Unknown,Transcriptomics,Genomics,Proteomics

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IL-6-mediated activation of STAT3M-NM-1 prevents trauma/hemorrhagic shock-induced liver inflammation

ABSTRACT: We previously demonstrated in rodents that T/HS results in liver injury that can be prevented by IL-6 administration at the start of resuscitation, however the mechanism(s) for the IL-6 protective effect is not fully known. We used microarrays to detail the global gene expression in response to T/HS and the effect of IL-6 on this model with and without pharmacologic blockade of Stat3-mediated IL-6 and identified distinct members of the inflammasome de-regulated during T/HS that normalized with IL-6 administration during resuscitation. Adult male rats were subjected to either sham procedure, a trauma plus hemorrhagic shock (T/HS) procedure, T/HS with IL-6 versus PBS during resuscitation, or pretreatment with pharmacologic blockade of Stat3-mediated IL6 via GQ-ODN and after procedural completion, animals were sacrificed and livers were taken and homegenized for RNA extraction and microarray analysis using Affymetrix Rat 230A GeneChipM-BM-. array

ORGANISM(S): Rattus norvegicus

SUBMITTER: Stephen Thacker

PROVIDER: E-GEOD-27978 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The etiology of trauma-hemorrhage shock-induced acute lung injury has been difficult to elucidate due, at least in part, to the inability of in vivo studies to separate the non-injurious pulmonary effects of trauma-hemorrhage from the tissue injurious ones. To circumvent this in vivo limitation, we utilized a model of trauma-hemorrhagic shock (T/HS) in which T/HS-lung injury was abrogated by dividing the mesenteric lymph duct. In this way, it was possible to separate the pulmonary injurious response from the non-injurious systemic response to T/HS by comparing the pulmonary molecular response of rats subjected to T/HS which did and did not develop lung injury as well as to non-shocked rats. Utilizing high-density oligonucleotide arrays and treatment group comparisons of whole lung tissue collected at 3 hours after the end of the shock or sham-shock period, 139 of the 8,799 assessed genes were differentially expressed. Experiment Overall Design: Four groups of rats (n=3) were studied in order to identify changes in pulmonary gene expression associated with T/HS, both in the presence and absence of lung injury. These included trauma-sham shock (T/SS) rats which had a laparotomy (trauma) but were not subjected to hemorrhagic shock. These rats had no lung injury and served as controls for rats which were subjected to T/HS (laparotomy plus 90 min of shock) and had lung injury. Differences in gene expression between these two groups would represent both the effects of hemorrhagic shock as well as lung injury. To distinguish the gene response of hemorrhagic shock from the gene response associated with lung injury, gene expression was also compared between T/HS rats (hemorrhage and lung injury) and rats subjected to T/HS plus lymph duct ligation (T/HS-LDL), since the T/HS-LDL rats experienced hemorrhagic shock but had no measurable lung injury. Lastly, to identify hemorrhagic shock- modified genes, the pulmonary gene response of T/HS-LDL (hemorrhage without lung injury) were compared to rats subjected to T/SS plus LDL (no hemorrhage or lung injury). Three hours after the end of the 90 min shock or sham-shock period (i.e. 4.5 hrs after the induction of T/HS), the rats were sacrificed and specimens harvested for genechip analysis and histology.

Project description:Objective of the study is to find out the differentially regulated genes of Mycobacterium BCG strain in extended stationary phase comparison to log phase growth and resuscitation phase. Mycobacterium BCG culture was grown in Sauton medium at 37o C without shaking. Cells at A600 0.6-0.8 were harvested as log phase culture. The cells harvested after 5 months incubation at 37oC without shaking became non culturable and were harvested as extended stationary phase cells. The extended stationary phase cells were treated with resuscitation promoting factor (Rpf) from Micrococcus luteus and harvested after 8 days. Cells of this stage were treated as resuscitation phase cells. Gene expression profiling was carried out using Agilent microarray platform. Keywords: Extended stationary phase, Log phase growth and Resuscitation phase. â¢ Organism: Mycobacterium tuberculosis BCG â¢ Slides: Agilentâs Mycobacterium tuberculosis custom array 4x44k (G2514F) AMADID No: 016013 â¢ Labeling kit: Ambion Message Amp II Bacteria a RNA Amplification Kit Cat# 1790 â¢ Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA â¢ Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 â¢ Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 â¢ RNA quality was checked using Bioanalyzer. Hybridization protocol 825ng each of labeled control cRNA was mixed with 825ng of treated labelled cRNA, mixed with appropriate amount of blocking buffer and hybridization buffer (Agilent Technologies). The hybridization mix was applied on to the backings and hybridized to custom designed 60mer Mycobacterium tuberculosis microarray using sure hyb chambers at 65degC for 17 hours. Slides were washed with gene expression wash buffer 1 and 2 (Agilent Technologies) followed by Acetonitrile. Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Slides were scanned at 5 micron resolution using Agilent scanner.Automated feature extraction was done using Agilentâs Feature Extraction Software. Data processing Analysis of feature extracted data was done using Agilentâs GeneSpring GX V 7.3.1 software. Pre-processing of the replicate experiment data was done to flag the outliers. Normalization of the data was done using per spot per chip intensity dependant lowess normalization and dye swap experiment was data transformed. Genes with log2 ratio of 1.29 and above in replicate experiments were considered as up regulated and 0.81 and below was considered down regulated. p-value was calculated by GeneSpring GX for each gene on the basis of replicate probes to indicate statistical significance. Biological significance of differentials were analyzed using Genotypics Biointerpreter - Web based tool for biological interpretation of given genelist.

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IL-6-mediated activation of STAT3M-NM-1 prevents trauma/hemorrhagic shock-induced liver inflammation

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