The aim of the present study was to investigate whether the physiological features of Ano1 were affected by enhanced green fluorescent protein (EGFP) fusing at Ano1 C-terminal. The eukaryotic expression vectors of Ano1 and EGFP-Ano1 were constructed, and these plasmids were transfected into Fischer rat thyroid follicular epithelial (FRT) cells using liposome. The expression and location of Ano1 were examined by using inverted fluorescence microscope. The ability of Ano1 to transport iodide was detected by kinetics experiment of fluorescence quenching. The results showed that both Ano1 and EGFP-Ano1 were expressed on FRT cell membrane and could be activated by Ca(2+). There was no significant difference of the ability to transport iodide between Ano1 and EGFP-Ano1. These results suggest Ano1 and EGFP-Ano1 have similar physiological feature.
Animals ; Anoctamin-1 ; Cell Membrane ; physiology ; Chloride Channels ; metabolism ; Epithelial Cells ; physiology ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Microscopy, Fluorescence ; Plasmids ; Rats ; Recombinant Fusion Proteins ; metabolism ; Thyroid Gland ; cytology ; Transfection

The present study was aimed to investigate the effects of exercise and nutrition intervention on rat immune function. Flor-Essence is a kind of health food produced by FLORA company in Canada and certified by Quality Assurance International (QAI). Its main components are burdock root, cress leaves of grass, kelp, Turkish rhubarb root, et al. Flor-Essence has been shown to activate the body detoxification path, improve the physical environment, and inhibit cancer cell growth and proliferation. Sixty male Sprague-Dawley (SD) rats were randomly divided into six groups: control, NS + training, low-dose Flor-Essence + training, low-dose Flor-Essence, high-dose Flor-Essence + training, high-dose Flor-Essence groups. The rats in NS + training, low-dose Flor-Essence + training, high-dose Flor-Essence + training groups swam 35 min per day in the water tank for 6 days a week. One hour before exercise, the rats were given low- (2.5 mg/mL) or high-dose (5 mg/mL) Flor-Essence daily by intragastric administration, and the rats in NS + training group were given equivalent volume of NS. On the last day of four training weeks, all rats took part in a bout of exhaustive exercise, and then were sacrificed immediately. Arterial blood serum samples were taken for the assays of IL-6, IL-12 and TNF-α contents, spleens for natural killer (NK) cells activity. The results showed that serum IL-6 content in NS + training group was decreased compared with that in control group. Low- and high-dose Flor-Essence groups showed decreased IL-6, IL-12 and TNF-α serum contents, as well as longer exhaustive time, compared with control group. The improving effects of high-dose Flor- Essence on IL-6, TNF-α and exhaustive time were greater than those of low dose. Compared with NS + training, low- and high-dose Flor-Essence + training reduced serum contents of IL-6 and TNF-α, and prolonged exhaustive time; only high-dose Flor-Essence + training decreased serum IL-12 content and enhanced NK cells activity. These results suggest Flor-Essence in combination with exercise can improve rat immune function and sports performance, and the effect of high-dose Flor-Essence is better than that of low dose.
Animals ; Interleukin-12 ; blood ; Interleukin-6 ; blood ; Killer Cells, Natural ; drug effects ; Male ; Physical Conditioning, Animal ; Plant Extracts ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Swimming ; Tumor Necrosis Factor-alpha ; blood

The aim of this study was to investigate the effects of digoxin on the chemoresistance of human breast cancer cell line MCF-7/adriamycin (ADR) and its underlying mechanism. MCF-7 and MCF-7/ADR cells were designated as control and ADR groups, respectively. MCF-7/ADR cells in ADR + digoxin group received 48 h of digoxin (10 nmol/L) treatment; MCF-7/ADR cells transfected with pLKO.1-shHIF-1α and pLKO.1-shcontrol plasmids were named shHIF-1α and shcontrol groups, respectively. CCK-8 assay was employed to detect the cytotoxic effect of ADR on MCF-7/ADR cells, and IC50 value and resistance index were calculated according to CCK-8. RT-PCR was used to measure the mRNA levels of hypoxia inducible factor-1α (HIF-1α) and multidrug resistance-1 (MDR1). Western blot was used to analyze the protein levels of HIF-1α and MDR1. Flow cytometry was used to determine the apoptosis. The result showed that the resistance index of MCF-7/ADR cells was 115.6, and it was reduced to 47.2 under the action of digoxin (P < 0.05). In comparison with control group, ADR groups showed increased protein and mRNA levels of HIF-1α and MDR1 (P < 0.05). Digoxin reduced the protein levels of HIF-1α and MDR1, as well as the mRNA level of MDR1, but did not affect the mRNA level of HIF-1α. After HIF-1α gene was silenced, the protein levels of HIF-1α and MDR1 were down-regulated (P < 0.05), and the pro-apoptotic effect of ADR on MCF-7/ADR cells was enhanced. Although it was also observed that digoxin promoted cell apoptosis in both shcontrol and shHIF-1α groups, the difference between the two groups was not significant. In conclusion, the results suggest that digoxin may partially reverse the ADR resistance in human breast cancer cell line MCF-7/ADR by means of down-regulating the expression levels of HIF-1α and MDR1 and promoting apoptosis via HIF-1α-independent pathway.
ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Apoptosis ; Breast Neoplasms ; metabolism ; pathology ; Digoxin ; pharmacology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; MCF-7 Cells ; drug effects ; RNA, Messenger ; Transfection

