Evaluation of the anti-inflammatory potential of Solidago microglossa (Arnica-brasileira) in vivo and its effects on PPARγ activity.

Solidago microglossa is used as an anti-inflammatory agent in traditional Brazilian medicine, and this work evaluated the anti-inflammatory potential of the crude ethanolic extract of the flowers of S. microglossa in vivo, as assayed by paw edema models induced by carrageenan, prostaglandin E2, bradykinin and compound 48/80. In the chemical profile, we identified compounds by electrospray ionization mass spectrometry and quantified them by HPLC-DAD. Additionally, this study analyzed the potential to activate the in vitro transcriptional activity of PPARγ, which is a nuclear receptor linked to the anti-inflammatory response. It was possible to identify five compounds: quinic acid, quercetin, chlorogenic acid, hyperoside, and rutin. In the paw edema evaluation, it was possible to show the potential of reducing edema during the inflammatory process. The crude ethanolic extract of the flowers of S. microglossa activated PPARγ compared to the full agonist rosiglitazone and in a dose-response manner. It is possible to conclude that the extract of the flowers of S. microglossa showed anti-inflammatory activity, and the phenolic compounds present in this species might be responsible for this activity.

Dibutyltin Compounds Effects on PPARγ/RXRα Activity, Adipogenesis, and Inflammation in Mammalians Cells.

Organotins are a group of chemical compounds that have a tin atom covalently bound to one or more organic groups. The best-studied organotin is tributyltin chloride, which is an environmental pollutant and an endocrine disruptor. Tributyltin chloride has been shown to bind to PPARγ/RXRα and induces adipogenesis in different mammalian cells. However, there are few studies with other organotin compounds, such as dibutyltins. The aim of this study was to investigate the effect of dibutyltins diacetate, dichloride, dilaurate, and maleate on the transcriptional activity of the nuclear PPARγ and RXRα receptors, and on adipogenesis and inflammation. Analogous to tributyltin chloride, in reporter gene assay using HeLa cells, we observed that dibutyltins diacetate, dichloride, dilaurate, and maleate are partial agonists of PPARγ. Unlike tributyltin chloride, which is a full agonist of RXRα, dibutyltins dichloride and dilaurate are partial RXRα agonists. Additionally, the introduction of the C285S mutation, which disrupts tributyltin chloride binding to PPARγ, abrogated the dibutyltin agonistic activity. In 3T3-L1 preadipocytes, all dibutyltin induced adipogenesis, although the effect was less pronounced than that of rosiglitazone and tributyltin chloride. This adipogenic effect was confirmed by the expression of adipogenic markers Fabp4, Adipoq, and Glut4. Exposure of 3T3-L1 cells with dibutyltin in the presence of T0070907, a specific PPARγ antagonist, reduced fat accumulation, suggesting that adipogenic effect occurs through PPARγ. Furthermore, dibutyltins dichloride, dilaurate, and maleate inhibited the expression of proinflammatory genes in 3T3-L1 cells, such as Vcam1, Dcn, Fn1, S100a8, and Lgals9. Additionally, in RAW 264.7 macrophages, tributyltin chloride and dibutyltin dilaurate reduced LPS-stimulated TNFα expression. Our findings indicate that dibutyltins diacetate, dichloride, dilaurate, and maleate are PPARγ partial agonists and that dibutyltins dichloride and dilaurate are also partial RXRα agonists. Furthermore, dibutyltins induce adipogenesis in a PPARγ-dependent manner and repress inflammatory genes in 3T3-L1 and RAW 264.7 cells. Although dibutyltins display some partial PPARγ/RXRα agonistic effects, the translation of cell-based results assays into in vivo effects on inflammation and insulin resistance is not entirely known. Nevertheless, further studies are necessary to address their effects in different periods of life and to elucidate the actions of organostanic compounds in whole-body context.

Chronic ethanol intake leads to structural and molecular alterations in the rat endometrium.

