Proximity hybridization based "turn-on" DNA tweezers for accurate and enzyme-free small extracellular vesicle analysis.

Small extracellular vesicles (sEVs) are a type of extracellular vesicle that carries many types of molecular information. The identification of sEVs is essential for the non-invasive detection and treatment of illnesses. Hence, there is a significant need for the development of simple, sensitive, and precise methods for sEV detection. Herein, a DNA tweezers-based assay utilizing a "turn-on" mechanism and proximity ligation was suggested for the efficient and rapid detection of sEVs through amplified fluorescence. The target facilitates the proximity combination of the C1 probe and C2 probe, resulting in the formation of a complete extended sequence. The elongated sequence can cyclically initiate the hairpin probe (HP), leading to the activation of DNA tweezers. An excellent linear correlation was achieved, with a limit of detection of 57 particles per µL. Furthermore, it has been effectively employed to analyze sEVs under intricate experimental conditions, demonstrating a promising and pragmatic prospect for future applications. Given that the identification of sEVs was successfully accomplished using a single-step method that exhibited exceptional sensitivity and strong resistance to interference, the proposed technique has the potential to provide a beneficial platform for accurate recognition of sEVs and early detection of diseases.

Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

Mouse Model of Fragile X Syndrome Analyzed by Quantitative Proteomics: A Comparison of Methods.

Multiple methods for quantitative proteomics are available for proteome profiling. It is unclear which methods are most useful in situations involving deep proteome profiling and the detection of subtle distortions in the proteome. Here, we compared the performance of seven different strategies in the analysis of a mouse model of Fragile X Syndrome, involving the knockout of the fmr1 gene that is the leading cause of autism spectrum disorder. Focusing on the cerebellum, we show that data-independent acquisition (DIA) and the tandem mass tag (TMT)-based real-time search method (RTS) generated the most informative profiles, generating 334 and 329 significantly altered proteins, respectively, although the latter still suffered from ratio compression. Label-free methods such as BoxCar and a conventional data-dependent acquisition were too noisy to generate a reliable profile, while TMT methods that do not invoke RTS showed a suppressed dynamic range. The TMT method using the TMTpro reagents together with complementary ion quantification (ProC) overcomes ratio compression, but current limitations in ion detection reduce sensitivity. Overall, both DIA and RTS uncovered known regulators of the syndrome and detected alterations in calcium signaling pathways that are consistent with calcium deregulation recently observed in imaging studies. Data are available via ProteomeXchange with the identifier PXD039885.

Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant.

Epilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in total membrane protein expression. Super-resolution and FRET imaging confirmed heteromeric channel formation and a higher expression density of Kv7.2mut. Cannabidiol (1 µM) offset the effects of Kv7.2mut by inducing a hyperpolarizing shift in voltage for activation independent of CB1 or CB2 receptors. These data reveal that the ability for cannabidiol to reduce the effects of a pathogenic Kv7.2 variant supports its use as a potential therapeutic to reduce seizure activity.

Increased RyR2 open probability induces neuronal hyperactivity and memory loss with or without Alzheimer's disease-causing gene mutations.

INTRODUCTION: Neuronal hyperactivity is an early neuronal defect commonly observed in familial and sporadic Alzheimer's disease (AD), but the underlying mechanisms are unclear. METHODS: We employed a ryanodine receptor 2 (RyR2) mutant mouse model harboring the R4496C+/- mutation that markedly increases the channel's open probability (Po) to determine the impact of increased RyR2 activity in neuronal function without AD gene mutations. RESULTS: Genetically increasing RyR2 Po induced neuronal hyperactivity in vivo in anesthetized and awake mice. Increased RyR2 Po induced hyperactive behaviors, impaired learning and memory, defective dendritic spines, and neuronal cell death. Increased RyR2 Po exacerbated the onset of neuronal hyperexcitability and learning and memory impairments in 5xFAD mice. DISCUSSION: Increased RyR2 Po exacerbates the onset of familial AD-associated neuronal dysfunction, and induces AD-like defects in the absence of AD-causing gene mutations, suggesting that RyR2-associated neuronal hyperactivity represents a common target for combating AD with or without AD gene mutations.