Vascular adventitial fibroblasts (AF) may play an important role in vascular inflammation. This study was aimed to investigate the expression pattern of inflammatory mediators in AF induced by angiotensin II (AngII) and to explore the effects of AF-derived inflammatory mediators on the adhesion and migration of macrophages both in vitro and in vivo. We used real-time RT-PCR to detect the mRNA expression of inflammatory mediators in cultured AF. The results showed that AngII (1 × 10(-7) mol/L) up-regulated mRNA expression of 4 inflammatory mediators, including P-selectin, ICAM-1, IL-6 and MCP-1, in cultured AF. Western blot analysis or ELISA revealed that AngII up-regulated P-selectin and ICAM-1 protein expression and IL-6 secretion in cultured AF, but did not alter MCP-1 secretion. We further detected the effects of AF-derived inflammatory mediators on the adhesion and chemotaxis of RAW264.7, a macrophage cell line. We found that AF stimulated with AngII could enhance the adhesion of RAW264.7 and the conditioned medium from AngII-stimulated AF could enhance the migration of RAW264.7. Immunofluorescence study showed an enhanced accumulation of CD68 positive cells and the up-regulation of P-selectin, ICAM-1, IL-6 and MCP-1 in aortic adventitia of AngII-infused (200 ng/kg per min for 2 weeks) rats. We concluded that AF may contribute to vascular inflammation via expression of certain inflammatory mediators and the subsequent adhesion and chemotaxis of macrophages.
Adventitia ; drug effects ; Angiotensin II ; pharmacology ; Animals ; Cell Line ; Chemokine CCL2 ; metabolism ; Culture Media, Conditioned ; Fibroblasts ; drug effects ; immunology ; Inflammation ; immunology ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-6 ; metabolism ; Macrophages ; drug effects ; immunology ; Mice ; P-Selectin ; metabolism ; RAW 264.7 Cells ; Rats ; Up-Regulation

To explore the relationship between the epithelial adhesion molecules and immune responses of airway epithelium, we observed the expression of integrin β4 and intercellular adhesion molecule-1 (ICAM-1) in the mice airway epithelium after sensitization with allergens. BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) or house dust mite (HDM) and then developed airway hyper-responsiveness as determined by barometric whole-body plethysmography. Both OVA and HDM sensitization led to increases of the number of peripheral leukocytes as well as inflammatory cells infiltration in lungs. OVA sensitized mice showed more severe inflammatory cells infiltration than HDM sensitized mice. Immunohistochemistry analysis of mice lung tissues revealed that sensitization with both allergens also led to a decrease of integrin β4 expression and an increase of ICAM-1 expression in airway epithelia. OVA sensitized mice showed a more significant increase of ICAM-1 expression compared with HDM sensitized mice. siRNA mediated silencing of integrin β4 gene in 16HBE cells resulted in an up-regulation of ICAM-1 expression. Our results indicate a possible role of airway epithelial adhesion molecules in allergen-induced airway immune responses.
Allergens ; pharmacology ; Animals ; Integrin beta4 ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Lung ; metabolism ; physiopathology ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; Pyroglyphidae ; Respiratory Hypersensitivity ; metabolism

