MiR-195 promotes myocardial fibrosis in MI rats via targeting TGF-ß1/Smad.

The aim of this study was to investigate the effect of micro ribonucleic acid (miR)-195 on myocardial infarction (MI) in rats via regulating the transforming growth factor-ß1 (TGF-ß1)/Smad signaling pathway. A total of 36 Sprague-Dawley rats were randomly divided into a normal group (n=12), a model group (n=12) and an miR-195 antagomir group (n=12). In the normal group, the heart was exposed only, and normal saline was intraperitoneally injected after operation. In the model group, the acute MI model was established. In the miR-195 antagomir group, the acute MI model was also established, and miR- 195 antagomir was intraperitoneally injected. The samples were collected at 2 weeks after surgery. Then cardiac function was detected via echocardiography, and the morphology of heart tissues was observed via hematoxylin and eosin (H&E) staining. Moreover, the expression of Collagen I was determined using immunohistochemistry, the protein expressions of TGF-ß1, Smad3 and Smad7 were detected using Western blotting, and the expression of miR-195 was detected via quantitative polymerase chain reaction (qPCR). It was found by echocardiography that, compared with those in the normal group, left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) significantly declined, while left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs) significantly rose in the other two groups (P<0.05). In comparison with the model group, the miR-195 antagomir group had significantly increased LVEF and LVFS, and significantly decreased LVDd and LVDs (P<0.05). The immunohistochemistry results showed that the mean optical density of tissues with positively expressed Collagen I was obviously higher in the other two groups than that in the normal group (P<0.05), while it was obviously lower in the miR-195 antagomir group than that in the model group (P<0.05). According to the results of Western blotting, the protein expressions of TGF-ß1 and Smad3 were evidently increased, while the protein expression of Smad7 was evidently decreased in the other two groups compared with those in the normal group (P<0.05). The opposite results were found in the miR-195 antagomir group compared with those in the model group (P<0.05). The results of qPCR manifested that the expression of miR-195 was markedly higher in the other two groups than that in the normal group (P<0.05), while it was markedly lower in the miR-195 antagomir group than in the model group (P<0.05). Moreover, it was observed using H&E staining that the myocardial fibers in the normal group had normal arrangement and intact structure, without obvious morphological abnormalities. In the model group, the myocardial fibers were arranged disorderly, and there were massive proliferating fibrous tissues, with a high degree of fibrosis. In themiR-195 antagomir group, the myocardial fibers were damaged and arranged less disorderly, and proliferation and fibrosis could be seen in some fibrous tissues, but to a lesser extent than the model group. In conclusion, miR-195 promotes myocardial fibrosis in MI rats via up-regulating the TGF-ß1/Smad signaling pathway.

High thyroid-stimulating hormone level is associated with the risk of developing atherosclerosis in subclinical hypothyroidism.

The aim of our study was to assess the potential role of thyroid-stimulating hormone (TSH) in the risk of developing atherosclerosis in subclinical hypothyroidism (SCH). A cohort of 240 SCH patients and 150 euthyroid volunteers were recruited for the study. SCH patients were stratified into 2 groups according to TSH levels (group A: TSH<10 mIU/l; group B: TSH>10 mIU/l). All subjects were examined for clinical and biochemical parameters. Visfatin, omentin-1, and circulating endothelial biomarkers were measured. Patients in group B received l-thyroxine replacement to achieve euthyroidism; after 6 months of euthyroidism all measurements were repeated. Patients with SCH had higher total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and C-reactive protein (CRP) levels and lower nitric oxide (NO) and omentin-1 levels compared to euthyroid subjects (all p<0.05). TC, LDL-C, and CRP decreased significantly, while NO and omentin-1 levels increased significantly after l-thyroxine replacement. Based on multivariate liner stepwise regression analysis, omentin-1 was independently correlated with BMI and TSH; NO was independently correlated with age, TSH, LDL-C, and omentin-1. High TSH level contributes to endothelial dysfunction in SCH, while TSH-induced decrease of omentin-1 provides a new link between SCH and atherogenic risk.