Limiting RyR2 Open Time Prevents Alzheimer's Disease-Related Neuronal Hyperactivity and Memory Loss but Not ß-Amyloid Accumulation.

Neuronal hyperactivity is an early primary dysfunction in Alzheimer's disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued ß-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-ß-amyloid-targeted anti-AD strategy.

FMRP(1-297)-tat restores ion channel and synaptic function in a model of Fragile X syndrome.

Fragile X Syndrome results from a loss of Fragile X Mental Retardation Protein (FMRP). We now show that FMRP is a member of a Cav3-Kv4 ion channel complex that is known to regulate A-type potassium current in cerebellar granule cells to produce mossy fiber LTP. Mossy fiber LTP is absent in Fmr1 knockout (KO) mice but is restored by FMRP(1-297)-tat peptide. This peptide further rapidly permeates the blood-brain barrier to enter cells across the cerebellar-cortical axis that restores the balance of protein translation for at least 24 h and transiently reduces elevated levels of activity of adult Fmr1 KO mice in the Open Field Test. These data reveal that FMRP(1-297)-tat can improve function from the levels of protein translation to synaptic efficacy and behaviour in a model of Fragile X syndrome, identifying a potential therapeutic strategy for this genetic disorder.

Long-Term Potentiation at the Mossy Fiber-Granule Cell Relay Invokes Postsynaptic Second-Messenger Regulation of Kv4 Channels.

Mossy fiber afferents to cerebellar granule cells form the primary synaptic relay into cerebellum, providing an ideal site to process signal inputs differentially. Mossy fiber input is known to exhibit a long-term potentiation (LTP) of synaptic efficacy through a combination of presynaptic and postsynaptic mechanisms. However, the specific postsynaptic mechanisms contributing to LTP of mossy fiber input is unknown. The current study tested the hypothesis that LTP induces a change in intrinsic membrane excitability of rat cerebellar granule cells through modification of Kv4 A-type potassium channels. We found that theta-burst stimulation of mossy fiber input in lobule 9 granule cells lowered the current threshold to spike and increases the gain of spike firing by 2- to 3-fold. The change in postsynaptic excitability was traced to hyperpolarizing shifts in both the half-inactivation and half-activation potentials of Kv4 that occurred upon coactivating NMDAR and group I metabotropic glutamatergic receptors. The effects of theta-burst stimulation on Kv4 channel control of the gain of spike firing depended on a signaling cascade leading to extracellular signal-related kinase activation. Under physiological conditions, LTP of synaptically evoked spike output was expressed preferentially for short bursts characteristic of sensory input, helping to shape signal processing at the mossy fiber-granule cell relay. SIGNIFICANCE STATEMENT: Cerebellar granule cells receive mossy fiber inputs that convey information on different sensory modalities and feedback from descending cortical projections. Recent work suggests that signal processing across multiple cerebellar lobules is controlled differentially by postsynaptic ionic mechanisms at the level of granule cells. We found that long-term potentiation (LTP) of mossy fiber input invoked a large increase in granule cell excitability by modifying the biophysical properties of Kv4 channels through a specific signaling cascade. LTP of granule cell output became evident in response to bursts of mossy fiber input, revealing that Kv4 control of intrinsic excitability is modified to respond most effectively to patterns of afferent input that are characteristic of physiological sensory patterns.

[B Cell Activating Transcription Factor Regulates Acute Airway Inflammation in Asthmatic Mice].