The present study was aimed to investigate the role of cerebrospinal fluid-contacting nucleus (CSF-CN) neurons in modulation of inflammatory pain and underlying mechanism. The inflammatory pain model was made by subcutaneous injection of the complete Freund's adjuvant (CFA) into the left hind paw of rats. The phosphorylation level of PKC (p-PKC) was examined by Western blot. Thermal withdrawal latency (TWL) of the rats was measured to assess inflammatory pain. The results showed that, compared with the sham controls, the inflammatory pain model rats showed shortened TWL on day 1, 3, and 7 after CFA injection, as well as increased level of p-PKC in CSF-CN neurons at 24 h after CFA injection. The administration of GF109203X, a PKC inhibitor, into lateral ventricle decreased the level of p-PKC protein expression and increased TWL in the model rats. These results suggest that blocking the PKC pathway in CSF-CN neurons may be an effective way to reduce or eliminate the inflammatory pain.
Animals ; Freund's Adjuvant ; Inflammation ; enzymology ; Neurons ; enzymology ; Pain ; enzymology ; Phosphorylation ; Protein Kinase C ; cerebrospinal fluid ; chemistry ; Rats ; Rats, Sprague-Dawley

The present study was designed to investigate the inhibitory effects of intravenous general anesthetic propofol (0.1-3.0 mmol/L) on excitatory synaptic transmission in supraoptic nucleus (SON) neurons of rats, and to explore the underlying mechanisms by using intracellular recording technique and hypothalamic slice preparation. It was observed that stimulation of the dorsolateral region of SON could elicit the postsynaptic potentials (PSPs) in SON neurons. Of the 8 tested SON neurons, the PSPs of 7 (88%, 7/8) neurons were decreased by propofol in a concentration-dependent manner, in terms of the PSPs' amplitude (P < 0.01), area under curve, duration, half-width and 10%-90% decay time (P < 0.05). The PSPs were completely and reversibly abolished by 1.0 mmol/L propofol at 2 out of 7 tested cells. The depolarization responses induced by pressure ejection of exogenous glutamate were reversibly and concentration-dependently decreased by bath application of propofol. The PSPs and glutamate-induced responses recorded simultaneously were reversibly and concentration-dependently decreased by propofol, but 0.3 mmol/L propofol only abolished PSPs. The excitatory postsynaptic potentials (EPSPs) of 7 cells increased in the condition of picrotoxin (30 µmol/L, a GABA(A) receptor antagonist) pretreatment. On this basis, the inhibitory effects of propofol on EPSPs were decreased. These data indicate that the presynaptic and postsynaptic mechanisms may be both involved in the inhibitory effects of propofol on excitatory synaptic transmission in SON neurons. The inhibitory effects of propofol on excitatory synaptic transmission of SON neurons may be related to the activation of GABA(A) receptors, but at a high concentration, propofol may also act directly on glutamate receptors.
Anesthetics, Intravenous ; pharmacology ; Animals ; Excitatory Postsynaptic Potentials ; drug effects ; GABA-A Receptor Antagonists ; pharmacology ; Glutamic Acid ; pharmacology ; In Vitro Techniques ; Neurons ; drug effects ; Propofol ; pharmacology ; Rats ; Receptors, Glutamate ; metabolism ; Supraoptic Nucleus ; cytology

Spinal microglia and astrocytes play an important role in mediating behavioral hypersensitive state following peripheral nerve injury. However, little is known about the expression patterns of activated microglia and astrocytes in the spinal dorsal horn. The aim of the present study was to investigate the spatial distribution of microglial and astrocytic activation in cervical, thoracic, lumbar and sacral segments of spinal dorsal horn following chronic constriction injury (CCI) of sciatic nerve. The hind paw withdrawal threshold (PWT) of wild type (WT), CX3CR1(YFP) and GFAP(YFP) transgenic mice to mechanical stimulation was determined by von Frey test. Immunofluorescence staining was used to examine the spatial distribution of microglial and astrocytic activation in the spinal dorsal horn. Following CCI, all the WT, CX3CR1(YFP) and GFAP(YFP) mice developed robust allodynia in the ipsilateral paw on day 3 after CCI, and the allodynia was observed to last for 14 days. In comparison with sham groups, the PWTs of CCI group animals were significantly decreased (P < 0.01, n = 6). On day 14 after CCI, CX3CR1(YFP)-GFP immunofluorescence intensity was significantly increased in the ipsilateral lumbar spinal dorsal horn of the CX3CR1(YFP) mice (P < 0.01, n = 6), but no detectable changes were observed in other spinal segments. Increased GFAP(YFP)-GFP immunofluorescence intensity was observed in the ipsilateral thoracic, lumbar and sacral spinal segments of the GFAP(YFP) mice on day 14 after CCI. Iba-1 and GFAP immunofluorescence staining in WT mice showed the same result of microglia and astrocyte activation on day 14 after CCI. CX3CR1(YFP)-GFP and GFAP(YFP)-GFP immunofluorescence signal was colocalized with microglial marker Iba-1 and astrocytic marker GFAP, respectively. Interestingly, on day 3 after CCI, Iba-1-immunoreactivity was significantly increased in the ipsilateral thoracic, lumbar and sacral spinal segments of WT mice, whereas the significant upregulation of GFAP-immunoreactivity restrictedly occurred in the ipsilateral lumbar spinal segment. These results suggest that microglial and astrocytic activation may be involved in the development and maintenance of secondary allodynia in mice with neuropathic pain.
Animals ; Astrocytes ; physiology ; Disease Models, Animal ; Hyperalgesia ; Mice ; Mice, Transgenic ; Microglia ; physiology ; Neuralgia ; Peripheral Nerve Injuries ; Sciatic Nerve ; injuries ; Spinal Cord Dorsal Horn ; cytology ; Up-Regulation

