Running exercise alleviates hippocampal neuroinflammation and shifts the balance of microglial M1/M2 polarization through adiponectin/AdipoR1 pathway activation in mice exposed to chronic unpredictable stress.

Running exercise has been shown to alleviate depressive symptoms. However, the mechanism underlying the antidepressant effects of running exercise is not fully understood. The imbalance of M1/M2 microglia phenotype/polarization and concomitant dysregulation of neuroinflammation play crucial roles in the pathogenesis of depression. Running exercise increases circulating levels of adiponectin which is known to cross the blood‒brain barrier and suppress inflammatory responses. AdipoR1 is an adiponectin receptor that is involved in regulating microglial phenotypes and activation states. However, whether running exercise regulates hippocampal microglial phenotypes and neuroinflammation through adiponectin/AdipoR1 to exert its antidepressant effects remains unclear. In the current study, 4 weeks of running exercise significantly alleviated the depressive-like behaviors of chronic unpredictable stress (CUS)-exposed mice. Moreover, running exercise decreased the microglial numbers and altered microglial morphology in three subregions of the hippocampus to restore the M1/M2 balance; these effects were accompanied by regulation of pro-/anti-inflammatory cytokine production and secretion in CUS-exposed mice. These effects may involve elevation of peripheral tissue (adipose tissue and muscle) and plasma adiponectin levels, and hippocampal AdipoR1 levels as well as activation of the AMPK-NF-κB/STAT3 signaling pathway by running exercise. When an adeno-associated virus was used to knock down hippocampal AdipoR1, mice showed depressive-like behaviors and alterations in microglia and inflammatory factor expression in the hippocampus that were similar to those observed in CUS-exposed mice. Together, these results suggest that running exercise maintains the M1/M2 balance and inhibits neuroinflammation in the hippocampus of CUS-exposed mice. These effects might occur via adiponectin/AdipoR1-mediated activation of the AMPK-NF-κB/STAT3 signaling pathway.

Spatially Confined Radical Addition Reaction for Electrochemical Synthesis of Carboxylated Graphene and its Applications in Water Desalination and Splitting.

Due to the chemical stability of graphene, synthesis of carboxylated graphene still remains challenging during the electrochemical exfoliation of graphite. In this work, a spatially confined radical addition reaction which occurs in the sub-nanometer scaled interlayers of the expanded graphene sheets for the electrochemical synthesis of highly stable carboxylated graphene is reported. Here, formate anions act as both intercalation ions and co-reactant acid for the confinement of electro-generated carboxylic radical (●COOH) in the sub-nanometer scaled interlayers, which facilitates the radical addition reaction on graphene sheets. The controllable carboxylation of graphene is realized by tuning the concentration of formate anions in the electrolyte solution. The high crystallinity of the obtained product indicates the occurrence of spatially confined ●COOH addition reaction between the sub-nanometer interlayers of expanded graphite. In addition, the carboxylated graphene have been used for water desalination and hydrogen/oxygen reduction reaction. Therefore, this work provides a new method for the in situ preparation of functionalized graphene through the electrolysis and its applications in water desalination and hydrogen/oxygen reduction reactions.

Sheet-Isolated MoS2 Used for Dispersing Pt Nanoparticles and its Application in Methanol Fuel Cells.

It is highly challenging to activate the basal plane and minimize the π-π stacking of MoS2 sheets, thus enhancing its catalytic performance. Here, we display an approach for making well-dispersed MoS2 . By using the N-doped multi-walled carbon nanotubes (NMWCNTs) as an isolation unit, the aggregation of MoS2 sheets was effectively reduced, favoring the dispersion of Pt nanoparticles (noted as Pt/NMWCNTs-isolated-MoS2 ). Excellent bifunctional catalytic performance for methanol oxidation and oxygen reduction reaction (MOR/ORR) were demonstrated by the produced Pt/NMWCNTs-isolated-MoS2 . In comparison to Pt nanoparticles supported on MoS2 (Pt/MoS2 ), the MOR activity (2314.14 mA mgpt -1 ) and stability (317.69 mA mgpt -1 after 2 h of operation) on Pt/NMWCNTs-isolatedMoS2 were 24 and 232 times higher, respectively. As for ORR, Pt/NMWCNTs-isolated-MoS2 holds large half-wave potential (0.88 V) and high stability (92.71 % after 22 h of operation). This work presents a tactic for activating the basal planes and reducing the π-π stacking of 2D materials to satisfy their applications in electrocatalysis. In addition, the proposed sheet-isolation method can be used for fabricating other 2D materials to promote the dispersion of nanoparticles, which assist its application in other fields of energy as well as the environment.