OBJECTIVE: To investigate the regulatory effect of B cell activating transcription factor (BATF) on acute airway inflammation and its association with retinoic acid orphan nuclear receptors gammat (RORyt) in asthmatic mice. METHODS: 24 female BALB/c mice were randomly and equally divided into three groups (n 8): normal saline (NS) treated, asthma (AS) control and dexamethasone (DEX) treated. AS mice were sensitized and challenged with OVA to establish murine asthma model. Histological changes in lung tissues of the mice were observed by HE staining. Numbers of white blood cell (WBC), polymorphonuclear leukocyte (PMN) and eosinophils (EOS) in the bronchoalveolar lavage fluid (BALF) of the mice were counted. The concentration of interleukin-17 (IL-17) in BALF was measured by ELISA. Quantitative real-time PCR (RT-PCR) was performed to assess the mRNA expressions of BATF, IL-17 and RORγt in the lung tissues. RESULTS: The HE staining showed a higher level of inflammatory cell infiltration around the bronchi of AS mice compared with those treated with NS, predominantly in the forms of EOS, PMN and lymphocytes. The AS and DEX treated mice had higher levels of EOS, PMN, WBC and mRNA expressions of BATF, IL-17 and RORγt in BALF than those treated with NS (P < 0.05). DEX reduced the levels of EOS, PMN, WBC and IL-17 in BALF significantly (P < 0.05). The mRNA expression of BATF in lung tissues of mice was positively correlated with the expression of IL-17, RORγt and the counts of WBC,EOS and PMN in BALF (P < 0.05). CONCLUSION: Asthmatic mice have increased expressions of BATF, IL-17 and RORγt in bronchial and lung tissues. BATF can, through regulating the secretion of Th17 cells, readjust the airway inflammatory. The regulatory function may take effect through synergy with RORγt .

Aß40 modulates GABA(A) receptor α6 subunit expression and rat cerebellar granule neuron maturation through the ERK/mTOR pathway.

In addition to their neurotoxic role in Alzheimer's disease (AD), ß-amyloid peptides (Aßs) are also known to play physiological roles. Here, we show that recombinant Aß40 significantly increased the outward current of the GABA(A) receptor containing (GABA(A)α6) in rat cerebellar granule neurons (CGNs). The Aß40-mediated increase in GABA(A)α6 current was mediated by an increase in GABA(A)α6 protein expression at the translational rather than the transcriptional level. The exposure of CGNs to Aß40 markedly induced the phosphorylation of ERK (pERK) and mammalian target of rapamycin (pmTOR). The increase in GABA(A)α6 current and expression was attenuated by specific inhibitors of ERK or mTOR, suggesting that the ERK and mTOR signaling pathways are required for the effect of Aß40 on GABA(A)α6 current and expression in CGNs. A pharmacological blockade of the p75 neurotrophin receptor (p75(NTR)), but not the insulin or α7-nAChR receptors, abrogated the effect of Aß40 on GABA(A)α6 protein expression and current. Furthermore, the expression of GABA(A)α6 was lower in CGNs from APP(-/-) mice than in CGNs from wild-type mice. Moreover, the internal granule layer (IGL) in APP(-/-) mice was thinner than the IGL in wild-type mice. The injection of Aß40 into the cerebellum reversed this effect, and the application of p75(NTR) blocking antibody abolished the effects of Aß40 on cerebellum morphology in APP(-/-) mice. Our results suggest that low concentrations of Aß40 play a role in regulating CGN maturation through p75(NTR).

CXCL12/CXCR4 signal axis plays an important role in mediating bone morphogenetic protein 9-induced osteogenic differentiation of mesenchymal stem cells.

