n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Reperfusion-Injury

Diabetes mellitus can exacerbate renal ischemia‑reperfusion (I/R) injury (RI/RI) in diabetic rats. Previous studies have shown that Notch signaling is involved in renal disorders. The aim of the present study was to evaluate the protective effect of the Notch inhibitor γ‑secretase N‑[N‑(3,5‑difluorophenacetyl)‑L‑alanyl]‑S‑phenylglycine t‑butyl ester (DAPT) on RI/RI in a streptozocin (STZ)‑induced diabetic rat model. STZ‑induced diabetic rats were randomly grouped for different treatments. Cisplatin was used to trigger the Notch signaling pathway and the animals were preconditioned with DAPT to block the signaling pathway. Renal function, oxidative stress and inflammatory factors were examined. DAPT‑treated diabetic rats demonstrated mitigated renal injury and function, antioxidative activity was significantly improved and HIF‑1a was upregulated. Notch inhibitor DAPT is a potential therapeutic target to improve the outcome of RI/RI in STZ‑induced diabetic rats in part via the regulation of anti‑oxidation and HIF‑1a.

Topics: Animals; Blood Urea Nitrogen; Cisplatin; Creatinine; Diabetes Mellitus, Experimental; Dipeptides; Disease Models, Animal; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Kidney; Male; Oxidation-Reduction; Rats, Sprague-Dawley; Receptors, Notch; Reperfusion Injury; RNA, Messenger; Signal Transduction; Streptozocin

Intestinal ischemia/reperfusion (I/R) induces disruption of the intestinal barrier function. Aryl hydrocarbon receptor (AhR) has a vital role in maintaining the intestinal barrier function. However, the precise mechanism by which AhR maintains intestinal barrier function remains unclear. Notch1 signaling is downstream of AhR, and has also been reported to have a role in the development of tight junctions (TJs) and maintenance of intestinal homeostasis. Therefore, we hypothesized that AhR activation may attenuate the intestinal barrier dysfunction through increased activation of Notch1 signaling. Adult C57BL/6J mice were divided into three groups: Sham, I/R and I/R + 6-formylindolo(3,2-b)carbazole (Ficz) groups. Mice were sacrificed after I/R for 6 h and the intestine was harvested for histological examination, mRNA and protein content analysis, and mucosal permeability investigation. Additionally, a hypoxic Caco‑2 cell culture model was used to evaluate the role of AhR‑Notch1 signaling in the development of TJs and epithelial permeability in vitro. The AhR‑Notch1 signaling components and TJ proteins were assessed by reverse transcription‑quantitative polymerase chain reaction, western blotting, immunohistochemistry or immunofluorescence staining. Epithelial permeability was detected by transepithelium electrical resistance. The data demonstrated that Ficz significantly attenuated the intestinal tissue damage and the disrupted distribution of TJs, increased the expression of TJ proteins, reversed the decrease in TER and upregulated epithelial Notch1 signaling following intestinal I/R in vivo and hypoxia in vitro. Furthermore, inhibition of Notch1 signaling by N‑[N‑(3,5‑difluorophenacetyl)‑L‑alanyl]‑S‑phenylglycine t‑butyl ester (inhibitor of Notch signaling) counteracted the effects of Ficz on the development of TJs in hypoxic Caco‑2 cells. In conclusion, AhR activation ameliorated epithelial barrier dysfunction following intestinal I/R and hypoxia through upregulation of Notch1 signaling, which suggests that AhR may be a potential pharmaceutical agent to combat this condition.

