The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain. Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury, as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke. The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex. A stably transfected cell line was observed by florescence microscope, and identified by RT-qPCR and Western blotting. The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed. Subsequently, the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques. It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.
Analgesics/therapeutic use* ; Animals ; Drug Discovery ; HEK293 Cells ; Humans ; Neuralgia/metabolism* ; Receptors, N-Methyl-D-Aspartate/genetics*

OBJECTIVE: To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25-VitD3) supplementation on cerebral injury after ischemia/reperfusion (I/R) in mice with middle cerebral artery occlusion (MCAO). METHODS: Male C57BL6 mice were randomly divided into Sham group, Vehicle group and 1,25-VitD3 group, with 10 mice in each group. Vehicle group and 1,25-VitD3 group were given MCAO for 1 hour, and then killed after reperfusion for 24 hours. Mice in 1,25-VitD3 group were treated with 1,25-VitD3 at the dose of 100 ng/(kg·d) by injected intraperitoneally for 5 days before MCAO operation. Cerebral ischemic penumbra areas of each group were collected for TTC staining, RT-PCR, TTC staining and immunohistochemistry assay. The function defect of mice was evaluated by using neurological function score. RESULTS: Compared with the sham group, the volume of cerebral infarction in Vehicle group was increased significantly, and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues were increased significantly (P＜0.05); compared with Vehicle group, supplementation of 1,25-VitD3 reduced the volume of cerebral infarction by about 50% in I/R mice (P＜0.05), and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues of 1,25-VitD3 group were decreased significantly (P＜0.05). The expression of Foxp3, a T-regulatory cell marker, was significantly increased in the brain of mice (P＜0.05), while the expression of Rorc, a transcription factor, was significantly decreased (P＜0.05), suggesting that Th17/gamma Delta T-cell response was reduced and the number of neutrophils in the brain injury site of mice was significantly reduced (P＜0.05). CONCLUSION Vitamin D could alleviate the development of cerebral infarction after arterial occlusion (MCAO) reperfusion, and its mechanism may be through regulating the inflammatory response in mouse brain I/R.
Animals ; Brain ; Cytokines ; metabolism ; Infarction, Middle Cerebral Artery ; drug therapy ; Inflammation ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase 2 ; metabolism ; Protective Agents ; pharmacology ; Random Allocation ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; T-Lymphocytes ; Th17 Cells ; Vitamin D ; pharmacology

Recurrent pregnancy loss (RPL) is a common complication in obstetrics, affecting about 5% of women of childbearing age. An increase in the number of abortions results in escalation in the risk of miscarriage. Although concentrated research has identified numerous causes for RPL, about 50% of them remain unexplained. Pregnancy is a complex process, comprising fertilization, implantation, organ and tissue differentiation, and fetal growth, which is effectively controlled by a number of both maternal and fetal factors. An example is the immune response, in which T cells and natural killer cells participate, and inflammation mediated by tumor necrosis factor or colony-stimulating factor, which hinders embryo implantation. Furthermore, vitamin D affects glucose metabolism and inhibits embryonic development, whereas microRNA has a negative effect on the gene expression of embryo implantation and development. This review examines the causes of RPL from multiple perspectives, and focuses on the numerous factors that may result in RPL.
Abortion, Habitual ; Abortion, Spontaneous ; Colony-Stimulating Factors ; Embryo Implantation ; Embryonic Development ; Female ; Fertilization ; Fetal Development ; Gene Expression ; Glucose ; Humans ; Inflammation ; Killer Cells, Natural ; Metabolism ; MicroRNAs ; Obstetrics ; Pregnancy ; Proteomics ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Vitamin D

The aim of the present study was to observe whether dopamine receptor (DR) was involved in the effects of sodium salicylate (SS) on the expressions of N-methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid (GABA) receptors in rat cochlear spiral ganglion neurons (SGNs). Forty-eight hours after primary culture of rat SGNs, immunofluorescence technique was applied to detect expressions of DR1 and DR2, the two subtypes of dopamine receptors. Western blot was performed to assess NMDA receptor NR1 subunit and GABAreceptor subunit α2 (GABRα2) protein expressions in the SGNs after the treatments of SS alone or in combination with DR antagonists. The results demonstrated that: (1) The DR1 and DR2 were expressed in the bodies and axons of the SGN; (2) After the treatment with SS, the surface protein expressions of GABRα2 and NR1 were decreased by 44.69% and 21.57%, respectively, while the total protein expressions showed no significant changes; (3) Neither SS + SCH23390 (DR1 antagonist) group nor SS + Eticlopride (DR2 antagonist) group showed significant differences in GABRα2 and NR1 surface protein expressions compared with the control group. These results suggest that SS regulates the surface GABAand NMDA receptors trafficking on SGN, and the mechanism may involve DR mediation.
Animals ; Benzazepines ; pharmacology ; Cells, Cultured ; Cochlea ; cytology ; Neurons ; drug effects ; Rats ; Receptors, Dopamine ; metabolism ; Receptors, GABA-A ; metabolism ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Sodium Salicylate ; toxicity ; Spiral Ganglion ; drug effects

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.
Animals ; Blood Proteins/chemistry/*metabolism ; Endocytosis ; Female ; HEK293 Cells ; HT29 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Mice, Nude ; Neoplasms/genetics/metabolism/pathology ; Phospholipase D/chemistry/*metabolism ; Phosphoproteins/chemistry/*metabolism ; Protein Structure, Tertiary ; Receptor, Epidermal Growth Factor/*metabolism ; Signal Transduction ; *Up-Regulation ; Vesicular Transport Proteins/*genetics/metabolism ; rab5 GTP-Binding Proteins/*metabolism

OBJECTIVEThe aim of this study is to investigate whether microwave exposure would affect the N-methyl-D-aspartate receptor (NMDAR) signaling pathway to establish whether this plays a role in synaptic plasticity impairment.