Mesenchymal progenitor stem cells (MPCs) are a group of bone marrow stromal progenitor cells processing osteogenic, chondrogenic, adipogenic and myogenic lineages differentiations. Previous studies have demonstrated that bone morphogeneic protein 9(BMP9) is one of the most osteogenic BMPs both in vitro and in vivo, however, the underlying molecular mechanism of osteogenesis induced by BMP9 is needed to be deep explored. Here, we used the recombinant adenoviruses assay to introduce BMP9 into C3H10T1/2 mesenchymal stem cells to elucidate the role of CXCL12/CXCR4 signal axis during BMP9-incuced osteogenic differentiation. The results showed that CXCL12 and CXCR4 expressions were down-regulated at the stage of BMP9-induced osteogenic differentiation, in a dose- and time-dependent. Pretreatment of C3H10T1/2 cells with CXCL12/CXCR4 could significantly affect the early and mid osteogenic markers alkaline phosphatase (ALP), osteocalcin (OCN), the transcription factors of Runx2, Osx, Plzf and Dlx5 expression, through activating the Smad, MAPK signaling pathway. Addition of exogenous CXCL12 did not affect the changes of the late osteogenic marker calcium deposition. Thus, our findings suggest a co-requirement of the CXCL12/CXCR4 signal axis in BMP9-induced the early- and mid-process of osteogenic differentiation of MSCs.

[Effects of combination of Salvia and Ligustrazine on TNF-α and TGF-ß1 in serum and BALF of rats with pulmonary fibrosis].

OBJECTIVE: To observe the effects of Salvia combined with Ligustrazine on the TNF-α and TGF-ß1 in sera and bronchoalveolar lavage fluid (BALF) of rats with pulmonary fibrosis induced by bleomycin (BLM). METHODS: Male SD rats (n=90) were randomly assigned into 6 groups: normal saline (NS) group, BLM group, dexamethasone (DXM) group and three Chinese traditional herbal groups (C1: small dose group, C2: medium dose group, C3: large dose group). Rats were challenged intratracheally with BLM to establish pulmonary fibrosis rat models, while the NS group with 0.9% NaCl solution. Rats were given intra-peritoneal injections of normal saline (both the NS group and the BLM group), dexamethasone (the DXM group), or Salvia combined with Ligustrazine (the C1, C2, C3 groups). At the same time, 5 rats from the 6 groups were sacrificed at day 7, 14 and 28 after administration respectively. HE and Masson staining were performed to observe the extent of alveolitis and fibrosis; the levels of TNF-α and TGF-ß1 in both sera and BALF were detected by ELISA. RESULTS: Compared with NS group, a great deal of inflammatory cell infiltration into the alveolar was observed in BLM group at day 7, the alveolitis was lessened at day 14, while fibrosis was found; at day 28, the alveolitis became relieved and the fibrosis developed seriously. The extent of alveolitis and pulmonary fibrosis in DXM group was milder than that in BLM group at day 7, 14 and 28 (P<0.05). Compared with BLM group, the extent of pulmonary fibrosis in group C1 didn't remarkably change (P>0.05). The extent of alveolitis and pulmonary fibrosis in group C2 and C3 was significantly milder than that in BLM group at day 14, and the extent of pulmonary fibrosis in group C2 and C3 was remarkably reduced at day 28 (P<0.05). Compared with NS group, the levels of TNF-α and TGF-ß1 in both sera and BALF of the other groups were obviously higher (P<0.05). Compared with BLM group, the levels in DXM, C2 and C3 groups were lower at day 7, 14, 28 (P<0.05). Compared with DXM group, the levels in C1, C2 and C3 groups were lower at day 7, 14, 28 (P<0.05), and the difference between C2 and C3 groups was statistically significant (P>0.05). The levels of of TNF-α and TGF-ß1 were consistent in rat sera and BALF. CONCLUSION: Medium and large doses of Salvia combined with Ligustrazine could abate the alveolitis and ameliorate the extent of pulmonary fibrosis by decreasing the levels of TNF-α and TGF-ß1 in both serum and BALF.

Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion.

Transforming growth factor-ß (TGF-ß) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-ß family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.

Exposure to extremely low-frequency electromagnetic fields modulates Na+ currents in rat cerebellar granule cells through increase of AA/PGE2 and EP receptor-mediated cAMP/PKA pathway.

Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

Abnormal expression of the mitotic checkpoint protein BubR1 contributes to the anti-microtubule drug resistance of esophageal squamous cell carcinoma cells.

