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Abstract Background Previously, we had identified a specific whole blood–derived microRNAs (miRNAs) signature in mice following in vivo injection of lipopolysaccharide (LPS) originated from Gram-negative bacteria. This study was designed to profile the circulating miRNAs expression in mice exposed to lipoteichoic acid (LTA) which is a major component of the wall of Gram-positive bacteria. Results C57BL/6 mice received intraperitoneal injections of 100 μg of LTA originated from Bacillus subtilis, Streptococcus faecalis, and Staphylococcus aureus were killed 6 h and the whole blood samples were obtained for miRNA expression analysis using a miRNA array (Phalanx miRNA OneArray® 1.0). Up-regulated expression of miRNA targets in the whole blood, serum and white blood cells (WBCs) of C57BL/6 and Tlr2−/− mice upon LTA treatment in 10, 100, or 1000 ug concentrations was quantified at indicated time (2, 6, 24, and 72 h) using real-time RT-PCR and compared with that in the serum of C57BL/6 mice injected with 100 ug of LPS. A significant increase of 4 miRNAs (miR-451, miR-668, miR-1902, and miR-1904) was observed in the whole blood and the serum in a dose- and time-dependent fashion following LTA injection. Induction of miRNA occurred in the serum after 2 h and persisted for at least 6 h. No increased expression of these 4 miRNAs was found in the WBCs. Higher but not significant expression level of these 4 miRNAs were observed following LTA treatment in the serum of Tlr2−/−against that of C57BL6 mice. In contrast, LPS exposure induced moderate expression of miR-451 but not of the other 3 miRNA targets. Conclusions We identified a specific circulating miRNA signature in mice exposed to LTA. That expression profile is different from those of mice exposed to LPS. Those circulating miRNAs induced by LTA or LPS treatment may serve as promising biomarkers for the differentiation between exposures to Gram-positive or Gram-negative bacteria.

Abstract Background Lipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS. Methods C57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10–1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4−/− mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus. Results Following LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4−/− mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets. Conclusions We identified a specific whole blood–derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.

Abstract Background To compare the microRNA (miRNA) expression profiles in neurons and innervated muscles after sciatic nerve entrapment using a non-constrictive silastic tube, subsequent surgical decompression, and denervation injury. Methods The experimental L4-L6 spinal segments, dorsal root ganglia (DRGs), and soleus muscles from each experimental group (sham control, denervation, entrapment, and decompression) were analyzed using an Agilent rat miRNA array to detect dysregulated miRNAs. In addition, muscle-specific miRNAs (miR-1, -133a, and -206) and selectively upregulated miRNAs were subsequently quantified using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Results In the soleus muscles, 37 of the 47 miRNAs (13.4% of the 350 unique miRNAs tested) that were significantly downregulated after 6 months of entrapment neuropathy were also among the 40 miRNAs (11.4% of the 350 unique miRNAs tested) that were downregulated after 3 months of decompression. No miRNA was upregulated in both groups. In contrast, only 3 miRNAs were upregulated and 3 miRNAs were downregulated in the denervated muscle after 6 months. In the DRGs, 6 miRNAs in the entrapment group (miR-9, miR-320, miR-324-3p, miR-672, miR-466b, and miR-144) and 3 miRNAs in the decompression group (miR-9, miR-320, and miR-324-3p) were significantly downregulated. No miRNA was upregulated in both groups. We detected 1 downregulated miRNA (miR-144) and 1 upregulated miRNA (miR-21) after sciatic nerve denervation. We were able to separate the muscle or DRG samples into denervation or entrapment neuropathy by performing unsupervised hierarchal clustering analysis. Regarding the muscle-specific miRNAs, real-time RT-PCR analysis revealed an ~50% decrease in miR-1 and miR-133a expression levels at 3 and 6 months after entrapment, whereas miR-1 and miR-133a levels were unchanged and were decreased after decompression at 1 and 3 months. In contrast, there were no statistical differences in the expression of miR-206 during nerve entrapment and after decompression. The expression of muscle-specific miRNAs in entrapment neuropathy is different from our previous observations in sciatic nerve denervation injury. Conclusions This study revealed the different involvement of miRNAs in neurons and innervated muscles after entrapment neuropathy and denervation injury, and implied that epigenetic regulation is different in these two conditions.

Abstract Background To profile the expression of microRNAs (miRNAs) and their potential target genes in the gracilis muscles following ischemic injury in rats by monitoring miRNA and mRNA expression on a genome-wide basis. Methods Following 4 h of ischemia and subsequent reperfusion for 4 h of the gracilis muscles, the specimens were analyzed with an Agilent rat miRNA array to detect the expressed miRNAs in the experimental muscles compared to those from the sham-operated controls. Their expressions were subsequently quantified by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to determine their expression pattern after different durations of ischemia and reperfusion. In addition, the expression of the mRNA in the muscle specimens after 4 h of ischemia and reperfusion for 1, 3, 7, and 14 d were detected with the Agilent Whole Rat Genome 4 × 44 k oligo microarray. A combined approach using a computational prediction algorithm that included miRanda, PicTar, TargetScanS, MirTarget2, RNAhybrid, and the whole genome microarray experiment was performed by monitoring the mRNA:miRNA association to identify potential target genes. Results Three miRNAs (miR-21, miR-200c, and miR-205) of 350 tested rat miRNAs were found to have an increased expression in the miRNA array. Real-time RT-PCR demonstrated that, with 2-fold increase after 4 h of ischemia, a maximum 24-fold increase at 7 d, and a 7.5-fold increase at 14 d after reperfusion, only the miR-21, but not the miR-200c or miR-205 was upregulated throughout the experimental time. In monitoring the target genes of miR-21 in the expression array at 1, 3, 7, 14 d after reperfusion, with persistent expression throughout the experiment, we detected the same 4 persistently downregulated target genes (Nqo1, Pdpn, CXCL3, and Rad23b) with the prediction algorithms miRanda and RNAhybrid, but no target gene was revealed with PicTar, TargetScanS, and MirTarget2. Conclusions This study revealed 3 upregulated miRNAs in the gracilis muscle following ischemic injury and identified 4 potential target genes of miR-21 by examining miRNAs and mRNAs expression patterns in a time-course fashion using a combined approach with prediction algorithms and a whole genome expression array experiment.