METHODS48 male Wistar rats were exposed to 30 mW/cm2 microwave for 10 min every other day for three times. Hippocampal structure was observed through H&E staining and transmission electron microscope. PC12 cells were exposed to 30 mW/cm2 microwave for 5 min and the synapse morphology was visualized with scanning electron microscope and atomic force microscope. The release of amino acid neurotransmitters and calcium influx were detected. The expressions of several key NMDAR signaling molecules were evaluated.

RESULTSMicrowave exposure caused injury in rat hippocampal structure and PC12 cells, especially the structure and quantity of synapses. The ratio of glutamic acid and gamma-aminobutyric acid neurotransmitters was increased and the intracellular calcium level was elevated in PC12 cells. A significant change in NMDAR subunits (NR1, NR2A, and NR2B) and related signaling molecules (Ca2+/calmodulin-dependent kinase II gamma and phosphorylated cAMP-response element binding protein) were examined.

CONCLUSION30 mW/cm2 microwave exposure resulted in alterations of synaptic structure, amino acid neurotransmitter release and calcium influx. NMDAR signaling molecules were closely associated with impaired synaptic plasticity.

Animals ; Gene Expression Regulation ; radiation effects ; Hippocampus ; cytology ; Microwaves ; Neuronal Plasticity ; radiation effects ; Neurons ; radiation effects ; Neurotransmitter Agents ; metabolism ; PC12 Cells ; Rats ; Receptors, N-Methyl-D-Aspartate ; genetics ; metabolism ; Signal Transduction ; physiology ; radiation effects ; Time Factors

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcriptional mediator that coordinates the expression of various genes involved in tumorigenesis in response to changes in oxygen tension. The stability of HIF-1alpha protein is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that PLD2 protein itself interacts with HIF-1alpha, prolyl hydroxylase (PHD) and VHL to promote degradation of HIF-1alpha via the proteasomal pathway independent of lipase activity. PLD2 increases PHD2-mediated hydroxylation of HIF-1alpha by increasing the interaction of HIF-1alpha with PHD2. Moreover, PLD2 promotes VHL-dependent HIF-1alpha degradation by accelerating the association between VHL and HIF-1alpha. The interaction of the pleckstrin homology domain of PLD2 with HIF-1alpha also promoted degradation of HIF-1alpha and decreased expression of its target genes. These results indicate that PLD2 negatively regulates the stability of HIF-1alpha through the dynamic assembly of HIF-1alpha, PHD2 and VHL.
Cell Line ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*metabolism ; Phospholipase D/*metabolism ; Prolyl Hydroxylases/metabolism ; Proteasome Endopeptidase Complex/*metabolism ; *Protein Interaction Maps ; Proteolysis ; Ubiquitin-Protein Ligases/metabolism ; Von Hippel-Lindau Tumor Suppressor Protein/metabolism

OBJECTIVETo investigate the dynamic changes in the expression of clara cell protein (CC16) and surfactant protein D (SP-D) in the lung tissues and bronchoalveolar lavage fluid (BALF) of silica-treated rats.

METHODSEighty-four Wistar rats were randomly divided into control group (n = 42) and silica group (n = 42). The silica group was subsequently divided into 3, 7, 14, 21, 28, and 60 d subgroups. The silicotic model was made by instilling silica suspension directly through the trachea into rat lungs. At 3, 7, 14, 21, 28, and 60 d after silica instillation, 8 rats in each group were sacrificed and their lung tissues and BALF were collected. The expression of SP-D and CC16 in lung tissues was detected by immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of SP-D and CCl6 in BALF.

RESULTSThe immunohistochemical assay indicated that CCl6 and SP-D were expressed in lung cells. The ELISA found that in 7, 14, 21, 28, and 60 d silica subgroups, the content of CCl6 in rat BALF was 8.14±0.70, 7.15±0.66, 7.00±0.69, 6.34 ± 0.59, and 5.27±0.49 ng/L, respectively; CCl6 expression decreased gradually with the silica exposure time prolonged, indicating a negative correlation (ra = -0.953, P < 0.01). Compared with the control group, all silica subgroups had significantly decreased CCl6 levels (P < 0.05). The content of SP-D in BALF was 12.20 ± 1.57, 14.41 ± 0.65, and 12.18 ± 0.74 ng/L, respectively, in the 7, 14, and 21 d silica subgroups, significantly higher than that in the control group (P < 0.05).

CONCLUSIONThe dynamic changes in SP-D and CCl6 protein levels in the lung tissues and BALF of rats could be induced by silica exposure and are related to silica exposure time. With the extension of silica exposure, CCl6 levels are gradually reduced, while the SP-D levels first increase and then fall.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Epithelial Cells ; metabolism ; Lung ; metabolism ; Pulmonary Surfactant-Associated Protein D ; metabolism ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity ; Uteroglobin ; metabolism

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.
Autophagy/*drug effects ; Cell Line, Tumor ; Colorectal Neoplasms/enzymology/*genetics/*therapy ; Genetic Therapy ; HCT116 Cells ; Humans ; Phospholipase D/*antagonists & inhibitors/*genetics/metabolism ; Quinolines/*pharmacology ; *RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Signal Transduction/drug effects ; Spiro Compounds/*pharmacology