Esophageal cancer is a common malignancy with a high mortality rate. The lack of effective chemotherapy and a means to overcome drug resistance leads to the predictable failure of esophageal cancer treatment. Mitotic checkpoint proteins play a critical role in regulating the cell cycle and proliferation. Abnormal expression of the mitotic checkpoint protein BubR1 has been reported in several types of cancers. In this study, we investigated the role of BubR1 in conferring resistance of esophageal cancer cells to anti-microtubule drugs. Using quantitative real-time PCR analysis on 50 samples of paired esophageal squamous cell cancer (ESC) tissues and adjacent non-cancerous tissues, we found that 72% (36 of 50) of the analyzed ESC samples exhibited high expression levels of BubR1, which was also confirmed in ESC cell lines. ESC cells with high levels of BubR1 were less sensitive to the anti-microtubule drugs paclitaxel and nocodazole. Recombinant adenovirus-mediated enforced expression of BubR1 in relatively sensitive ESC cell lines resulted in increased resistance to paclitaxel. Conversely, RNAi-mediated knockdown of BubR1 restored ESC cell sensitivity to paclitaxel. Cell cycle analysis indicated that the sub-G1 population increased in the ESC cells with reduced BubR1 levels. Taken together, our results suggest that upregulation of BubR1 expression may be associated with ESC resistance to paclitaxel treatment. Thus, BubR1 may serve as a potential chemosensitizing target to overcome chemoresistance.

MicroRNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bub1 expression in mouse.

Bub1 is a critical component of the spindle assembly checkpoint (SAC) and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3'-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF) cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.

Neuritin activates insulin receptor pathway to up-regulate Kv4.2-mediated transient outward K+ current in rat cerebellar granule neurons.

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K(+) currents (I(A)) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I(A) is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I(A). Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I(A) and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I(A) amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.

[Effect of Budesonide on Smad4, PDGF-A and PAI-1 in a rat model of pulmonary fibrosis].

AIM: To observe the effect of Budesonide (BUD) on TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue in pulmonary fibrosis rats. METHODS: Forty-five adult female Wistar rats were randomly divided into normal solution (NS) group, BUD group and bleomycin (BLM) group. 9 g/L NaCl solution was instilled into the tratracheaes in NS group, and BLM were used in BUD group and BLM group. NS group and BLM group were inhaled with 9 g/L NaCl solution once everyday at day 0-6, and BUD group were used with BUD. Five rats in every group were killed at 7th, 14th 28th day. The appearances of alveolitis and fibrosis were displayed in HE and Masson staining. The expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue were detected by immunohistochemistry. RESULTS: The extent of the alveolitis in BUD group at 7th, 14th day were significantly lower than that in BLM group(P<0.05). The fibrosis and the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissues at 7th, 14th, 28th day, BUD group were significantly lower than BLM group(P<0.05). CONCLUSION: After inhaled BUD, the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue could be decreased, and the extent of alveolitis and pulmonary fibrosis could be improved in bleomycin-induced pulmonary fibrosis rats.

The antidepressant citalopram inhibits delayed rectifier outward K⁺ current in mouse cortical neurons.

Citalopram, a selective serotonin (5-HT) reuptake inhibitor (SSRI) as well as an antidepressant, is thought to exert its effects by increasing synaptic 5-HT levels. However, few studies have addressed the possibility that citalopram has other molecular mechanisms of action. We examined the effects of citalopram on delayed rectifier outward K(+) current (I(K) ) in mouse cortical neurons. Extracellular citalopram reversibly inhibited I(K) in a dose-dependent manner and significantly shifted both steady-state activation and inactivation curves toward hyperpolarization. Neither 5-HT itself nor antagonists of 5-HT and dopamine receptors could abolish citalopram-induced inhibition of I(K) . In addition, intracellular application of GTPγ-S similarly failed to prevent the inhibition of I(K) by citalopram. When applied intracellularly, citalopram had no effect on I(K) and did not influence the reduction of I(K) induced by extracellular citalopram. The effect of citalopram was use dependent, but not frequency dependent, and it did not require channel opening. Electrophysiological recordings in acute cortical slice showed that citalopram significantly reduced the action potential (AP) firing frequency of cortical neurons and increased action potential duration (APD). The selective Kv2.1 subunit blocker Jingzhaotoxin-III (JZTX-III) did not abolish citalopram-induced I(K) inhibition. Transfection of HEK293 cells with Kv2.1 or Kv2.2 constructs indicated that citalopram mainly inhibited Kv2.2 current. We suggest that citalopram-induced inhibition of I(K) in mouse cortical neurons is independent of G-protein-coupled receptors and might exert its antidepressant effects by enhancing presynaptic efficiency. Our results may help to explain some of the unknown therapeutic effects of citalopram.