Background. The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury. Methods. Thigh skeletal muscles of C57BL/6, Tlr4−/−, and NF-κB−/− mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression. Results. Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway. Conclusions. This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

This study was designed to determine the in vitro angiogenic ability of far-infrared (FIR) radiation in the skin-derived cultured human microvascular endothelial cells and to elucidate the role of mitogen-activated protein kinases (MAPKs) in this process. The results revealed that FIR radiation from a WS TY301 FIR emitter activated p38 and extracellular signal-regulated kinase (ERK), but not Akt or c-Jun N-terminal protein kinases (JNK), and significantly promoted angiogenesis by increasing tube formation in Matrigel and the migration of cells across an eight micron polyester filter. The addition of 50 μ PD98059, a MEK inhibitor, significantly inhibited the activation of ERK and the enhanced angiogenesis; in contrast, the inhibition of p38 phosphorylation did not inhibit the enhanced angiogenesis. After FIR radiation, there was no increase in vascular endothelial growth factor (VEGF) isoforms (VEGF-A, -B, -C and -D) mRNA and VEGF protein, no increase phosphorylation of endothelial nitric oxide synthase (eNOS) detected using Western blotting, and no increase in NO production detected using flow cytometry in cells pre-incubated with the cell-permeable NO-binding dye diluted 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate (DAF-FM DA). This study revealed that FIR radiation possesses in vitro angiogenic activity via the activation of the MEK/ERK but not the VEGF/Akt/eNOS-dependent signaling pathways. [ABSTRACT FROM AUTHOR]

Heme oxygenase-1 (HO-1) expression, in response to various stimuli, has been generally reported to protect against cellular stress. However, we previously demonstrated simvastatin-induced HO-1-exaggerated nuclear factor kappa beta (NF-κβ) activation and superoxide production on exposure to lipopolysaccharide (LPS). The addition of the iron chelator, desferrioxamine, to reduce the accumulation of ferric iron from heme by HO-1 resulted in a blockade of aggravated superoxide production. In this study, we demonstrated that, unlike simvastatin, neither atorvastatin- nor rosuvastatin-induced HO-1 upregulation resulted in increased NF-κβ activation and superoxide production of Neuro-2A cells against LPS. Moreover, increased superoxide formation by either atorvastatin or rosuvastatin, in the presence or absence of LPS, could not be reduced by the addition of desferrioxamine, unlike simvastatin. In contrast to our previous experiment, in which simvastatin activated ERK and p38, but not JNK or Akt, both atorvastatin and rosuvastatin phosphorylated ERK, but not p38. Inhibition of p38 activation by 5 μM of SB203580 effectively reduced exaggerated HO-1 upregulation in cells pretreated with simvastatin, but not atorvastatin or rosuvastatin, plus exposure to LPS. The addition of an ERK inhibitor (50 μM of PD98059) did not decrease HO-1 upregulation in cells following any statin pretreatment. Further, the effect of simvastatin on primary cultures of cortical neurons was also different from that on Neuro-2A cells, as HO-1 upregulation did not result in exaggerated NF-κβ activation and superoxide production. This study demonstrated the involvement of p38 in the differential induction of HO-1 by different statins and showed the different physiological relevance of experiments on different cell types. [ABSTRACT FROM AUTHOR]

With potentially neuroprotective properties, heme oxygenase-1 (HO-1) has been suggested to be the main mediator of cholesterol-independent anti-inflammatory and antioxidant actions of statins. However, we had demonstrated that simvastatin-induced HO-1 increased apoptosis of Neuro 2A cells in glucose deprivation, and iron production from HO-1 activity may be responsible for the toxicity. This study was designed to explore the effect of simvastatin-induced HO-1 on cultured Neuro 2A and C6 cells exposed to lipopolysaccharide (LPS). We found that the HO-1 upregulation was significantly associated with increased nuclear factor kappa B (NF-kappaB) activation, manifested as IkappaBalpha phosphorylation and p65 nuclear translocation, as well as increased production of superoxides. Inhibition of the induced HO-1 by zinc protoporphyrin reduced the increased NF-kappaB activation and superoxides production. RNA interference with HO-1 siRNA reduced the expression of HO-1 transcripts and protein as well as oxygen radical production. Addition of the iron chelator desferrioxamine to reduce the accumulation of ferric iron from heme by HO-1 resulted in blockade of the aggravated oxygen radical production. There was no significant effect on production of oxygen radicals under these conditions in the presence of a CO donor (RuCO) or a CO scavenger (hemoglobin). In addition, the viable cells were significantly decreased in 48 h in those cells receiving simvastatin pretreatment plus LPS compared to those in control or exposed to simvastatin or LPS alone. This study revealed that simvastatin-induced HO-1 led to increased NF-kappaB activation and superoxides production in the neuronal cells when exposed to LPS, and iron production may play a role in such a response.