In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury ; chemically induced ; drug therapy ; genetics ; metabolism ; Adaptor Proteins, Vesicular Transport ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Cardamine ; chemistry ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Flowers ; chemistry ; Humans ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Plant Extracts ; administration & dosage ; chemistry ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Although extensively studied, the exact role of sleep in learning and memory is still not very clear. Sleep deprivation has been most frequently used to explore the effects of sleep on learning and memory, but the results from such studies are inevitably complicated by concurrent stress and distress. Furthermore, it is not clear whether there is a strict time-window between sleep and memory consolidation. In the present study we were able to induce time-locked slow-wave sleep (SWS) in mice by optogenetically stimulating GABAergic neurons in the parafacial zone (PZ), providing a direct approach to analyze the influences of SWS on learning and memory with precise time-windows. We found that SWS induced by light for 30 min immediately or 15 min after the training phase of the object-in-place task significantly prolonged the memory from 30 min to 6 h. However, induction of SWS 30 min after the training phase did not improve memory, suggesting a critical time-window between the induction of a brief episode of SWS and learning for memory consolidation. Application of a gentle touch to the mice during light stimulation to prevent SWS induction also failed to improve memory, indicating the specific role of SWS, but not the activation of PZ GABAergic neurons itself, in memory consolidation. Similar influences of light-induced SWS on memory consolidation also occurred for Y-maze spatial memory and contextual fear memory, but not for cued fear memory. SWS induction immediately before the test phase had no effect on memory performance, indicating that SWS does not affect memory retrieval. Thus, by induction of a brief-episode SWS we have revealed a critical time window for the consolidation of hippocampus-dependent memory.
Animals ; Cues ; Electroencephalography ; Electromyography ; Evoked Potentials, Motor ; physiology ; Fear ; psychology ; Glutamate Decarboxylase ; metabolism ; Hippocampus ; physiology ; Light ; Luminescent Proteins ; genetics ; metabolism ; Maze Learning ; physiology ; Memory Consolidation ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Sleep Deprivation ; Sleep, Slow-Wave ; physiology ; Time Factors ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

The GABAergic neurons in the parafacial zone (PZ) play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus EnvA-ΔG-DsRed combined with a Cre/loxP gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain; and the intermediate reticular nucleus and medial vestibular nucleus (parvocellular part) in the pons and medulla. We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newly-found inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.
Animals ; Axons ; physiology ; Brain ; anatomy & histology ; Brain Mapping ; Brain Stem ; cytology ; GABAergic Neurons ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neural Pathways ; physiology ; Peptide Elongation Factor 1 ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Transduction, Genetic ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2) neurons. CCL2 increased NMDA-induced currents in CCR2/VGLUT2 neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.
Animals ; Benzoxazines ; pharmacology ; therapeutic use ; Chemokine CCL2 ; antagonists & inhibitors ; genetics ; metabolism ; pharmacology ; Excitatory Amino Acid Agents ; pharmacology ; Excitatory Amino Acid Agonists ; pharmacology ; Female ; Freund's Adjuvant ; toxicity ; Hyperalgesia ; chemically induced ; metabolism ; prevention & control ; Long-Term Potentiation ; drug effects ; physiology ; Luminescent Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Myelitis ; chemically induced ; drug therapy ; metabolism ; Neurons ; drug effects ; Pain Management ; Somatostatin ; genetics ; metabolism ; Spinal Cord ; cytology ; Spiro Compounds ; pharmacology ; therapeutic use ; Vesicular Glutamate Transport Protein 2 ; genetics ; metabolism ; Vesicular Inhibitory Amino Acid Transport Proteins ; genetics ; metabolism

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

Animals ; COS Cells ; Cercopithecus aethiops ; Endoplasmic Reticulum ; GTP Phosphohydrolases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; GTP-Binding Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Gene Expression ; Genetic Complementation Test ; HeLa Cells ; Humans ; Kinetics ; Membrane Fusion ; genetics ; Membrane Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Protein Multimerization ; RNA, Small Interfering ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Vesicular Transport Proteins ; genetics ; metabolism

OBJECTIVETo observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia.

METHODSA total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats' memory learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSIn comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX.

CONCLUSIONRepeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals' neuromemory ability.