Anti-LINGO-1 antibody ameliorates cognitive impairment, promotes adult hippocampal neurogenesis, and increases the abundance of CB1R-rich CCK-GABAergic interneurons in AD mice.

In view of the negative regulatory effect of leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 1 (LINGO-1) on neurons, an antibody against LINGO-1 (anti-LINGO-1 antibody) was herein administered to 10-month-old APP/PS1 transgenic Alzheimer's disease (AD) mice for 2 months as an experimental intervention. Behavioral, stereology, immunohistochemistry and immunofluorescence analyses revealed that the anti-LINGO-1 antibody significantly improved the cognitive abilities, promoted adult hippocampal neurogenesis (AHN), decreased the amyloid beta (Aß) deposition, enlarged the hippocampal volume, and increased the numbers of total neurons and GABAergic interneurons, including GABAergic and CCK-GABAergic interneurons rich in cannabinoid type 1 receptor (CB1R), in the hippocampus of AD mice. In contrast, this intervention significantly reduced the number of GABAergic interneurons expressing LINGO-1 and CB1R in the hippocampus of AD mice. More importantly, we also found a negative correlation between LINGO-1 and CB1R on GABAergic interneurons in the hippocampus of AD mice, while the anti-LINGO-1 antibody reversed this relationship. These results indicated that LINGO-1 plays an important role in the process of hippocampal neuron loss in AD mice and that antagonizing LINGO-1 can effectively prevent hippocampal neuron loss and promote AHN. The improvement in cognitive abilities may be attributed to the improvement in AHN, and in the numbers of GABAergic interneurons and CCK-GABAergic interneurons rich in CB1Rs in the hippocampus of AD mice induced by the anti-LINGO-1 antibody. Collectively, the double target effect (LINGO-1 and CB1R) initiated by the anti-LINGO-1 antibody may provide an important basis for the study of drugs for the prevention and treatment of AD in the future.

Activation of dorsal horn cannabinoid CB2 receptor suppresses the expression of P2Y12 and P2Y13 receptors in neuropathic pain rats.

BACKGROUND: More evidence suggests that dorsal spinal cord microglia is an important site contributing to CB2 receptor-mediated analgesia. The upregulation of P2Y12 and P2Y13 purinoceptors in spinal dorsal horn microglia is involved in the development of pain behavior caused by peripheral nerve injury. However, it is not known whether the expression of P2Y12 and P2Y13 receptors at spinal dorsal horn will be influenced after CB2 receptor activation in neuropathic pain rats. METHODS: Chronic constriction injury (CCI) and intrathecal ADPbetaS injection were performed in rats to induce neuropathic pain. The paw withdrawal latency (PWL) was used to evaluate thermal hyperalgesia in neuropathic rats. The expression of P2Y12 and P2Y13 receptors, p-p38MAPK, and NF-kappaBp65 was detected with RT-PCR and western blotting analysis. RESULTS: Treatment with AM1241 produces a pronounced inhibition of CCI-induced thermal hyperalgesia and significantly inhibited the increased expression of P2Y12 and P2Y13 receptors at the mRNA and protein levels, which open up the possibility that P2Y12 and P2Y13 receptor expression are downregulated by CB2 receptor agonist AM1241 in CCI rats. Western blot analysis demonstrated that AM1241 reduced the elevated expression of p-p38MAPK and NF-κBp65 in the dorsal spinal cord induced by CCI. After administration with either SB203580 (p38MAPK inhibitor) or PDTC (NF-kappaB inhibitor), the levels of P2Y13 receptor expression in the dorsal spinal cord were lower than those in the CCI group. However, in CCI rats, the increased expression of P2Y12 receptor was prevented by intrathecal administration of PDTC but not by SB203580. In addition, minocycline significantly decreased the increased expression of P2Y12 and P2Y13 receptors. The similar results can be observed in ADPbetaS-treated rats. Intrathecal injection of ADPbataS causes thermal hyperalgesia and increased expression of P2Y12 and P2Y13 receptors in the dorsal spinal cord of naive rats. Moreover, intrathecal injection of AM1241 alleviates pain response and reduces the elevated expression of P2Y12 and P2Y13 receptors, p-p38MAPK, and NF-κBp65 in the dorsal spinal cord of ADPbetaS-treated rats. Intrathecal injection of SB203580 significantly inhibited the ADPbetaS-induced P2Y13 receptor expression, without affecting P2Y12 receptor expression. However, treatment with either SB203580 or PDTC effectively inhibited P2Y13 receptor expression compared to ADPbetaS-treated rats. CONCLUSIONS: In CCI- and ADPbetaS-treated rats, AM1241 pretreatment could efficiently activate CB2 receptor, while inhibiting p38MAPK and NF-kappaB activation in the dorsal spinal cord. CB2 receptor stimulation decreased P2Y13 receptor expression via p38MAPK/NF-kappaB signaling. On the other hand, CB2 receptor activation decreased P2Y12 receptor expression via p38MAPK-independent NF-kappaB signaling pathway.

