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Electronegative LDL (LDL(-)) and free fatty acids (FFAs) are circulating risk factors for cardiovascular diseases (CVDs) and have been associated with inflammation. Interleukin-1 beta (IL-1β) represents a key cytokine in the development of CVD; however, the initial trigger of IL-1β in CVD remains to be explored. In this study, we investigated the combined effects of LDL(-) from the plasma of ST-segment elevation myocardial infarction (STEMI) patients or diet-induced hypercholesterolemic rabbits and bovine serum albumin bound palmitic acid (PA-BSA) on IL-1β production in macrophages. Macrophages derived from THP-1 cells or human peripheral blood mononuclear cells were independently treated with LDL(-), PA-BSA or cotreated with LDL(-) and PA-BSA. The results showed that nLDL and/or PA-BSA had no effect on IL-1β, and LDL(-) slightly increased IL-1β; however, cotreatment with LDL(-) and PA-BSA resulted in abundant secretion of IL-1β in macrophages. Rabbit LDL(-) induced the elevation of cellular pro-IL-1β and p-Iκ-Bα, but PA-BSA had no effect on pro-IL-1β or p-Iκ-Bα. In potassium-free buffer, LDL(-)-induced IL-1β reached a level similar to that induced by cotreatment with LDL(-) and PA-BSA. Moreover, LDL(-) and PA-BSA-induced IL-1β was inhibited in lectin-type oxidized LDL receptor-1 (LOX-1) knockdown cells and by blockers of voltage-gated potassium (Kv) channels. LDL(-) from diet-induced hypercholesterolemic rabbit had a similar effect as STEMI LDL(-) on IL-1β in macrophages. These results show that PA-BSA cooperates with LDL(-) to trigger IL-1β production in macrophages via a mechanism involving the LOX-1 and Kv channel pathways, which may play crucial roles in the regulation of inflammation in CVD., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In response to environmental stimuli, monocytes undergo polarization into classically activated (M1) or alternatively activated (M2) states. M1 and M2 macrophages exert opposing pro- and anti-inflammatory properties, respectively. Electronegative low-density lipoprotein (LDL) (LDL(-)) is a naturally occurring mildly oxidized LDL found in the plasma of patients with hypercholesterolemia, diabetes, and acute myocardial infarction, and has been shown to involve in the pathogenesis of atherosclerosis. In this study, we examined the effects of LDL(-) on macrophage polarization and the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) in this process. THP-1 macrophages were treated with native LDL (nLDL) or LDL(-), and then the expression of M1/M2-related surface markers and cytokines were evaluated. The results show that treatment with LDL(-) resulted in profound increase in proinflammatory cytokines, IL-1β, IL-6, and TNF-α, and M1-surface marker CD86; however, M2-related cytokines, IL-10 and TGF-β, and M2-surface marker CD206 were not changed by LDL(-). Untreated or nLDL-treated cells were used as control. LDL(-)-induced M1 polarization and secretion of proinflammatory cytokines were diminished in LOX-1 knockdown cells. Taken together, the results show that LDL(-) promotes differentiation of human monocytes to M1 macrophages through a LOX-1-dependent pathway, and explore the contribution of LDL(-) and LOX-1 to the development of chronic inflammation in atherosclerosis.

Background: 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages., Results: Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 μM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells., Conclusion: YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.

Fourteen oxygenated compounds were isolated from the preparative-scale biotransformation of isostevic acid (ent-beyeran-19-oic acid). Incubation of it with Aspergillus niger BCRC 32720 produced eight metabolites, four with Bacillus megaterium ATCC 14581, and another four with Mortierella isabellina ATCC 38063. In addition to their structural elucidation by NMR spectroscopy and HRMS, structures of four of these were further confirmed by X-ray diffraction studies. Real-time reverse transcription PCR analysis found that 15 of these compounds displayed significant in vitro anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophages by reducing the levels of both TNF-α and COX-2 mRNA relative to control cells stimulated by LPS alone. The activity of one metabolite was similar to that of dexamethasone in inhibiting the expression of TNF-α mRNA, while all test compounds except two of them were more potent than dexamethasone in inhibiting the expression of the COX-2 mRNA., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

From the screening of 21 microbial strains, Absidia pseudocylindrospora ATCC 24169 and Aspergillus niger BCRC 32720 were found to reproducibly transform isosteviol lactam (4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam) (3) into various compounds. Preparative-scale transformation of 3 with Abs. pseudocylindrospora yielded two new hydroxylated compounds (4 and 5), with conservation of the lactam ring. Preparative-scale transformation of 3 with Asp. niger afforded seven new compounds, 6 and 9-14, together with the known compounds 7 and 8. A single-crystal X-ray diffraction experiment confirmed the structure of 14. The suppressive effects of compounds 1-14 on the lipopolysaccharide-induced expression of the inducible nitric oxide synthase gene in RAW 264.7 macrophages were examined by a reverse-transcription real-time PCR analysis. With the exception of 7, all other compounds significantly reduced levels of iNOS mRNA relative to control cells, which were induced by LPS alone. Compounds 2, 3, and 5 were similar in activity to dexamethasone, while 9 was more potent.