Topics: Animals; Caco-2 Cells; Carbazoles; Cell Hypoxia; Cell Membrane Permeability; Cell Shape; Dipeptides; Epithelial Cells; Humans; Intestinal Mucosa; Male; Mice, Inbred C57BL; Receptor, Notch1; Receptors, Aryl Hydrocarbon; Reperfusion Injury; Signal Transduction; Tight Junction Proteins; Tight Junctions; Up-Regulation

Inhalational anesthetic preconditioning can induce neuroprotective effects, and the notch signaling pathway plays an important role in neural progenitor cell differentiation and the inflammatory response after central nervous system injury. This study evaluated whether the neuroprotective effect of isoflurane preconditioning is mediated by the activation of the notch signaling pathway. Mice were divided into two groups consisting of those that did or did not receive preconditioning with isoflurane. The expression levels of notch-1, notch intracellular domain (NICD), and hairy and enhancer of split (HES-1) were measured in mice subjected to transient global cerebral ischemia-reperfusion injury. The notch signaling inhibitor DAPT and conditional notch-RBP-J knockout mice were used to investigate the mechanisms of isoflurane preconditioning-induced neuroprotection. Immunohistochemical staining, real-time polymerase chain reaction assays, and Western blotting were performed. Isoflurane preconditioning induced neuroprotection against global cerebral ischemia. Preconditioning up-regulated the expression of notch-1, HES-1, and NICD after ischemic-reperfusion. However, these molecules were down-regulated at 72 h after ischemic-reperfusion. The inhibition of notch signaling activity by DAPT significantly attenuated the isoflurane preconditioning-induced neuroprotection, and similar results were obtained using notch knockout mice. Our results demonstrate that the neuroprotective effects of isoflurane preconditioning are mediated by the pre-activation of the notch signaling pathway.

Topics: Animals; Apoptosis; Ataxia; Basic Helix-Loop-Helix Transcription Factors; CA1 Region, Hippocampal; Carotid Artery, Common; Cerebrovascular Circulation; Dipeptides; Drug Evaluation, Preclinical; Homeodomain Proteins; Ischemic Attack, Transient; Isoflurane; Ligation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neuroprotective Agents; Nitrogen; Premedication; Protein Structure, Tertiary; Random Allocation; Receptor, Notch1; Reperfusion Injury; Signal Transduction; Transcription Factor HES-1; Up-Regulation

The aging kidney has a diminished regenerative potential and an increased tendency to develop tubular atrophy and fibrosis after acute injury. In this study, we found that activation of tubular epithelial Notch1 signaling was prolonged in the aging kidney after ischemia/reperfusion (IR) damage. To analyze the consequences of sustained Notch activation, we generated mice with conditional inducible expression of Notch1 intracellular domain (NICD) in proximal tubules. NICD kidneys were analyzed 1 and 4 wk after renal IR. Conditional NICD expression was associated with aggravated tubular damage, a fibrotic phenotype, and the expression of cellular senescence markers p21 and p16(INK4a). In wild-type mice pharmacological inhibition of Notch using the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) improved tubulo-interstitial damage and antagonized the prosenescent pathway activation after IR. In vitro, activation of Notch signaling with delta-like-ligand-4 caused prosenescent changes in tubular cells while inhibition with DAPT attenuated these changes. In conclusion, our data suggest that sustained epithelial Notch activation after IR might contribute to the inferior outcome of old kidneys after injury. Sustained epithelial activation of Notch is associated with a prosenescent phenotype and maladaptive repair.

Topics: Acute Kidney Injury; Adaptor Proteins, Signal Transducing; Aging; Animals; Calcium-Binding Proteins; Cellular Senescence; Dipeptides; Fibrosis; Intracellular Signaling Peptides and Proteins; Kidney Tubules; Kidney Tubules, Proximal; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Receptor, Notch1; Reperfusion Injury

