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Thyroxine Affects Lipopolysaccharide-Induced Macrophage Differentiation and Myocardial Cell Apoptosis via the NF-?B p65 Pathway Both In Vitro and In Vivo.

ABSTRACT: Background:Numerous studies have demonstrated that the inflammatory response is involved in the progression of lipopolysaccharide- (LPS-) induced myocardial cell apoptosis. Accumulating evidence has shown that thyroxine participates in diseases by downregulating the inflammatory response. This study aimed at investigating whether thyroxine alleviates LPS-induced myocardial cell apoptosis. Methods:Bone marrow-derived macrophages (Mø) were treated with LPS and thyroxine, and Mø differentiation and Mø-related cytokine expression were measured. The effect of Mø differentiation on mouse cardiomyocyte (MCM) apoptosis was also detected in vitro. In addition, C57BL/6 mice underwent thyroidectomy and were treated with LPS 35 days later; subsequently, Mø differentiation and myocardial cell apoptosis in hearts were analyzed. To determine whether the nuclear factor-kappa B (NF-?B) p65 pathway mediates the effect of thyroxine on Mø differentiation and myocardial cell apoptosis, the specific NF-?B p65 pathway inhibitor JSH-23 was administered to mice that underwent a thyroidectomy. Results:Levothyroxine treatment significantly reduced the activation of the NF-?B p65 pathway, decreased M1 macrophage (Mø1) differentiation and Mø1-related cytokine mRNA levels in LPS-treated Mø, and increased M2 macrophage (Mø2) differentiation and Mø2-related cytokine mRNA expression. The protective effects of levothyroxine on MCM apoptosis mediated by LPS-treated Mø were alleviated by JSH-23. In mice, thyroidectomy aggravated LPS-induced cardiac injury and cardiac dysfunction, further promoted NF-?B p65 activation, and increased cardiac Mø1 expression and myocardial cell apoptosis but decreased cardiac Mø2 expression. JSH-23 treatment significantly ameliorated the thyroidectomy-induced increases in myocardial cell apoptosis and Mø differentiation. Conclusions:Thyroxine alleviated the Mø1/Mø2 imbalance, reduced the inflammatory response, decreased myocardial cell apoptosis, and protected against cardiac injury and cardiac dysfunction in LPS-treated mice. Thyroxine may be a novel therapeutic strategy to prevent and treat LPS-induced cardiac injury.

SUBMITTER: Zhu S

PROVIDER: S-EPMC6537024 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Thyroxine Affects Lipopolysaccharide-Induced Macrophage Differentiation and Myocardial Cell Apoptosis via the NF-<i>κ</i>B p65 Pathway Both In Vitro and In Vivo.

Zhu Shan S Wang Yuan Y Liu Hongtao H Wei Wen W Tu Yi Y Chen Chuang C Song Junlong J Xu Zhiliang Z Li Juanjuan J Wang Changhua C Sun Shengrong S

Mediators of inflammation 20190514

<h4>Background</h4>Numerous studies have demonstrated that the inflammatory response is involved in the progression of lipopolysaccharide- (LPS-) induced myocardial cell apoptosis. Accumulating evidence has shown that thyroxine participates in diseases by downregulating the inflammatory response. This study aimed at investigating whether thyroxine alleviates LPS-induced myocardial cell apoptosis.<h4>Methods</h4>Bone marrow-derived macrophages (Mø) were treated with LPS and thyroxine, and Mø diff ...[more]

PMID: 31217746

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Project description:Measuring post-translational modifications on transcription factors by targeted mass spectrometry is hampered by low protein abundance and inefficient isolation. Here, we utilized HaloTag technology to overcome these limitations and evaluate various top down mass spectrometry approaches for measuring NF-κB p65 proteoforms isolated from human cells. We show isotopic resolution of N-terminally acetylated p65 and determined it is the most abundant proteoform expressed following transfection in 293T cells. We also show MS(1) evidence for monophosphorylation of p65 under similar culture conditions and describe a high propensity for p65 proteoforms to fragment internally during beam-style MS(2) fragmentation; up to 71% of the fragment ions could be matched as internals in some fragmentation spectra. Finally, we used native spray mass spectrometry to measure proteins copurifying with p65 and present evidence for the native detection of p65, 71kDa heat shock protein, and p65 homodimer. Collectively, our work demonstrates the efficient isolation and top down mass spectrometry analysis of p65 from human cells, and we discuss the perturbations of overexpressing tagged proteins to study their biochemistry. This article is part of a Special Issue entitled: Protein Species.Biological significanceCharacterizing transcription factor proteoforms in human cells is of high value to the field of molecular biology; many agree that post-translational modifications and combinations thereof play a critical role in modulating transcription factor activity. Thus, measuring these modifications promises increased understanding of molecular mechanisms governing the regulation of complex gene expression outcomes. To date, comprehensive characterization of transcription factor proteoforms within human cells has eluded measurement, owing primarily-with regard to top down mass spectrometry-to large protein size and low relative abundance. Here, we utilized HaloTag technology and recombinant protein expression to overcome these limitations and show top down mass spectrometry characterization of proteoforms of the 60kDa NF-kB protein, p65. By optimizing the analytical procedure (i.e. purification, MS(1), and MS(2)), our results make important progress toward the ultimate goal of targeted transcription factor characterization from endogenous loci.

Dataset Information

Thyroxine Affects Lipopolysaccharide-Induced Macrophage Differentiation and Myocardial Cell Apoptosis via the NF-?B p65 Pathway Both In Vitro and In Vivo.

Publications

Thyroxine Affects Lipopolysaccharide-Induced Macrophage Differentiation and Myocardial Cell Apoptosis via the NF-<i>κ</i>B p65 Pathway Both In Vitro and In Vivo.

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