MiRNA-875-3p alleviates the progression of colorectal cancer via negatively regulating PLK1 level.

OBJECTIVE: To clarify the expression pattern of miRNA-875-3p in CRC and its potential regulatory effect on the progression of CRC. MATERIALS AND METHODS: MiRNA-875-3p level in 56 matched CRC tissues and adjacent normal tissues were determined. The correlation between the miRNA-875-3p level and pathological indexes of CRC patients was analyzed. Prognostic potential of miRNA-875-3p in CRC patients was assessed by introducing the Kaplan-Meier curves. Influences of miRNA-875-3p on viability, migration, and wound closure were assessed through a series of functional experiments. The interaction between miRNA-875-3p and PLK1 in regulating the progression of CRC was finally uncovered by Dual-Luciferase reporter gene and rescue experiments. RESULTS: MiRNA-875-3p was downregulated in CRC tissues and cell lines. CRC patients with low level of miRNA-875-3p suffered a higher rate of distant metastasis and worse prognosis. Overexpression of miRNA-875-3p attenuated proliferative and migratory capacities of SW480 and HT29 cells. PLK1 was confirmed to be the target gene of miRNA-875-3p. PLK1 was upregulated in CRC tissues and cell lines, which was negatively regulated by miRNA-875-3p. MiRNA-875-3p alleviated the malignant progression of CRC via negatively regulating PLK1. CONCLUSIONS: MiRNA-875-3p is downregulated in CRC, which is closely related to distant metastasis and poor prognosis of CRC patients. MiRNA-875-3p alleviates the progression of CRC through targeting and downregulating PLK1.

Valproic acid inhibits the depolarizing rectification in neurons of rat amygdala.

The actions of valproic acid (VPA) on neuronal membrane properties and synaptic transmission were studied using intracellular recording techniques in rat basolateral neurons of the amygdala slices. In therapeutically attainable concentrations (10-100 microM), VPA decreased synaptically-induced epileptiform bursting in the presence of bicuculline. Additionally, the frequency of repetitive discharge induced by direct superthreshold depolarizing current pulses was decreased by VPA. However, evoked excitatory and inhibitory postsynaptic potentials were not affected at this level of drug concentration. The current-voltage relationship of untreated neurons revealed rectification of membrane potential when neuronal membrane was depolarized with cathodal current pulses. This depolarizing rectification was blocked by VPA. High medium calcium or addition of the sodium channel blocker tetrodotoxin (TTX) also blocked the depolarizing rectification, whereas the calcium channel antagonist diltiazem had no effect on the rectification. Elevation of medium calcium concentration also blocked the bicuculline-induced bursting. These results indicate that the inhibition by VPA of subthreshold slow sodium current and membrane depolarizing rectification results in suppression of neuronal membrane excitability which is probably a major mechanism for its anticonvulsant action.

Veratridine-enhanced persistent sodium current induces bursting in CA1 pyramidal neurons.

The mechanism of veratridine-induced bursting activity was studied in rat hippocampal CA1 pyramidal neurons. Veratridine (0.1-0.3 microM) induces bursting in previously normal pyramidal neurons. The current-voltage curves of untreated neurons show a slight deviation from the linear Ohmic relation; this deviation is known as the "depolarizing rectification". Veratridine markedly accentuates the depolarizing rectification so that a zero slope or negative slope appears in the current-voltage curve of these neurons. Both the veratridine-induced bursting activity and negative slope resistance are blocked by small concentrations of tetrodotoxin or by raising the calcium concentration of the superfusion medium. Under single-electrode voltage clamping, a subthreshold persistent (slowly inactivating) sodium current, which can be recorded in untreated neurons, is found to be enhanced in the veratridine-treated neurons. This current is thought to be responsible for the slow depolarizing phase of bursting activity and the development of negative slope resistance in the current-voltage relationship. The present results demonstrate that veratridine enhances the slowly inactivating sodium current, leading to the development of negative slope resistance and induction of bursting in rat hippocampal CA1 pyramidal neurons.

