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TLR4/NF-?B-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

ABSTRACT: The aim of this study was to profile TLR4/NF-?B-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury.Thigh skeletal muscles of C57BL/6, Tlr4(-/-), and NF-?B(-/-) 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.Three TLR4/NF-?B-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.This study profiled TLR4/NF-?B-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.

SUBMITTER: Yang JC

PROVIDER: S-EPMC4321099 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

Yang Johnson Chia-Shen JC Wu Shao-Chun SC Rau Cheng-Shyuan CS Chen Yi-Chun YC Lu Tsu-Hsiang TH Wu Yi-Chan YC Tzeng Siou-Ling SL Wu Chia-Jung CJ Hsieh Ching-Hua CH

BioMed research international 20150126

<h4>Background</h4>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.<h4>Methods</h4>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- ( ...[more]

PMID: 25692136

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Project description:TLR4/NF-ÎºB signaling plays a central mediator in response to danger signals released in the muscle ischemia-reperfusion injury (IRI). This study was designed to profile TLR4/NF-ÎºB-responsive microRNAs (miRNAs) in the skeletal muscles following IRI. Following 2 h of ischemia and subsequent reperfusion for indicated times (0 h, 4 h, 1 d, and 7 d) of the isolated thigh skeletal muscles based on femoral artery perfusion of C57BL/6, Tlr4â/â, and NF-ÎºBâ/âmice, the muscle specimens were analyzed with an miRNA array to detect the TLR4/NF-ÎºB-responsive miRNAs. Male C57BL/6 mice (10â12 weeks of age, 22â35 g) were purchased from BioLasco (Taiwan). Tlr4â/â (C57BL/10ScNJ) and NF-ÎºBâ/â (B6.Cg-Nfkb1tm1Bal/J) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All housing conditions were established and surgical procedures, analgesia, and assessments were performed in an AAALAC-accredited, SPF facility following national and institutional guidelines. Animal protocols were approved by the IACUC of Chang Gung Memorial Hospital. Mice were anesthetized with an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine, given as an intraperitoneal injection (0.01 ml/g body weight). After the induction of anesthesia, the mice were restrained in a supine position on a heating pad to maintain body temperature at 37Â°C. The quadriceps muscle perfused based on the femoral artery of the mouse was carefully isolated away from the femoral bone and the underlying adductor muscle group. In the ischemic group, ischemia was induced by placing a microvascular clamp carefully across the proximal site of vascular pedicle for 2 h and then the microvascular clamp was removed. Good vascular inflow and outflow through the pedicle was verified under direct magnified vision. In the sham-operated control group, the muscle was isolated without microvascular clamp being applied. The incision wound was closed with interrupted sutures (4-0 nylon) and the animals were allowed to awaken in the remaining periods of reperfusion. The harvested muscles were frozen in isopentane chilled in liquid nitrogen and stored at -80Â°C. For the miRNA array experiments, the isolated skeletal muscles of C57BL/6 mice after 2 h of ischemia and 0 h, 4 h, 1 d, and 7 d of reperfusion as well as of Tlr4â/âand NF-ÎºBâ/â mice after 2 h of ischemia and 1 d of reperfusion were used in 3 replicate experiments.

Project description:BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an important mediator of neuroinflammatory responses that regulates inflammatory injury following cerebral ischemia and may be a potential target. Salidroside (Sal) has good anti-inflammatory effects; however, it remains unclear whether Sal can regulate NLRP3 inflammasome activation through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway after cerebral ischemia to alleviate inflammatory injury.MethodsWe established an oxygen-glucose deprivation and reoxygenation (OGD/R) model of BV2 cells and a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model. Cell Counting Kit-8 (CCK-8), flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to detect the viability and apoptosis of BV2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factors. 2,3,5-triphenyltetrazolium chloride (TTC) staining and modified Neurological Severity Score (mNSS) were used to detect cerebral infarction volume and neurological deficit in rats. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the protein expression levels.ResultsOur results showed that Sal increased viability, inhibited lactate dehydrogenase (LDH) release, and reduced apoptosis in OGD/R-induced BV2 cells. Sal reduced the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8. Following induction by OGD/R, BV2 cells exhibited NLRP3 inflammasome activation and increased protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, IL-1β, and IL-18. Protein levels of key TLR4 signaling pathway elements, such as TLR4, myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa B p65 (p-NF-κB p65)/NF-κB p65 were upregulated. Interestingly, it was revealed that Sal could reverse these changes. In addition, TAK242, a specific inhibitor of TLR4, had the same effect as Sal treatment on BV2 cells following induction by OGD/R. In the MCAO/R rat model, Sal was also observed to inhibit NLRP3 inflammasome activation in microglia, reduce cerebral infarction volume, and inhibit apoptosis.ConclusionsIn summary, we found that Sal inhibited NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, thus playing a protective role. Therefore, Sal may be a promising drug for the clinical treatment of ischemic stroke.

Dataset Information

TLR4/NF-?B-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

Publications

TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

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