We described the effects of low- and high-dose ethanol intake on the structure and apoptosis signaling of the uterine endometrium of UChA and UChB rats (animals with voluntary ethanol consumption). Thirty adult female rats, 90 days old, were divided into three groups (n = 10/group): UChA rats fed with 10% (v/v) ethanol ad libitum (free choice for water or ethanol) drinking < 1.9 g/kg/day; UChB rats fed with 10% (v/v) ethanol ad libitum (free choice for water or ethanol) drinking from 2 to 5 g/kg/day; control rats without ethanol (only water). After 120 days of treatment, rats displaying estrus were euthanized. Uterine epithelial cells of the UCh rats showed dilated cisterns of the rough endoplasmic reticulum, presence of lipid droplets, altered nuclear chromatin, and disrupted mitochondria. The UCh rats exhibited intense atrophied epithelial cells with smaller areas and perimeters of cytoplasm and nuclei. The endometrium of UChA rats showed higher levels of caspase-3 while Xiap and Bcl2 varied from moderate to weak. Both UChA and UChB rats exhibited a stronger immunoreaction to Ki-67 and IGFR-1 on epithelial and stromal cells. Chronic ethanol intake leads to structural and molecular alterations in the uterine endometrium of UCh rats, regardless of low- or high-dose consumption, promoting reproductive disorders.

Mechanisms of peroxisome proliferator activated receptor γ regulation by non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) display anti-inflammatory, antipyretic and analgesic properties by inhibiting cyclooxygenases and blocking prostaglandin production. Previous studies, however, suggested that some NSAIDs also modulate peroxisome proliferator activated receptors (PPARs), raising the possibility that such off target effects contribute to the spectrum of clinically relevant NSAID actions. In this study, we set out to understand how peroxisome proliferator activated receptor-γ (PPARγ/PPARG) interacts with NSAIDs using X-ray crystallography and to relate ligand binding modes to effects on receptor activity. We find that several NSAIDs (sulindac sulfide, diclofenac, indomethacin and ibuprofen) bind PPARγ and modulate PPARγ activity at pharmacologically relevant concentrations. Diclofenac acts as a partial agonist and binds to the PPARγ ligand binding pocket (LBP) in typical partial agonist mode, near the ß-sheets and helix 3. By contrast, two copies of indomethacin and sulindac sulfide bind the LBP and, in aggregate, these ligands engage in LBP contacts that resemble agonists. Accordingly, both compounds, and ibuprofen, act as strong partial agonists. Assessment of NSAID activities in PPARγ-dependent 3T3-L1 cells reveals that NSAIDs display adipogenic activities and exclusively regulate PPARγ-dependent target genes in a manner that is consistent with their observed binding modes. Further, PPARγ knockdown eliminates indomethacin activities at selected endogenous genes, confirming receptor-dependence of observed effects. We propose that it is important to consider how individual NSAIDs interact with PPARγ to understand their activities, and that it will be interesting to determine whether high dose NSAID therapies result in PPAR activation.

A thyroid hormone receptor/KLF9 axis in human hepatocytes and pluripotent stem cells.

Biological processes require close cooperation of multiple transcription factors that integrate different signals. Thyroid hormone receptors (TRs) induce Krüppel-like factor 9 (KLF9) to regulate neurogenesis. Here, we show that triiodothyronine (T3) also works through TR to induce KLF9 in HepG2 liver cells, mouse liver, and mouse and human primary hepatocytes and sought to understand TR/KLF9 network function in the hepatocyte lineage and stem cells. Knockdown experiments reveal that KLF9 regulates hundreds of HepG2 target genes and modulates T3 response. Together, T3 and KLF9 target genes influence pathways implicated in stem cell self-renewal and differentiation, including Notch signaling, and we verify that T3 and KLF9 cooperate to regulate key Notch pathway genes and work independently to regulate others. T3 also induces KLF9 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC) and this effect persists during differentiation to definitive endoderm and hiPSC-derived hepatocytes. Microarray analysis reveals that T3 regulates hundreds of hESC and hiPSC target genes that cluster into many of the same pathways implicated in TR and KLF9 regulation in HepG2 cells. KLF9 knockdown confirms that TR and KLF9 cooperate to regulate Notch pathway genes in hESC and hiPSC, albeit in a partly cell-specific manner. Broader analysis of T3 responsive hESC/hiPSC genes suggests that TRs regulate multiple early steps in ESC differentiation. We propose that TRs cooperate with KLF9 to regulate hepatocyte proliferation and differentiation and early stages of organogenesis and that TRs exert widespread and important influences on ESC biology.