This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)-induced expression of both inducible nitric oxide synthase (iNOS) and granulocyte-colony stimulating factor (G-CSF) in macrophages and its underlying mechanism. The study arose from an observation that rapamycin inhibited the LPS-induced increase in octamer-binding factor-2 (Oct-2) protein levels through a mammalian target of rapamycin (mTOR)-dependent pathway in mouse RAW264.7 macrophages. As both iNOS and G-CSF are potential Oct-2 target genes, we tested the effect of rapamycin on their expression and found that it reduced the LPS-induced increase in iNOS and G-CSF mRNA levels and iNOS and G-CSF protein levels. Blocking of mTOR-signaling using a dominant-negative mTOR expression plasmid resulted in inhibition of the LPS-induced increase in iNOS and G-CSF protein levels, supporting the essential role of mTOR. Forced expression of Oct-2 using the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LPS-induced increase in iNOS and G-CSF mRNA levels. Chromatin immunoprecipitation assays showed that LPS enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that this effect was inhibited by pretreatment with rapamycin. Moreover, RNA interference knockdown of Oct-2 reduced iNOS and G-CSF expression in LPS-treated cells. The inhibitory effect of rapamycin on the LPS-induced increase in Oct-2 protein levels and on the iNOS and G-CSF mRNA levels was also detected in human THP-1 monocyte-derived macrophages. This study demonstrates that rapamycin reduces iNOS and G-CSF expression at the transcription level in LPS-treated macrophages by inhibiting Oct-2 expression., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Microbial transformation of isosteviol oxime (ent-16-E-hydroxyiminobeyeran-19-oic acid) (2) with Aspergillus niger BCRC 32720 and Absidia pseudocylindrospora ATCC 24169 yielded several compounds. In addition to bioconverting the d-ring to lactone and lactam moieties, 4alpha-carboxy-13alpha-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (7) and 4alpha-carboxy-13alpha-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam (10), one known compound, ent-1beta,7alpha-dihydroxy-16-oxo-beyeran-19-oic acid (6), and five new compounds, ent-7alpha-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (3), ent-1beta,7alpha-dihydroxy-16-E-hydroxyiminobeyeran-19-oic acid (4), ent-1beta-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (5), ent-8beta-cyanomethyl-13-methyl-12-podocarpen-19-oic acid (8), and ent-8beta-cyanomethyl-13-methyl-13-podocarpen-19-oic acid (9), were isolated from the microbial transformation of 2. Elucidation of the structures of these isolated compounds was primarily based on 1D and 2D NMR, and HRESIMS data, and 3-5 were further confirmed by X-ray crystallographic analyses. Additionally, the inhibitory effects of all of these compounds were evaluated on NF-kappaB and AP-1 activation in LPS-stimulated RAW 264.7 macrophages. Among the compounds tested, 5 and 10 significantly inhibited NF-kappaB activation, with 5 showing equal potency to dexamethasone; 3 and 6-9 significantly inhibited AP-1 activation, particularly 8, which showed more inhibitory activity than dexamethasone.

This article reports on a study of the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) in RAW 264.7 macrophages and its underlying mechanisms. TSA pretreatment potently diminishes LPS-stimulated nitric oxide (NO) release and both mRNA and protein levels of iNOS in macrophages. The effects of TSA and LPS on transcription factors binding to two LPS-responsive elements within the iNOS promoter, one binding the NF-kappaB site and the other the octamer element, were investigated. Results show that TSA did not alter the LPS-activated NF-kappaB activity demonstrated by the nuclear translocation of p50 and p65 and by a NF-kappaB-driven reporter gene expression system. In addition, neither TSA nor LPS changed the expression of Oct-1, a ubiquitously expressed octamer binding protein. However, TSA suppressed the LPS-induced expression of Oct-2, another octamer binding protein, at both mRNA and protein levels. Chromatin immunoprecipitation assays revealed that binding of Oct-2 to the iNOS promoter was enhanced by LPS treatment; however, pretreatment with TSA resulted in loss of this binding. Moreover, forced expression of Oct-2 by transfection of pCG-Oct-2 plasmid restored the TSA-suppressed iNOS expression elevated by LPS stimulation, further indicating that Oct-2 activation is a crucial step for transcriptional activation of the iNOS gene in response to LPS stimulation in macrophages. This study demonstrates that TSA diminishes iNOS expression in LPS-treated macrophages by inhibiting Oct-2 expression and thus reducing the production of NO.