Notch signaling plays a critical role in the maintenance of intestinal crypt epithelial cell proliferation. The aim of this study was to investigate the role of Notch signaling in the proliferation and regeneration of intestinal epithelium after intestinal ischemia reperfusion (I/R) injury.. Male Sprague-Dawley rats were subjected to sham operation or I/R by occlusion of the superior mesenteric artery (SMA) for 20 min. Intestinal tissue samples were collected at 0, 1, 2, 4, and 6 h after reperfusion. Proliferation of the intestinal epithelium was evaluated by immunohistochemical staining of proliferating nuclear antigen (PCNA). The mRNA and protein expression levels of Notch signaling components were examined using Real-time PCR and Western blot analyses. Immunofluorescence was also performed to detect the expression and location of Jagged-2, cleaved Notch-1, and Hes-1 in the intestine. Finally, the γ-secretase inhibitor DAPT and the siRNA for Jagged-2 and Hes-1 were applied to investigate the functional role of Notch signaling in the proliferation of intestinal epithelial cells in an in vitro IEC-6 culture system.. I/R injury caused increased intestinal crypt epithelial cell proliferation and increased mRNA and protein expression of Jagged-2, Notch-1, and Hes-1. The immunofluorescence results further confirmed increased protein expression of Jagged-2, cleaved Notch-1, and Hes-1 in the intestinal crypts. The inhibition of Notch signaling with DAPT and the suppression of Jagged-2 and Hes-1 expression using siRNA both significantly inhibited the proliferation of IEC-6 cells.. The Jagged-2/Notch-1/Hes-1 signaling pathway is involved in intestinal epithelium regeneration early after I/R injury by increasing crypt epithelial cell proliferation.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Cell Proliferation; Dipeptides; Disease Models, Animal; Gene Silencing; Homeodomain Proteins; Intestinal Mucosa; Jagged-2 Protein; Male; Membrane Proteins; Proliferating Cell Nuclear Antigen; Rats; Receptor, Notch1; Regeneration; Reperfusion Injury; RNA Interference; Signal Transduction; Transcription Factor HES-1

This study aims to investigate the role of Notch pathway in the renal ischemia/reperfusion injury (IRI)-associated inflammation and apoptosis.. Male Sprague-Dawley rats were divided into three groups: normal saline (NS)-treated sham rats, NS-treated ischemia/reperfusion (I/R) rats, and N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) (a γ-secretase inhibitor) treated I/R rats. I/R rat model underwent nephrectomy of the right kidney and was subjected to 60 min of left renal pedicle occlusion followed by 24 h, 48 h, and 72 h of reperfusion, respectively. The levels of creatinine, urea nitrogen (BUN), interleukin (IL)-6, tumor necrosis factor (TNF)-α in serum samples and urinary N-acety-β-d-glucosaminidase (NAG) were assayed. Histological examinations were performed. The protein expression of Notch2, hairy/enhancer of split 1 (hes-1), NF-κB2, monocyte chemoattractant protein (MCP)-1, B-cell lymphoma 2 (bcl-2), and bcl-2-associated X (bax) were detected and the degree of apoptosis of tubular cells was evaluated.. Renal IR induced severe tubular damage, caused significant increases in the Scr, BUN, IL-6, TNF-α, urinary NAG, Notch2, hes-1, NF-κB2, MCP-1, ratio of tubule cells apoptosis, and reduction in the ratio of bcl-2 to bax. However, DAPT treatment significantly reduced the level of Scr, BUN, IL-6, TNF-α, and NAG. Thus, I/R activates Notch2/hes-1 signaling and DAPT treatment can ameliorate the severity of tubular damage after renal IRI, lower the expression of NF-κB2, MCP-1, and bax protein, increase the expression of bcl-2 protein, and reduce the ratio of terminal 2-deoxyuridine 5-triphosphate nick end-labeling-positive cells.. Notch signaling plays an important role in the renal IRI-associated inflammation and apoptosis. DAPT can protect against IRI through partly suppressing inflammation and apoptosis, which could constitute a new target for AKI.

Topics: Acute Kidney Injury; Amyloid Precursor Protein Secretases; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; bcl-2-Associated X Protein; Cytokines; Dipeptides; Epithelial Cells; Homeodomain Proteins; In Situ Nick-End Labeling; Inflammation; Kidney; Kidney Function Tests; Male; NF-kappa B; Rats; Rats, Sprague-Dawley; Receptor, Notch2; Reperfusion Injury; Signal Transduction; Transcription Factor HES-1