Empathy, a basic prosocial behavior, is referred to as an ability to understand and share others' emotional state. Generally, empathy is also a social-behavioral basis of altruism. In contrast, impairment of empathy development may be associated with autism, narcissism, alexithymia, personality disorder, schizophrenia and depression. Thus, study of the brain mechanisms of empathy has great importance to not only scientific and clinical advances but also social harmony. However, research on empathy has long been avoided due to the fact that it has been considered as a distinct feature of human beings from animals, leading to paucity of knowledge in the field. In 2006, a Canadian group from McGill University found that a mouse in pain could be shared by its paired cagemate, but not a paired stranger, showing decreased pain threshold and increased pain responses through emotional contagion while they were socially interacting. In 2014, we further found that a rat in pain could also be shared by its paired cagemate 30 min after social interaction, showing long-term decreased pain threshold and increased pain responses, suggesting persistence of empathy for pain (empathic memory). We also mapped out that the medial prefrontal cortex, including the anterior cingulate cortex, prelimbic cortex and infralimbic cortex, is involved in empathy for pain in rats, suggesting that a neural network may be associated with development of pain empathy in the CNS. In the present brief review, we give a brief outline of the advances and challenges in study of empathy for pain in humans and animals, and try to provide a novel bio-psychosocial-behavioral model for study of pain and its emotional comorbidity using laboratory animals.
Animals ; Cerebral Cortex ; physiology ; Emotions ; Empathy ; Gyrus Cinguli ; physiology ; Humans ; Mice ; Models, Animal ; Pain ; Pain Threshold ; Prefrontal Cortex ; physiology ; Rats

Neurotransmission begins with neurotransmitter being released from synaptic vesicles. To achieve this function, synaptic vesicles endure the dynamic "release-recycle" process to maintain the function and structure of presynaptic terminal. Synaptic transmission starts with a single action potential that depolarizes axonal bouton, followed by an increase in the cytosolic calcium concentration that triggers the synaptic vesicle membrane fusion with presynaptic membrane to release neurotransmitter; then the vesicle membrane can be endocytosed for reusing afterwards. This process requires delicate regulation, intermediate steps and dynamic balances. Accumulating evidence showed that the release ability and mobility of synapses varies under different stimulations. Synaptic vesicle heterogeneity has been studied at molecular and cellular levels, hopefully leading to the identification of the relationships between structure and function and understanding how vesicle regulation affects synaptic transmission and plasticity. People are beginning to realize that different types of synapses show diverse presynaptic activities. The steady advances of technology studying synaptic vesicle recycling promote people's understanding of this field. In this review, we discuss the following three aspects of the research progresses on synaptic vesicle recycling: 1) presynaptic vesicle pools and recycling; 2) research progresses on the differences of glutamatergic and GABAergic presynaptic vesicle recycling mechanism and 3) comparison of the technologies used in studying presyanptic vesicle recycling and the latest progress in the technology development in this field.
Action Potentials ; Axons ; physiology ; Calcium ; physiology ; Endocytosis ; Humans ; Presynaptic Terminals ; physiology ; Synapses ; physiology ; Synaptic Transmission ; Synaptic Vesicles ; physiology