Antagonizing LINGO-1 reduces activated microglia and alleviates dendritic spine loss in the hippocampus of APP/PS1 transgenic mice.

In Alzheimer's disease (AD), microglia are involved in synaptic pruning and mediate synapse loss. LINGO-1 is a negative regulator of nerve growth, and whether antagonizing LINGO-1 can attenuate synaptic pruning by microglia and rescue dendritic spines in the hippocampus in AD is still unclear. On this basis, the anti-LINGO-1 antibody, which binds to LINGO-1 protein and antagonizes the effects of LINGO-1, was administered to 10-month-old APP/PS1 transgenic mice for 2 months. The Morris water maze test, immunohistochemical and stereological methods, immunofluorescence and 3D reconstruction were used. Compared to wild-type mice, APP/PS1 transgenic mice had worse performance on behavioral tests, fewer dendritic spines but more microglia in the hippocampus. Meanwhile, the microglia in APP/PS1 transgenic mice had more branches of medium length (4-6 µm) and a cell body area with greater variability. Moreover, APP/PS1 transgenic mice had more postsynaptic termini colocalized with microglia in the hippocampus than wild-type mice. The anti-LINGO-1 antibody significantly reversed these changes in AD, indicating that the anti-LINGO-1 antibody can improve hippocampus-dependent learning and memory abilities and effectively rescue dendritic spines in the hippocampus of AD mice and that microglia might participate in this progression in AD. These results provide a scientific basis for further studying the mechanism of the anti-LINGO-1 antibody in AD and help to elucidate the role of LINGO-1 in the treatment of AD.

Running exercise decreases microglial activation in the medial prefrontal cortex in an animal model of depression.

BACKGROUND: Running exercise effectively ameliorates depressive symptoms in humans and depression-like behaviors in animals, but the underlying mechanisms remain unclear. Microglia-mediated neuroinflammation plays a major role in the development of depression. The medial prefrontal cortex (mPFC) is a key brain region involved in depression and is sensitive to physical activity. Whether the antidepressant effect of running exercise involves changes in mPFC microglia is not understood. METHODS: The animals were subjected to chronic unpredictable stress (CUS) intervention followed by treadmill running. The sucrose preference test and elevated plus maze test or tail suspension test were used for behavioral assessment of the animals. The number of microglia in the mPFC was quantified by immunohistochemistry and stereology. The density and morphology of microglia were analyzed via immunofluorescence staining combined with three-dimensional laser scanning techniques. The mRNA expressions of inflammatory cytokines in the mPFC were examined via quantitative real-time PCR. RESULTS: Running exercise effectively alleviated depressive-like behaviors in depression model animals. Running exercise reversed the increase in the number of microglia and the density of activated microglia in the mPFC of CUS animals. Running exercise effectively reversed the changes in microglia (reduced cell body area, total branch length and branch complexity) in the mPFC of CUS animals. Furthermore, running exercise regulated the gene expressions of pro-/antiinflammatory cytokines in the mPFC of CUS animals. CONCLUSIONS: Our results suggested that the antidepressant effects of running exercise may involve decreasing the number of activated microglia, reversing morphological changes in microglia in the mPFC, and reducing inflammatory responses.