[Effects of andrographolide on the concentration of cytokines in BALF and the expressions of type I and III collagen mRNA in lung tissue in bleomycin-induced rat pulmonary fibrosis].

AIM: To investigate the effects of andrographolide on the concentration of TNF-α and TGF-ß1 in bronchoalveolar lavage fluid (BALF) and the expressions of type I and III collagen mRNA in Lung tissue in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: 90 healthy SD male rats were randomly divided into 6 groups with 15 rats each group: normal saline (NS) group, BLM group, prednisone (Pred) group and different doses of andrographolide groups (andrographolide group A 62.5 mg/kg, andrographolide group B 125 mg/kg and andrographolide group C 250 mg/kg). BLM was given to BLM group, Pred group and andrographolide group A, B, C by intratracheal instillation, and same volume of NS was given to NS group in the same way. And then NS was given to NS group and BLM group, Pred was given to Pred group and different does of andrographolide were given to andrograoholide group A, B, C by gavage every day. Five rats of each group were killed respectively at day 7, 14, 28 after intratracheal instillation. Alveolitis and fibrosis were observed by HE and Masson staining. Real-time fluorescent quantitative reverse transcription- polymerase chain reaction (FQ RT-PCR) was performed to detect the expressions of type I and III collagen mRNA in lung tissue, and the concentration of TNF-α and TGF-ß1 in BALF was determined by enzyme-linked immunosorbnent assay. Blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also examined. RESULTS: (1) The AST, ALT, BUN and Cr in every group had no significant diference(P>0.05). (2) Alveolar septal edema, inflammatory cell infiltration and fibrosis were not found in NS group. In the BLM group, lots of inflammatory cells infiltration were observed in the alveolar at day 7; the alveolitis was still existed, but inflammatory cells were significantly reduced, and the number of the fibroblasts and matrix in alveolar septum were obviously increased at day 14, at the same time, alveolar structure was damaged and alveolar septum widened; the inflammation cells infiltration of the alveolar was relieved, pulmonary fibrosis was increased, and parts of alveolar space was disappeared , severe fibrosis was found at day 28. It was similar between andrographolide group A and BLM group in pathomorphology. A lot of inflammatory cells infiltration and local accumulation were observed at day 7 in andrographolide group B, C and Pred group. However, compared with andrographolide group A and BLM group, the fibrosis at day 14, 28 was significantly reduced.(3) The concentration of TGF-ß1, TNF-α in BALF of NS group was significantly lower than that of Pred group, BLM group, andrographolide group A, B, C at each time point(P<0.05). The concentration of TGF-ß1 and TNF-α in BALF of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the concentration of TGF-ß1 and TNF-α in BALF of andrographolide group A had no significant difference. (4) The expression of type I and III collagen mRNA in lung tissue of NS group was significantly lower than that of group Pred, BLM, andrographolide group A, B, C at each time point (P<0.05). The expression of type I and III collagen mRNA in lung tissue of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the expression of type I and III collagen mRNA in lung tissue of andrographolide group A had no significant difference. CONCLUSION: In BLM-induced rat pulmonary fibrosis, andrographolide could attenuate alveolitis and fibrosis, decrease mRNA expression of collagen I and III in lung tissue and decrease the concentration of TNF-α and TGF-ß1 in BALF. It had no side effects on liver and kidney function.