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Resveratrol Protects against TNF-?-Induced Injury in Human Umbilical Endothelial Cells through Promoting Sirtuin-1-Induced Repression of NF-KB and p38 MAPK.

ABSTRACT: Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-?)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches including a cell viability assay, reverse transcription and quantitative polymerase chain reaction, western blot, and immunofluorescence staining. We showed that TNF-? induced CD40 expression and ROS production in cultured HUVECs, which were attenuated by resveratrol treatment. Also, resveratrol increased the expression of sirtuin 1 (SIRT1); and repression of SIRT1 by small-interfering RNA (siRNA) and the SIRT1 inhibitor Ex527 reduced the inhibitory effects of resveratrol on CD40 expression and ROS generation. In addition, resveratrol downregulated the levels of p65 and phospho-p38 MAPK, but this inhibitory effect was attenuated by the suppression of SIRT1 activity. Moreover, the p38 MAPK inhibitor SD203580 and the nuclear factor (NF)-?B inhibitor pyrrolidine dithiocarbamate (PDTC) achieved similar repressive effects as resveratrol on TNF-?-induced ROS generation and CD40 expression. Thus, our study provides a mechanistic link between resveratrol and the activation of SIRT1, the latter of which is involved in resveratrol-mediated repression of the p38 MAPK/NF-?B pathway and ROS production in TNF-?-treated HUVECs.

SUBMITTER: Pan W

PROVIDER: S-EPMC4723256 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Resveratrol Protects against TNF-α-Induced Injury in Human Umbilical Endothelial Cells through Promoting Sirtuin-1-Induced Repression of NF-KB and p38 MAPK.

Pan Wei W Yu Huizhen H Huang Shujie S Zhu Pengli P

PloS one 20160122 1

Inflammation and reactive oxygen species (ROS) play important roles in the pathogenesis of atherosclerosis. Resveratrol has been shown to possess anti-inflammatory and antioxidative stress activities, but the underlying mechanisms are not fully understood. In the present study, we investigated the molecular basis associated with the protective effects of resveratrol on tumor necrosis factor-alpha (TNF-α)-induced injury in human umbilical endothelial cells (HUVECs) using a variety of approaches i ...[more]

PMID: 26799794

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Project description:Rationale: Many viral infections are known to activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. However, the role of p38 activation in viral infection and the underlying mechanism remain unclear. The role of virus-hijacked p38 MAPK activation in viral infection was investigated in this study. Methods: The correlation of hepatitis C virus (HCV) infection and p38 activation was studied in patient tissues and primary human hepatocytes (PHHs) by immunohistochemistry and western blotting. Coimmunoprecipitation, GST pulldown and confocal microscopy were used to investigate the interaction of p38α and the HCV core protein. In vitro kinase assays and mass spectrometry were used to analyze the phosphorylation of the HCV core protein. Plaque assays, quantitative real time PCR (qRT-PCR), western blotting, siRNA and CRISPR/Cas9 were used to determine the effect of p38 activation on viral replication. Results: HCV infection was associated with p38 activation in clinical samples. HCV infection increased p38 phosphorylation by triggering the interaction of p38α and TGF-β activated kinase 1 (MAP3K7) binding protein 1 (TAB1). TAB1-mediated p38α activation facilitated HCV replication, and pharmaceutical inhibition of p38α activation by SB203580 suppressed HCV infection at the viral assembly step. Activated p38α interacted with the N-terminal region of the HCV core protein and subsequently phosphorylated the HCV core protein, which promoted HCV core protein oligomerization, an essential step for viral assembly. As expected, SB203580 or the HCV core protein N-terminal peptide (CN-peptide) disrupted the p38α-HCV core protein interaction, efficiently impaired HCV assembly and impeded normal HCV replication in both cultured cells and primary human hepatocytes. Similarly, severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection also activated p38 MAPK. Most importantly, pharmacological blockage of p38 activation by SB203580 effectively inhibited SFTSV, HSV-1 and SARS-CoV-2. Conclusion: Our study shows that virus-hijacked p38 activation is a key event for viral replication and that pharmacological blockage of p38 activation is an antiviral strategy.

Project description:BackgroundThe cyclic AMP (cAMP) signaling pathway has been reported to either promote or suppress cell death, in a cell context-dependent manner. Our previous study has shown that the induction of dynein light chain (DLC) by cAMP response element-binding protein (CREB) is required for cAMP-mediated inhibition of mitogen-activated protein kinase (MAPK) p38 activation in fibroblasts, which leads to suppression of NF-kappaB activity and promotion of tumor necrosis factor-alpha (TNF-alpha)-induced cell death. However, it remains unknown whether this regulation is also applicable to fibroblastoma cells.MethodsIntracellular cAMP was determined in L929 fibroblastoma cells after treatment of the cells with various cAMP elevation agents. Effects of cAMP in the presence or absence of the RNA synthesis inhibitor actinomycin D or small interfering RNAs (siRNAs) against CREB on TNF-alpha-induced cell death in L929 cells were measured by propidium iodide (PI) staining and subsequent flow cytomety. The activation of p38 and c-Jun N-terminal protein kinase (JNK), another member of MAPK superfamily, was analyzed by immunoblotting. JNK selective inhibitor D-JNKi1 and p38 selective inhibitor SB203580 were included to examine the roles of JNK and p38 in this process. The expression of DLC or other mediators of cAMP was analyzed by immunoblotting. After ectopic expression of DLC with a transfection marker GFP, effects of cAMP on TNF-alpha-induced cell death in GFP+ cells were measured by PI staining and subsequent flow cytomety.ResultsElevation of cAMP suppressed TNF-alpha-induced necrotic cell death in L929 fibroblastoma cells via CREB-mediated transcription. The pro-survival role of cAMP was associated with selective unresponsiveness of L929 cells to the inhibition of p38 activation by cAMP, even though cAMP significantly inhibited the activation of JNK under the same conditions. Further exploration revealed that the induction of DLC, the major mediator of p38 inhibition by cAMP, was impaired in L929 cells. Enforced inhibition of p38 activation by using p38 specific inhibitor or ectopic expression of DLC reversed the protection of L929 cells by cAMP from TNF-alpha-induced cell death.ConclusionThese data suggest that the lack of a pro-apoptotic pathway in tumor cells leads to a net survival effect of cAMP.

Dataset Information

Resveratrol Protects against TNF-?-Induced Injury in Human Umbilical Endothelial Cells through Promoting Sirtuin-1-Induced Repression of NF-KB and p38 MAPK.

Publications

Resveratrol Protects against TNF-α-Induced Injury in Human Umbilical Endothelial Cells through Promoting Sirtuin-1-Induced Repression of NF-KB and p38 MAPK.

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