9-(tetrahydro-2-furyl)-adenine and Sepsis

Survivors of sepsis must often endure significant cognitive and behavioral impairments after discharge, but research on the relevant mechanisms and interventions remains lacking. TGR5, a member of the class A GPCR family, plays an important role in many physiological processes, and recent studies have shown that agonists of TGR5 show neuroprotective effects in a variety of neurological disorders. To date, no studies have assessed the effects of TGR5 on neuroinflammatory, cognitive, or behavioral changes in sepsis models.. A total of 267 eight-week-old male Sprague-Dawley rats were used in this study. Sepsis was induced via cecal ligation and puncture (CLP). All animals received volume resuscitation. The rats were given TGR5 CRISPR oligonucleotide intracerebroventricularly 48 h before CLP surgery. INT-777 was administered intranasally 1 h after CLP, and the cAMP inhibitor, SQ22536, was administered intracerebroventricularly 1 h after CLP. Survival rate, bodyweight change, and clinical scores were assessed, and neurobehavioral tests, western blot, and immunofluorescence staining were performed. The cognitive function of rats was measured using the Morris water maze during 15-20 days after CLP.. The expression of TGR5 in the rat hippocampus was upregulated, and peaked at 3 days after CLP. The survival rate of rats after CLP was less than 50%, and the growth rate, in terms of weight, was significantly decreased. While INT-777 treatment did not improve these changes, the treatment did reduce the clinical scores of rats at 24 h after CLP. On day 15 and later, the surviving mice completed a series of behavioral tests. CLP rats showed spatial and memory deficits and anxiety-like behaviors, but INT-777 treatment significantly improved these effects. Mechanistically, immunofluorescence analysis showed that INT-777 treatment reduced the number of microglia in the hippocampus, neutrophilic infiltration, and the expression of inflammatory factors after CLP in rats. Moreover, INT-777 treatment significantly increased the expression of TGR5, cAMP, p-PKA, and p-CREB, but downregulated the expression of IL-1β, IL-6, and TNF-α. CRISPR-mediated TGR5 knockdown and SQ22536 treatment abolished the neuroprotective effects of TGR5 activation after CLP.. This study demonstrates that INT-777 treatment reduced neuroinflammation and microglial cell activation, but improved cognitive impairment in the experimental sepsis rats. TGR5 has translational potential as a therapeutic target to improve neurological outcomes in sepsis survivors.

Topics: Adenine; Animals; Anxiety; Cholic Acids; Cognitive Dysfunction; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cytokines; Encephalitis; Male; Maze Learning; Nootropic Agents; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Sepsis; Signal Transduction; Survival Analysis

Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs.. Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 mumol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 mumol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 mumol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 mumol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 mumol/L, 100 mumol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes.. SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction.. The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hydrogen Peroxide; Isoquinolines; Lung; Male; Muscle Contraction; Oxadiazoles; Pericytes; Quinoxalines; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sepsis; Sulfonamides

The inducible nitric oxide synthase (iNOS) is strongly expressed following inflammatory stimuli. Adenosine 3',5'-cyclic monophosphate (cAMP) increases iNOS expression and activity in a number of cell types but decreases cytokine-stimulated iNOS expression in hepatocytes. The mechanisms for this effect are unknown.. Rat hepatocytes were stimulated with cytokines to induce iNOS and cultured with cAMP agonists dibutyryl-cAMP (dbcAMP), 8-bromo-cAMP, and forskolin (FSK). Nitric oxide synthesis was assessed by supernatant nitrite levels and iNOS expression was measured by Northern and Western blot analyses. Nuclear factor kappaB binding was assessed by electromobility shift assay.. Cyclic AMP dose dependently decreased NO synthesis in response to a combination of proinflammatory cytokines or interleukin-1beta (IL-1beta) alone. The adenylate cyclase inhibitor SQ 22,536 increased cytokine- or IL-1beta-stimulated NO synthesis. dbcAMP decreased iNOS mRNA expression and iNOS protein expression. Both dbcAMP and glucagon decreased iNOS promoter activity in rat hepatocytes transfected with the murine iNOS promoter and decreased DNA binding of the transcription factor NF-kappaB.. These data suggest that cAMP is important in hepatocyte iNOS expression and agents that alter cAMP levels may profoundly alter the response of hepatocytes to inflammatory stimuli through effects onthe iNOS promoter region and NF-kappaB.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Bucladesine; Cells, Cultured; Colforsin; Cyclic AMP; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glucagon; Hepatocytes; Interleukin-1; Male; Muscle, Smooth, Vascular; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Pulmonary Artery; Rats; Rats, Sprague-Dawley; RNA, Messenger; Second Messenger Systems; Sepsis; Transfection