MPFC PV+ interneurons are involved in the antidepressant effects of running exercise but not fluoxetine therapy.

Depression is a complex psychiatric disorder. Previous studies have shown that running exercise reverses depression-like behavior faster and more effectively than fluoxetine therapy. GABAergic interneurons, including the PV+ interneuron subtype, in the medial prefrontal cortex (MPFC) are involved in pathological changes of depression. It was unknown whether running exercise and fluoxetine therapy reverse depression-like behavior via GABAergic interneurons or the PV+ interneurons subtype in MPFC. To address this issue, we subjected mice with chronic unpredictable stress (CUS) to a 4-week running exercise or fluoxetine therapy. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that running exercise enriched GABAergic synaptic pathways in the MPFC of CUS-exposed mice. However, the number of PV+ interneurons but not the total number of GABAergic interneurons in the MPFC of CUS-exposed mice reversed by running exercise, not fluoxetine therapy. Running exercise increased the relative gene expression levels of the PV gene in the MPFC of CUS-exposed mice without altering other subtypes of GABAergic interneurons. Moreover, running exercise and fluoxetine therapy both significantly improved the length, area and volume of dendrites and the spine morphology of PV+ interneurons in the MPFC of mice exposed to CUS. However, running exercise but not fluoxetine therapy improved the dendritic complexity level of PV+ interneurons in the MPFC of CUS-exposed mice. In summary, the number and dendritic complexity level of PV+ interneurons may be important therapeutic targets for the mechanism by which running exercise reverses depression-like behavior faster and more effectively than fluoxetine therapy.

Anti-LINGO-1 antibody protects neurons and synapses in the medial prefrontal cortex of APP/PS1 transgenic mice.

The medial prefrontal cortex (mPFC), one of the most vulnerable brain regions in Alzheimer's disease (AD), plays a critical role in cognition. Leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein-1 (LINGO-1) negatively affects nerve growth in the central nervous system; however, its role in the pathological damage to the mPFC remains to be studied in AD. In this study, an anti-LINGO-1 antibody was administered to 10-month-old APP/PS1 mice, and behavioral tests, stereological methods, immunohistochemistry and immunofluorescence were used to answer this question. Our results revealed that LINGO-1 was highly expressed in the neurons of the mPFC of AD mice, and the anti-LINGO-1 antibody improved prefrontal cortex-related function and reduced the protein level of LINGO-1, atrophy of the volume, Aß deposition and massive losses of synapses and neurons in the mPFC of AD mice. Antagonizing LINGO-1 could effectively alleviate the pathological damage in the mPFC of AD mice, which might be an important structural basis for improving prefrontal cortex-related function. Abnormal expression of LINGO-1 in the mPFC may be one of the key targets of AD, and the effect initiated by the anti-LINGO-1 antibody may provide an important basis in the search for drugs for the prevention and treatment of AD.

A Study on Paradoxical Leadership and Multiple Path Mechanisms of Employees' Bootleg Innovation.

Purpose: Based on the social cognitive theory and cognitive-affective system theory, the purpose of this study is to explore how and when paradoxical leadership enhances employees' bootlegging innovation. To achieve this purpose, the authors proposed a double-chain mediation model in this study. Methods: Data with 342 questionnaires were collected for effective matching between employees and leaders at two time nodes. The hypotheses were validated by structural equation modeling and bootstrap approaches. Results: Results indicate that paradoxical leadership has a significant and positive impact on employees' bootleg innovation. In addition, psychological capital and thriving at work play a partial mediating role between paradoxical leadership and employees' bootlegging behaviors respectively and a chain mediating role between the two together. Moreover, there is no significant difference among the three mediating paths. Conclusion: The present research advances our understanding of bootleg innovation with a focus on the specific role of paradoxical leadership. Our findings, and especially those related to the role of psychological capital and thriving at work, reveal the influence mechanisms of paradoxical leadership on employees' bootleg innovation. At the same time, it is useful for understanding what leadership style can effectively stimulate employees' bootleg innovation.