Acetylcholinesterase ; genetics ; metabolism ; Acupuncture Analgesia ; Animals ; Choline O-Acetyltransferase ; genetics ; metabolism ; Cholinergic Agents ; metabolism ; Chronic Pain ; enzymology ; metabolism ; pathology ; Constriction, Pathologic ; Electroacupuncture ; Female ; Gene Expression Regulation ; Hypothalamus ; enzymology ; metabolism ; pathology ; Neuralgia ; enzymology ; metabolism ; pathology ; Ovariectomy ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Receptor, Muscarinic M1 ; genetics ; metabolism ; Vesicular Acetylcholine Transport Proteins ; genetics ; metabolism

BACKGROUNDInterferon-induced transmembrane protein 1 (IFITM1) has been identified as a molecular marker of the colorectal tumors; however its influences on the biological behaviors of the colorectal cancer cells are currently unknown. We aimed to study the influences of IFITM1 on the proliferation, invasion, and metastasis of the colorectal cancer SW480 cell lines.

METHODSWe constructed IFITM1/pEGFP-C3 recombinant plasmids and transfected them into the colorectal cancer SW480 cell lines. IFITM1/pEGFP-C3 recombinant plasmids were identified by means of immunofluorescence, laser confocal scanning microscopy, and reverse transcription polymerase chain reaction. IFITM1/SW480 cells with stable over-expression of IFITM1 were confirmed by G418 screening. The influences of IFITM1 on the proliferation of the SW480 cell lines were investigated by MTT assay and tumor transplantation experiments in nude mice. Cell invasion experiments were performed to determine the invasion capacity of the IFITM1/SW480 cells. Matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were detected by the gelatin zymographic analysis, and MMP-9 expression by the Western blotting analysis.

RESULTSIFITM1/pEGFP-C3 recombinant plasmids were successfully constructed in this study, and the IFITM1/SW480 cells with stable IFITM1 gene over-expression were confirmed by G418 screening. MTT results showed that the proliferation of the IFITM1/SW480 cells was significantly enhanced (P < 0.01). Tumors were harvested from four weeks old mice. Tumor volumes were (1347.00 ± 60.94) mm(3), (1032.40 ± 111.38) mm(3) and (1018.78 ± 28.83) mm(3); and tumor weights were (1522.34 ± 62.76) mg, (1137.78 ± 97.22) mg and (1155.76 ± 133.31) mg for mice inoculated with the IFITM1/SW480 cells, pEGFP-C3/SW480 cells and SW480 cells, respectively. Tumor volumes and weights from mice inoculated with the IFITM1/SW480 cells were significantly increased (P < 0.01). In addition, the numbers of the SW480 cells and IFITM1/SW480 cells that migrated through Matrigel were 448.64 ± 38.09 and 540.45 ± 44.61, respectively; so the invasive ability of the SW480 cells transfected with IFITM1 gene was significantly greater than that of the SW480 cells (P < 0.01). Gelatin zymographic analysis showed that MMP-9 and MMP-2 protein activities in the IFITM1/SW480 cells were significantly enhanced, and Western blotting analysis showed that MMP-9 expression in the IFITM1/SW480 cells was also increased.

CONCLUSIONIFITM1 can enhance the proliferation, invasion, and metastasis of the colorectal cancer SW480 cell lines.

Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Plasmids ; Vesicular Transport Proteins ; genetics ; metabolism

Amyotrophic Lateral Sclerosis ; genetics ; Animals ; Biological Transport, Active ; Bipolar Disorder ; genetics ; Glucose Transporter Type 4 ; metabolism ; Hepacivirus ; physiology ; Humans ; Neoplasm Metastasis ; Neoplasms ; metabolism ; Point Mutation ; Polymorphism, Single Nucleotide ; R-SNARE Proteins ; metabolism ; Tissue Distribution ; Transport Vesicles ; physiology ; Vesicular Transport Proteins ; chemistry ; genetics ; metabolism ; physiology ; Virus Replication

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.
Adaptor Proteins, Vesicular Transport/genetics/metabolism ; Arthritis, Rheumatoid/*metabolism ; Blotting, Western ; Cells, Cultured ; Fibroblasts/drug effects/*metabolism ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/*metabolism ; Interleukin-12/pharmacology ; Interleukin-16/pharmacology ; Interleukin-17/pharmacology ; Interleukin-23/pharmacology ; Lipopolysaccharides/pharmacology ; Myeloid Differentiation Factor 88/genetics/*metabolism ; Poly I-C/pharmacology ; Polymerase Chain Reaction ; RNA, Small Interfering/genetics/physiology ; Synovial Membrane/*cytology ; Toll-Like Receptor 4/genetics/metabolism ; Tumor Necrosis Factor-alpha/pharmacology