BackgroundMicroRNAs (miRNAs) are short non-coding RNAs that inhibit translation of target genes by binding to their mRNAs. The expression of numerous brain-specific miRNAs with a high degree of temporal and spatial specificity suggests that miRNAs play an important role in gene regulation in health and disease. Here we investigate the time course gene expression profile of miR-1, -16, and -206 in mouse dorsal root ganglion (DRG), and spinal cord dorsal horn under inflammatory and neuropathic pain conditions as well as following acute noxious stimulation.ResultsQuantitative real-time polymerase chain reaction analyses showed that the mature form of miR-1, -16 and -206, is expressed in DRG and the dorsal horn of the spinal cord. Moreover, CFA-induced inflammation significantly reduced miRs-1 and -16 expression in DRG whereas miR-206 was downregulated in a time dependent manner. Conversely, in the spinal dorsal horn all three miRNAs monitored were upregulated. After sciatic nerve partial ligation, miR-1 and -206 were downregulated in DRG with no change in the spinal dorsal horn. On the other hand, axotomy increases the relative expression of miR-1, -16, and 206 in a time-dependent fashion while in the dorsal horn there was a significant downregulation of miR-1. Acute noxious stimulation with capsaicin also increased the expression of miR-1 and -16 in DRG cells but, on the other hand, in the spinal dorsal horn only a high dose of capsaicin was able to downregulate miR-206 expression.ConclusionsOur results indicate that miRNAs may participate in the regulatory mechanisms of genes associated with the pathophysiology of chronic pain as well as the nociceptive processing following acute noxious stimulation. We found substantial evidence that miRNAs are differentially regulated in DRG and the dorsal horn of the spinal cord under different pain states. Therefore, miRNA expression in the nociceptive system shows not only temporal and spatial specificity but is also stimulus-dependent.

Treatment of neuropathic pain is a clinical challenge likely because of the time-dependent changes in many neurotransmitter systems, growth factors, ionic channels, membrane receptors, transcription factors, and recruitment of different cell types. Conversely, an increasing number of reports have shown the ability of extended and regular physical exercise in alleviating neuropathic pain throughout a wide range of mechanisms. In this study, we investigate the effect of swim exercise on molecules associated with initiation and maintenance of nerve injury-induced neuropathic pain. BALB/c mice were submitted to partial ligation of the sciatic nerve followed by a 5-week aerobic exercise program. Physical training reversed mechanical hypersensitivity, which lasted for an additional 4 weeks after exercise interruption. Swim exercise normalized nerve injury-induced nerve growth factor, and brain-derived neurotrophic factor (BDNF) enhanced expression in the dorsal root ganglion, but had no effect on the glial-derived neurotrophic factor. However, only BDNF remained at low levels after exercise interruption. In addition, exercise training significantly reduced the phosphorylation status of PLCγ-1, but not CREB, in the spinal cord dorsal horn in response to nerve injury. Finally, prolonged swim exercise reversed astrocyte and microglia hyperactivity in the dorsal horn after nerve lesion, which remained normalized after training cessation. Together, these results demonstrate that exercise therapy induces long-lasting analgesia through various mechanisms associated with the onset and advanced stages of neuropathy. Moreover, the data support further studies to clarify whether appropriate exercise intensity, volume, and duration can also cause long-lasting pain relief in patients with neuropathic pain.

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids.

Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non-coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non-vesicular circulating miRNAs exhibit a potential role as mediators of the cross-talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. J. Cell. Physiol. 232: 958-966, 2017. © 2016 Wiley Periodicals, Inc.

retinal neuron apoptosis is a key component of diabetic retinopathy (dr), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. apoptosis of retinal neurons serves a critical role in the pathogenesis of dr observed in patients with diabetes and streptozotocin (STZ)-induced diabetic rats. retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of dr. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of dr remains unclear. rna-dependent protein kinase (PKr) is a stress-sensitive pro-apoptotic kinase. our previous study indicated that PKR-associated protein X, a stress-sensitive activator of PKr, is upregulated in the early stage of STZ-induced diabetes. in order to assess the role of PKr in dr prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKr in the retina in the early stage of STZ-induced diabetes in rats. PKr activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p-eiF2-α) and the presence of apoptotic cells in the retina was investigated by Tunel assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. in addition, p-eiF2-α expression and cellular localization followed the trend of PKr, suggesting that this pro-apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKr may be a mechanism to sequester active PKr, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. apoptotic cells were not detected in the retina in the early stage of dr. a model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKr is suppressed in the early stage of dr. The possible role of mitochondrial rna (mtrna) and Alu rna in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtrna and transcription of Alu rna. Moreover, it mtrna activates PKr, whereas Alu rna inhibits the activation of this protein kinase.

Insulin resistance is associated to type II diabetes but its mechanism is not fully understood. Micro‐RNAs have been described as non‐coding molecules associated with post‐transcriptional regulation of several physiological processes including diabetes. We have observed that miR‐696 is regulated in an opposite manner compared to PGC‐1α leading to a proposition that the miR‐696 may be involved to insulin resistance. Mice mioblast cells (C2C12) were treated with palmitic acid (500μM) for 24 h. The insulin resistance was confirmed by evaluation of Akt phosphorylation after insulin incubation by western blot. Expression of mature miR‐696 was evaluated by real‐time RT‐PCR using TaqMan® MicroRNA Assay. The expression of microRNAs was normalized with the SnoR‐202. Gene expression of PGC1α was evaluated by real time RT‐PCR using Sybr Green. Analysis of oxygen consumption was performed by oxygraph (Hansatech) using CCCP and oligomycin as controls. We observed that PGC1α transcription was markedly reduced in insulin resistant cells, an effect that was reflected by a low mitochondrial respiration, reduced antioxidant capacity and elevated miR‐696 level. In contrast, the PGC1α transcription was markedly elevated after AICAR treatment as reflected by elevated mitochondrial respiration and antioxidant capacity and low miR‐696 level. Interestingly, the inhibition of miR‐696 transcription in insulin resistant cells re‐established the insulin response, PGC1α transcription and mitochondrial function indicating an association of miR‐696 and mitochondrial biogenesis. Therefore, we are suggesting that miR‐696 contributes to insulin resistance reducing mitochondrial biogenesis and mitochondrial function in skeletal muscle cells.

We examined whether the reduction of mitochondrial capacity and insulin response imposed by the high availability of fatty acids are associated with an increase of intracellular hydrogen peroxide (H2O2). We investigated whether the oxidative stress induced by excess fatty acids can be reversed with improved antioxidant capacity through overexpression of catalase. The catalase gene was successfully transfected in skeletal muscle cells through pcDNA3 plamid as indicated by catalase mRNA and content (p<0.05). The H2O2 production was increased after palmitic acid treatment. This effect markedly reduced the citrate sintase activity as well as the mRNA levels of PGC1α (p<0.05). This effect was accompanied by a significantly reduction of p‐AKt which further was demonstrated to down regulate p‐CREB and PPAR‐β. In addition, oxygen consumption and uptake glucose were markedly reduced in the presence of palmitic acid (p<0.05). However, the catalase transfection was observed to prevent reduction of p‐AKt level which further was demonstrated to up regulate p‐CREB and PPAR‐β (p<0.05). Our results showed that fatty acid reduced the p‐AKt level. This effect was associated with a low p‐CREB level and consequently PGC1‐α transcription. The reduced mitochondrial oxygen consumption favored ROS production and insulin resistance. The catalase‐ induced effect on p‐AKt increased the oxygen consumption and glucose and fatty acid metabolism suggesting that mitochondrial biogenesis might be regulated by phosphorylated level of AKt.