The "Double-Edged Sword" Effects of LMX Ambivalence---An Integrated Model of Two Approaches Based on Cognitive Flexibility and Job Anxiety.

Purpose: Bootleg Innovation helps enterprises create unconventional innovation achievements, and has gradually become an important approach for radical innovation under the background of open innovation. Through the integration of the cognitive-affective processing system and conservation of resources (COR) theory, the present study takes integrative complexity as a moderator, and explores the mechanism of LMX ambivalence on employees' bootleg innovation. Participants and Methods: A total of 387 employees and 110 supervising managers participated in the two-wave dyadic survey. To test our hypotheses, we performed the hierarchical regression and conducted bootstrapping analyses to test our hypotheses. Results: Findings indicated that LMX ambivalence not only improves employees' cognitive flexibility and promotes bootleg innovation, but also aggravates employees' job anxiety and hinders bootleg innovation. In addition, integrative complexity significantly moderates the above two mediating paths. Specifically, when integrative complexity was higher, the negative mediating effect of job anxiety was attenuated, and the positive mediating effect of cognitive flexibility was enhanced. Conclusion: Contrasting with previous research, this research analyzed the triggering mechanism of bootleg innovation combined with the dynamic interaction of environmental stimulus, emotion and cognition. The findings provide novel insight into how to achieve bootleg innovation.

Positive effects of running exercise on astrocytes in the medial prefrontal cortex in an animal model of depression.

Depression is one of the most common mental illnesses and seriously affects all aspects of life. Running exercise has been suggested to prevent or alleviate the occurrence and development of depression; however, the underlying mechanisms of these effects remain unclear. Independent studies have indicated that astrocytes play essential roles and that the medial prefrontal cortex (mPFC) is an important brain region involved in the pathology underlying depression. However, it is unknown whether running exercise achieves antidepressant effects by affecting the number of astrocytes and glutamate transport function in the mPFC. Here, animal models of depression were established using chronic unpredictable stress (CUS), and depression-like behavior was assessed by the sucrose preference test. After successfully establishing the depression model, experimental animals performed running exercise. Glial fibrillary acidic protein-positive (GFAP+ ) cell number in the mPFC was precisely quantified using immunohistochemical and stereological methods, and the densities of bromodeoxyuridine-positive (BrdU+ ) and BrdU+ /GFAP+ cells in the mPFC were measured using a semiquantitative immunofluorescence assay. Changes in glutamate transporter gene expression in mPFC astrocytes were detected by mRNA sequencing and qRT-PCR. We found that running exercise reversed CUS-induced decreases in sucrose preference, increased astrocyte number and the density of newborn astrocytes, and reversed decreases in gene expression levels of GFAP, S100b, and the glutamate transporters GLT-1 and GLAST in the mPFC of CUS animals. These results suggested that changes in astrocyte number and glutamate transporter function may be potential meditators of the effects of running exercise in the treatment of depression.

Crosslinking catalytic hairpin assembly for high-contrast imaging of multiple mRNAs in living cells.

A novel DNA nanotetrad mediated crosslinking catalytic hairpin assembly (CCHA) is reported to generate clumps of cross-linked mesh products for high-contrast and simultaneous imaging of multiple mRNAs in living cells.

TGF-ß Secretion by M2 Macrophages Induces Glial Scar Formation by Activating Astrocytes In Vitro.

Transforming growth factor-ß (TGF-ß) is a key factor that promotes fibrosis or scar formation, which could become an obstacle in the repair of impaired axons in the central nervous system (CNS) of the human body resulting from diseases or injuries. Considering that major pathological reactions occur during this process, we focused on TGF-secreting M2 macrophages to identify the interactions between M2 macrophages and astrocytes (AS) and verify the specific mechanism of fibrosis or glial scar formation. In the present study, we used the Transwell coculturing technique and found an increase in glial fibrillary acidic protein (GFAP), neurocan, IL-13, and TGF-ß expression after incubation for 48 h; the expression of these proteins decreased when additional inhibitors of the TGF-ß receptor were added. We concluded that fibrosis or glial scar formation would be enhanced by the secretion of neurocan from AS, resulting from the release of TGF-ß from M2 macrophages. We also used M2 macrophage-conditioned medium to further confirm this finding in a subsequent experiment. We hope that the findings in this research could provide a foundation for locating new targets for treating CNS diseases or injuries.

