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Selective extracellular signSelective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models

ABSTRACT: Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome

ORGANISM(S): Mus musculus

PROVIDER: GSE179554 | GEO | 2021/09/24

REPOSITORIES: GEO

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Project description:Background: We hypothesized that spleen microarray gene expression profiles analyzed with contemporary pathway analysis software would provide molecular pathways of interest and target genes that might help explain the affect of bcl-2 on improving survival during sepsis. Methods: Two mouse models of sepsis, cecal ligation and puncture and tracheal instillation of Pseudomonas aeruginosa, were tested in both wild-type mice and mice that overexpress bcl-2. Whole spleens were obtained 6 hours after septic injury. DNA microarray transcriptional profiles were obtained using the Affymetrix 430A GeneChip, containing 22,690 elements. Ingenuity Pathway Analysis software was used to construct hypothetical transcriptional networks that changed in response to sepsis and expression of the bcl-2 transgene. Results: A conservative approach was used wherein only changes induced by both abdominal and pulmonary sepsis were studied. At 6 hours, sepsis induced alterations in the abundance of hundreds of spleen genes, including a number of proinflammatory mediators (e.g., IL-6). These sepsis-induced alterations were blocked by expression of the bcl-2 transgene. Network analysis implicated a number of bcl-2-related apoptosis genes, including bcl2L11 (bim), bcl-2L2 (bcl-w), bmf, and mcl-1. Sepsis in bcl-2 transgenic animals resulted in alteration of RNA abundance for only a single gene, ceacam1. Conclusion: These findings are consistent with sepsis-induced alterations in the balance of pro- and anti-apoptotic transcriptional networks. In addition, our data suggest that the ability of bcl-2 overexpression to improve survival in sepsis in this model is related in part to prevention of sepsis-induced alterations in spleen transcriptional responses. Experiment Overall Design: To determine the splenic response in these lethal models of CLP and Pseudomonas pneumonia, microarray analysis was performed on each spleen harvested from wild-type animals 6 hours after CLP or tracheal instillation of bacteria. The responses of the CLP or Pseudomonas spleens were compared concurrently to those of the wild-type controls, sham laparotomy and tracheal instillation of saline, respectively. This study was repeated in animals overexpressing bcl-2. Thus, the splenocyte effect of sepsis secondary to CLP (n=6) or Pseudomonas pneumonia (n=5) could be determined compared to their controls (n=6 and 5, respectively), and the effect of bcl-2 overexpression in turn also could be determined in both CLP (n=5) and pneumonia models (n=5) compared to controls (n=5 and 5, respectively).

Project description:Dysregulated cardiac function after sepsis is common in intensive care unit (ICU) and known to predict poor long-term outcome and increase mortality. Effective therapeutic strategies are largely lacking. Moreover, the pathological feature and the molecular mechanism underlying cardiac dysfunction induced by sepsis remain unclear. Here, by performing echocardiograms on rodents after induction of polymicrobial sepsis with cecum ligation and puncture (CLP), we assessed the temporal dynamics of left ventricular ejection fraction (LVEF) and a serial of hemodynamics parameters on animals at different time point after CLP. Intriguingly, the mean LVEF is comparable in mice induced by CLP and sham, whereas survivors post CLP had stable LVEF and non-survivors had markedly fluctuated LVEF at early phase of CLP induction, suggesting LVEF away from normal range is highly associated with mortality. Consistent with clinical observations of depressed, preserved or hyperdynamic LVEF in septic patients from data compiled using our ICU cohort and from other studies, CLP-induced mice fall into three groups based on LVEF measured at 24 hours after surgery: high LVEF (HEF, LVEF>=90%), low LVEF (LEF, LVEF<65%), and normal LVEF (NEF, 65%=<LVEF<90%). We performed genome-wide transcriptomic and proteomic profiling on left ventricle samples collected from three CLP groups and sham mice. By implementing pathway analysis, gene set enrichment and coexpression network analysis, we identified jointly and distinctively changed genes, proteins and biologically-essential processes and pathways in three CLP groups with different LVEF. Notably, transmission electron microscopy examination shows remarkable mitochondrial and sarcomere defects in three CLP groups with different phenotypes associated with LVEF variances. Together, this study systematically characterizes the molecular, morphological, and functional alterations in CLP-induced cardiac injury, serving as a framework for future research into pathology and molecular mechanism of sepsis-induced cardiomyopathy.

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Selective extracellular signSelective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models

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