotoxicity.
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Autofagia/efeitos dos fármacos , Álcoois Benzílicos/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Glucosídeos/farmacologia , Metanfetamina/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/citologia , Humanos , Neuroblastoma , Transdução de SinaisRESUMO
Background: Addictive stimulant drugs, such as methamphetamine (METH), increase the risk of exposure to the human immunodeficiency virus-1 (HIV-1) infection and thus predispose individuals to the development of HIV-associated neurocognitive disorders (HANDs). Previous studies have indicated that HIV-Tat (the transactivator of transcription) and METH can synergistically induce autophagy in SH-SY5Y neuroblastoma cells and that autophagy plays a pivotal role in the neuronal dysfunction in HANDs. However, the underlying mechanism of METH-and HIV-Tat-induced neuronal autophagy remains unclear. Methods: We cultured primary midbrain neuronal cells of tree shrews and treated them with METH and HIV-Tat to study the role of METH and HIV-Tat in inducing autophagy. We evaluated the effects of the single or combined treatment of METH and HIV-Tat on the protein expressions of the autophagy-related genes, including Beclin-1 and LC3B, ATG5, and ATG7 in METH and HIV-Tat-induced autophagy. In addition, the presence of autophagosomes in the METH and/or HIV-Tat treatment was revealed using transmission electron microscopy. Results: The results indicated that METH increased the protein levels of LC3B and Beclin-1, and these effects were significantly enhanced by HIV-Tat. Moreover, the results suggested that ATG5 and ATG7 were involved in the METH and HIV-Tat-induced autophagy. In addition, it was found that mTOR inhibition via pharmacological intervention could trigger autophagy and promote METH and HIV-Tat-induced autophagy. Discussion: Overall, this study contributes to the knowledge of the molecular underpinnings of METH and HIV-Tat-induced autophagy in primary midbrain neuronal cells. Our findings may facilitate the development of therapeutic strategies for METH-and HIV-Tat-induced autophagy in HANDs.
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OBJECTIVES: MicroRNAs (miRNAs) are considered as the cellular regulators which post-transcriptionally modulate gene expression in diverse biological processes including cell development and immunity. In this study, we investigated functions of miR-181d in dendritic cells (DCs) maturation, and the underlying mechanisms were also explored. MATERIALS AND METHODS: Here we did the miRNA screening in human DCs in response to lipopolysaccharides (LPS) by quantitative real-time PCR (qRT-PCR). The expressions of DCs maturation markers were measured after miRNA mimics transfections. The pharmacological inhibitors of signalling pathways were applied to examine miR-181d effect on DCs maturation by Western blot. Luciferase assay and mixed lymphocyte reaction (MLR) were also performed to reveal the target gene of miR-181d and test the viability of T cells treated with miR-181d transfected DCs. RESULTS: Overexpression of miR-181d per se is sufficient to promote DCs maturation, and up-regulate CD80 and CD83 expressions without LPS. Besides, we showed that miR-181d activated NF-κB pathway and also promoted the expression of pro-inflammatory cytokine IL12 and TNF-α. Inhibition of NF-κB pathway suppressed DCs maturation. Luciferase reporter assay and target gene knockdown assay indicated that miR-181d targets regulator cylindromatosis (CYLD), a primary negative regulator of NF-κB pathway. MLR assay showed that miR-181d-transfected DCs could promote T-cell proliferation than iDCs in vitro. CONCLUSION: Our study demonstrates that miR-181d is required for DCs maturation through the activation of NF-κB pathway by targeting CYLD.
Assuntos
Células Dendríticas/citologia , Lipopolissacarídeos/imunologia , MicroRNAs/genética , NF-kappa B/imunologia , Transdução de Sinais , Regulação para Cima , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Enzima Desubiquitinante CYLD , Humanos , Interleucina-12/imunologia , MicroRNAs/imunologia , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologiaRESUMO
Comparative gene expression analysis by qRT-PCR is commonly used to detect differentially expressed genes in studies of PCOS pathology. Impaired GC function is strongly associated with PCOS pathogenesis, and a growing body of studies has been dedicated to identifying differentially expressed genes in GCs in PCOS patients and healthy women by qRT-PCR. It is necessary to validate the expression stability of the selected reference genes across the tested samples for target gene expression normalization. We examined the variability and stability of expression of the 15 commonly used reference genes in GCs from 44 PCOS patients and 45 healthy women using the GeNorm, BestKeeper, and NormFinder statistical algorithms. We combined the rankings of the three programs to produce a final ranking based on the geometric means of their stability scores. We found that HPRT1, RPLP0, and HMBS out of 15 examined commonly used reference genes are stably expressed in GCs in both controls and PCOS patients and can be used for normalization in gene expression profiling by qRT-PCR. Future gene-expression studies should consider using these reference genes in GCs in PCOS patients for more accurate quantitation of target gene expression and data interpretation.