Endogenous bursting due to altered sodium channel function in rat hippocampal CA1 neurons.

Intracellular recordings were obtained from pyramidal neurons in the rat hippocampal CA1 area in order to investigate membrane mechanisms involved in veratridine-induced epileptiform activity. Veratridine (0.03-0.2 microM) caused no changes in the passive membrane parameters including the resting potential, input resistance, and time constant. In the presence of small doses (0.03-0.1 microM) of veratridine, a single stimulus caused a relatively slow, large, synaptic-independent potential called the slow depolarizing after-potential (SDAP). When the hippocampal slice was treated with higher doses of veratridine (over 0.1 microM), bursting, or seizure-like activity (SLA) occurred in response to a brief super threshold intracellular stimulation. The duration of SLA bursting could be as long as ten seconds depending on the amplitude of SDAP, and was independent of the stimulus strength or duration. The frequency and configuration of SLA were sensitive to changes in membrane potential caused by applied DC current. At 0.3 microM or higher, veratridine induced spontaneous rhythmic bursting that was also sensitive to membrane potential changes. The evoked or spontaneous bursting is characterized by being: (1) independent of synaptic transmission in that it persisted after complete blockade of evoked synaptic potential with kynurenic acid (0.5 mM), (2) sensitive to selective inhibition by low doses of the specific sodium channel blockers tetrodotoxin (TTX) or cocaine with no apparent influence on the evoked action potential. These results indicate that endogenous SLA bursting can be induced in hippocampal CA1 pyramidal neurons when certain properties of sodium channels are altered by veratridine.

Veratridine-treated brain slices: a cellular model for epileptiform activity.

This study introduces veratridine-treated brain slices as a new in vitro synaptic-independent model for epileptiform discharge. Studies were performed on the hippocampus in rat brain slices using conventional electrophysiological intracellular recording techniques. Veratridine (0.3 microM) produced a time-dependent blockade of synaptic transmission as indicated by inhibition of the evoked population spike in the region CA1 of the hippocampus. However, in the same slices, intracellularly-evoked single action potentials were converted to epileptiform bursting shortly after exposure to veratridine. Additionally, in the veratridine model, spontaneous epileptiform activity developed after prolonged (more than 45 min) superfusion. The model was utilized to examine the action of two antiepileptic drugs: a sodium channel dependent and a synaptic dependent antiepileptic agents. Therapeutic concentrations of valproic acid (VPA, 10-100 microM) inhibited both evoked and spontaneous bursting induced by veratridine. However, therapeutic concentrations of the synaptic-dependent antiepileptic drug phenobarbital (20-40 microM) failed to inhibit veratridine-induced bursting. These results demonstrate that the veratridine-treated brain slice is a simple and reliable model for studying mechanisms of action and for screening of potential sodium channel-dependent antiepileptic drugs.

A protective effect of endomorphins on the oxidative injury of islet.

The antioxidative capacity of endomorphins (EMs), endogenous µ-opioid receptor agonists, has been demonstrated by IN VIVO assays. In this study, we attempt to evaluate the effects of endomorphin 1 (EM1) and endomorphin 2 (EM2) on pancreatic islet injuries induced by streptozotocin (STZ), alloxan (ALX) and H(2)O(2), respectively. Wistar rats' islets were isolated and purified. The function of the islet cells, the insulin response to glucose stimulation was examined by insulin Radio Immuno Assay and the cell viability was measured by MTT assay. DNA fragments were performed to evaluate the apoptosis, while the cell cycle distribution was analyzed by PI staining flow cytometric analysis. Furthermore, the islet were treated with EM1, EM2 or ALX for 24 h, and the expression of p53 and p21 protein were determined by Western blot. The results showed that STZ, ALX, and H(2)O(2) displayed clear concentration-dependent inhibitory effects on the pancreatic islet cells. While EMs improved the viability of islet induced by STZ, ALX or H(2)O(2), and EMs enhanced insulin accumulation of the cell supernatant after ALX and STZ stimulation. Our data also showed both that EMs inhibited cell apoptosis and cell cycle G1 arrest induced by STZ and ALX through down-regulaing p53 and p21 expression. Taken together, these results demonstrate that EMs can protect islet cells from STZ, ALX and H(2)O(2) induced injuries. Our observations imply that the endomorphins may have protective effects on islet cells oxidative injury.

