ABSTRACT
Listeria monocytogenes (LM) is a facultative intracellular bacterium that causes septicemia-associated acute hepatic injury. However, the pathogenesis of this process is still unclear, and there is still a lack of effective therapeutic strategy for the treatment of LM-induced liver injury. In this study, we attempted to explore the effects of necroptosis on bacterial-septicemia-associated hepatic disease and to explore the contribution of JQ1, a selective BRD4 inhibitor, to the suppression of necroptosis and inhibition of LM-triggered hepatic injury. The results indicated that hepatic BRD4 was primarily stimulated by LM both in vitro and in vivo, along with significantly up-regulated expression of receptor-interacting protein kinase (RIPK)-1, RIPK3, and p-mixed lineage kinase-like (MLKL), showing the elevated necroptosis. However, JQ1 treatment and RIPK1 knockout were found to significantly alleviate LM-induced acute liver injury. Histological alterations and cell death in hepatic samples in LM-infected mice were also alleviated by JQ1 administration or RIPK1 deletion. However, JQ1-improved hepatic injury by LM was abrogated by RIPK1 over-expression, suggesting that the protective effects of JQ1 took place mainly in an RIPK1-dependent manner. In addition, LM-evoked inflammatory response in liver tissues were also alleviated by JQ1, which was similar to the findings observed in mice lacking RIPK1. The anti-inflammatory effects of JQ1 were diminished by RIPK1 over-expression in LM-infected mice. Finally, both in vivo and in vitro experiments suggested that JQ1 dramatically improved hepatic mitochondrial dysfunction in LM-injected mice, but this effect was abolished by RIPK1 over-expression. In conclusion, these results indicated that suppressing BRD4 by JQ1 could ameliorate LM-associated liver injury by suppressing necroptosis, inflammation, and mitochondrial dysfunction by inhibiting RIPK1.
Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Listeria monocytogenes , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Apoptosis , Cell Death , Cytokines/metabolism , Inflammation , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondria/drug effects , Models, Animal , Necroptosis/drug effects , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , SepsisABSTRACT
Hypoxia is beneficial for the differentiation of stem cells transplanted for myocardial injury, but mechanisms underlying this benefit remain unsolved. Here, we report the impact of hypoxia-induced Jagged1 expression in cardiomyocytes (CMs) for driving the differentiation of cardiac stem cells (CSCs). Forced hypoxia-inducible factor 1 (HIF-1) expression and physical hypoxia (5% O) treatment could induce Jagged1 expression in neonatal rat CMs. Pharmacological inhibition of HIF-1 by YC-1 attenuated hypoxia-promoted Jagged1 expression in CMs. An ERK inhibitor (PD98059), but not inhibitors of JNK (SP600125), Notch (DAPT), NF-B (PTDC), JAK (AG490), or STAT3 (Stattic) suppressed hypoxia-induced Jagged1 protein expression in CMs. c-Kit CSCs isolated from neonatal rat hearts using a magnetic-activated cell sorting method expressed GATA4, SM22 or vWF, but not Nkx2.5 and cTnI. Moreover, 87.3% of freshly isolated CSCs displayed Notch1 receptor expression. Direct co-culture of CMs with BrdU-labeled CSCs enhanced CSCs differentiation, as evidenced by an increased number of BrdU/Nkx2.5 cells, while intermittent hypoxia for 21 days promoted co-culture-triggered differentiation of CSCs into CM-like cells. Notably, YC-1 and DAPT attenuated hypoxia-induced differentiation. Our results suggest that hypoxia induces Jagged1 expression in CMs primarily through ERK signaling, and facilitates early cardiac lineage differentiation of CSCs in CM/CSC co-cultures HIF-1/Jagged1/Notch signaling.
ABSTRACT
BACKGROUND:Activation of Notch signaling plays a critical role in stem cel differentiation, and this effect seems to be cel-type dependent. Little is reported on the role of activation of Notch1 signaling in the differentiation of c-Kit+ bone marrow mesenchymal stem cels. OBJECTIVE:To analyze the influence of activation of Notch1 signaling on the differentiation of c-Kit+ bone marrow mesenchymal stem cels. METHODS:The Notch1 intracelular domain (N1-ICD) was obtained from the cDNA library by PCR and cloned intoBamHI/AgeI digested adenoviral GV314 plasmid to construct N1-ICD overexpressing shuttle plasmid, and the positive clones were verified by sequencing. N1-ICD shuttle plasmid and helper plasmids pBHGloxΔE1,3 Cre were used to co-transfect HEK293T cels to obtain N1-ICD overexpressing adenoviral particles (N1-ICD-Ad). The c-Kit+ subpopulation were isolated from bone marrow mesenchymal stem cels of the Sprague-Dawley rat femurviamagnetic activated cel sorting. After transfection of the c-Kit+ BMSCs with N1-ICD-Ad adenovirus, we assessed the activation of Notch1 signaling and differentiation in c-Kit+ bone marrow mesenchymal stem cels by quantitative RT-PCR and immunofluorescent staining. RESULTS AND CONCLUSION:N1-ICD coding sequence was successfuly generated from the cDNA library, and then was cloned into the linearized adenoviral vectors GV314. The resistant clones were verified by sequencing. With the assistance of packaging plasmids, recombinant N1-ICD-Ad adenovirus plasmids were successful packaged in HEK293T cels, and its title was 2×1012 PFU/L. c-Kit+ bone marrow mesenchymal stem cels with the purity of 91.6% were successfuly isolated from the bone marrow mesenchymal stem cels of the Sprague-Dawley rat femur. Compared with the blank and negative controls, N1-ICD-Ad infection in the c-Kit+ bone marrow mesenchymal stem cels led to substantial accumulation of N1-ICD in the cytoplasm and nuclei, significantly unregulated expressions of Hes1 (a downstream gene of Notch) and cardiomyocyte differentiation genes Nkx2.5 and cTnT, significantly increased the expression of von Wilebrand factor, an endothelial cel differentiation gene, and mildly increased the expression of smooth muscle22α, a smooth muscle cel differentiation gene. These experimental results indicate that the activation of Notch1 signaling contributes to multi-lineages differentiation of c-Kit+ bone marrow mesenchymal stem cels, and the construction of N1-ICD overexpressing adenoviral vector makes the foundation for further research on the role of Notch1 signaling in stem cel biology.