Small molecule-linked programmable DNA for washing-free imaging of cell surface biomarkers.

Detection of specific biomarkers in cell membranes is critical for cell biology and disease theranostics. Here we develop a versatile terminal protection assay strategy for wash-free quantification and imaging of cell surface proteins using small molecule-linked DNA with programmable signal sequences. DNA probes are designed to link to a small molecule ligand at 3' end for specific recognition of the cell surface protein and a programmable signal sequence at 5' terminal for delivering detectable responses. Binding of the small molecule ligand to target protein enables protection of the DNA probes from exonuclease I mediated degradation, leaving the surface-binding probes intact while the non-binding probes degraded. This strategy thus allows wash-free detection of the cell surface protein via the selectively protected signal sequence. By programming the signal sequences as peroxidase-like DNAzyme, quantitative polymerase chain reaction (qPCR) targeting DNA and Ag nanoclusters (AgNCs) forming DNA template based on our new finding that the exonuclease I is able to quench the fluorescence of AgNCs, we can develop this strategy into a versatile platform for colorimetric detection, qPCR quantification and fluorescence imaging of the cell surface protein. This platform is demonstrated using a folate-linked DNA probe for folate receptor detection on tumor cell surface. The results revealed that this strategy enables highly selective and sensitive detection of the tumor cells as well as quantification and localization of the membrane protein on the cells, implying its potential in membrane protein based biomedical and clinical applications.

Activation of spinal dorsal horn P2Y13 receptors can promote the expression of IL-1ß and IL-6 in rats with diabetic neuropathic pain.

OBJECTIVE: The dorsal horn P2Y13 receptor is involved in the development of pain behavior induced by peripheral nerve injury. It is unclear whether the expression of proinflammatory cytokines interleukin (IL)-1ß and IL-6 at the spinal dorsal horn are influenced after the activation of P2Y13 receptor in rats with diabetic neuropathic pain (DNP). METHODS: A rat model of type 1 DNP was induced by intraperitoneal injection of streptozotocin (STZ). We examined the expression of P2Y13 receptor, Iba-1, IL-1ß, IL-6, JAK2, STAT3, pTyr1336, and pTyr1472 NR2B in rat spinal dorsal horn. RESULTS: Compared with normal rats, STZ-diabetic rats displayed obvious mechanical allodynia and the increased expression of P2Y13 receptor, Iba-1, IL-1ß, and IL-6 in the dorsal spinal cord that was continued for 6 weeks in DNP rats. The data obtained indicated that, in DNP rats, administration of MRS2211 significantly attenuated mechanical allodynia. Compared with DNP rats, after MRS2211 treatment, expression of the P2Y13 receptor, Iba-1, IL-1ß, and IL-6 were reduced 4 weeks after the STZ injection. However, MRS2211 treatment did not attenuate the expression of the P2Y13 receptor, Iba-1, IL-1ß, and IL-6 at 6 weeks after the STZ injection. MRS2211 suppressed JAK2 and STAT3 expression in the early stage, but not in the later stage. Moreover, pTyr1336 NR2B was significantly decreased, whereas pTyr1472 NR2B was unaffected in the dorsal spinal cord of MRS2211-treated DNP rats. CONCLUSION: Intrathecal MRS2211 produces an anti-nociceptive effect in early-stage DNP. A possible mechanism involved in MRS2211-induced analgesia is that blocking the P2Y13 receptor downregulates levels of IL-1ß and IL-6, which subsequently inhibit the activation of the JAK2/STAT3 signaling pathway. Furthermore, blocking the activation of the P2Y13 receptor can decrease NR2B-containing NMDAR phosphorylation in dorsal spinal cord neurons, thereby attenuating central sensitization in STZ-induced DNP rats.

P2Y6 receptor activation is involved in the development of neuropathic pain induced by chronic constriction injury of the sciatic nerve in rats.