Monosynaptic connections between identified A and B photoreceptors and interneurons in Hermissenda: evidence for labeled-lines.

The cellular and synaptic organization of the eye of the nudibranch mollusk Hermissenda is well-documented. The five photoreceptors within each eye are mutally inhibitory and can be classified into two types: A and B based on electrophysiological and anatomical criteria. Two of the three type B and two type A photoreceptors can be further identified according to their medial or lateral positions within each eye. In addition to reciprocal synaptic connections between photoreceptors, photoreceptors also project to second-order neurons in the cerebropleural ganglion. The second-order neurons receive convergent synaptic input from two additional sensory pathways; however, it has not been previously established if lateral A, lateral B, or medial B photoreceptors converge onto the same second-order neurons. To determine the specific synaptic organization of these components of the visual system, we have examined monosynaptic connections between identified lateral and medial type A and B photoreceptors and second-order cerebropleural (CP) interneurons. We found that monosynaptic connections between identified lateral A and lateral and medial B photoreceptors and CP interneurons follow a labeled-line principle. Illumination of the eyes or extrinsic depolarizing current applied to identified photoreceptors evoked excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in different CP interneurons. The PSPs in CP interneurons followed one-for-one spikes in the photoreceptors and could be elicited in artificial seawater solutions containing high divalent cations. Identified photoreceptors projected to more than one CP interneuron and expressed both excitatory and inhibitory connections with the different CP interneurons. In examples where a monosynaptic connection between a lateral B photoreceptor and a CP interneuron was identified, lateral A, medial A, or medial B photoreceptors did not project to the same CP interneuron. Moreover, when connections between medial B and CP interneurons were identified, lateral A, medial A, and lateral B connections were not found to project to the same CP interneuron. Similar results were obtained for a lateral A and CP interneuron connection. These results indicate that divergent labeled-lines exist between specific photoreceptors and second-order CP interneurons and potential convergence of synaptic input from primary and secondary elements of the visual system must occur at sites that are postsynaptic to the CP interneurons.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

Recovery of tail-elicited siphon-withdrawal reflex following unilateral axonal injury is associated with ipsi- and contralateral changes in gene expression in Aplysia californica.

Behavioral, cellular and molecular changes were examined following axonal injury in the marine mollusc Aplysia californica. Unilateral nerve injury was performed by crushing the pleural-pedal connective and the peripheral pedal nerves innervating one side of the posterior body wall and the tail. The injury procedure severs the axons of the pleural sensory neurons resulting in the blockade of the tail-elicited siphon-withdrawal reflex. Partial reflex recovery is observed within 3 d and reaches 50% of the pretest value by six weeks postinjury. Retrograde staining of injured nerves combined with electrophysiological recordings from siphon motor neurons show that axons can regenerate through the crushed site and reconnect with the tail by three weeks postinjury. Moreover, the behavioral and electrophysiological measurements suggest that the contralateral sensory neurons may contribute to the early recovery of the siphon-withdrawal reflex. The levels of mRNAs coding for actin and calreticulin are elevated while the mRNAs coding for intermediate filament protein, sensorin A, FMRFamide are reduced in the ipsilateral pleural ganglia as detected by Northern blots. In the contralateral pleural ganglia, the levels of mRNAs coding for actin, sensorin A and FMRFamide are elevated. These molecular changes in both the ipsi- and contralateral sides are consistent with the hypothesis that both sides are participating in the behavioral recovery following unilateral axonal injury.