Purinergic signaling in spinal cord microglia plays an important role in the pathogenesis of neuropathic pain. Among all P2 receptors, P2Y6 receptor is expressed in rat dorsal spinal cord. However, it's not clear that the role of P2Y6 receptor in the chronic constriction injury (CCI) model of neuropathic pain rats. We evaluated the effect of repeated intrathecal administration of MRS2578 (selective P2Y6 receptor antagonist) on CCI-induced nociceptive behaviors in rats. After CCI, MRS2578 (10-11-10-4 M) was administration. The thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were assessed. The expression of P2Y6 receptor and Iba-1 at rat dorsal spinal cord was observed by using RT-PCR. We found that intrathecal injection of MRS2578 suppressed CCI-induced mechanical allodynia and thermal hyperalgesia with a dose-dependent manner. The CCI rats presented increased expression of P2Y6 receptor and Iba-1 at the mRNA level in the ipsilateral dorsal spinal cord than that in sham group. Treatment with either minocycline or SB203580 effectively inhibited P2Y6 receptor expression compared to CCI rats. Intrathecal injection of UDP enhanced mechanical and thermal allodynia than that in CCI group. To the further study, intrathecal injection of UDP causes mechanical allodynia and thermal hyperalgesia in naive rats. The increased expression of P2Y6 receptor and Iba-1 were observed in UDP-treated rats. Intrathecal injection of MRS2578 alleviates pain response in UDP-treated rats. These observations suggested that P2Y6 receptor in dorsal spinal cord contribute to mechanical allodynia and thermal hyperalgesia in CCI-induced neuropathic pain.

Protein scaffolded DNA tetrads enable efficient delivery and ultrasensitive imaging of miRNA through crosslinking hybridization chain reaction.

Efficient intracellular delivery of nucleic acids to achieve sensitive detection and gene regulation is essential for chemistry and biology. Here we developed a novel protein scaffolded DNA tetrad, a four-arm DNA nanostructure constructed using streptavidin (SA) protein and four biotinylated hairpin DNA probes for efficient nucleic acid delivery and ultrasensitive miRNA imaging through crosslinking hybridization chain reaction (cHCR). DNA tetrads were easy to prepare and allowed precise control of the structure of the probes. DNA tetrads showed rapid intracellular delivery of DNA probes and high efficiency in lysosome escape by using confocal images for individual cells and flow cytometry for a large population of cells. cHCR allowed generating clumps of crosslinked hydrogel networks specifically to target miRNA, affording high sensitivity and spatial resolution for imaging. To our knowledge, this is the first time that HCR amplification has been realized in situ on nanostructures. Moreover, the FRET based design of cHCR conferred improved precision with the use of dual-emission ratiometric imaging to avoid false signals in biological systems. Intracellular imaging experiments further showed that DNA tetrad based cHCR could realize ultrasensitive and accurate miRNA imaging in living cells. Moreover, DNA tetrad based cHCR provided a potential tool for quantitative measurement of intracellular miRNA. The results suggested that this developed strategy provided a useful platform for nucleic acid delivery and low level biomarker imaging.

P2Y12 and P2Y13 receptors involved in ADPßs induced the release of IL-1ß, IL-6 and TNF-α from cultured dorsal horn microglia.

OBJECTIVE: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y12 and P2Y13 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y12 and P2Y13 receptors activation is associated with the expression and the release of interleukin-1B (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPßs (ADP analog) on the expression and the release of IL-1ß, IL-6, and TNF-α. METHODS AND RESULTS: In this study, we observed the effect of P2Y receptor agonist ADPßs on the expression and release of IL-1ß, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPßs induced the increased expression of Iba-1, IL-1ß, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPßs-evoked increase in Iba-1, IL-1ß, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y12 receptor antagonist MRS2395 or P2Y13 receptor antagonist MRS2211, respectively. Similarly, ADPßs-evoked release of IL-1ß, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPßs-evoked increased expression of Iba-1, IL-1ß, IL-6 and TNF-α mRNA, and release of IL-1ß, IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y12 and P2Y13 receptor-evoked increased gene expression of IL-1ß, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y12 and P2Y13 receptor-evoked release of IL-1ß, IL-6 and TNF-α, were also inhibited by Y-27632, SB203580 and PDTC, respectively. CONCLUSION: These observations suggest that P2Y12 and P2Y13 receptor-evoked gene expression and release of IL-1ß, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.