Juxiong Liu

Anti-inflammatory effects of BHBA in both in vivo and in vitro Parkinson’s disease models are mediated by GPR109A-dependent mechanisms

BackgroundAccumulating evidence suggests that neuroinflammation plays an important role in the progression of Parkinson’s disease (PD). Excessively activated microglia produce several pro-inflammatory enzymes and pro-inflammatory cytokines, leading to damage to surrounding neurons and eventually inducing neurodegeneration. Therefore, the inhibition of microglial overactivation may be a potential therapeutic strategy to prevent the further progression of PD. β-Hydroxybutyric acid (BHBA) has been shown to suppress lipopolysaccharide (LPS)-induced inflammation in BV-2 cells and to protect dopaminergic neurons in previous studies, but the underlying mechanisms remain unclear. Thus, in this study, we further investigated this mechanism in LPS-induced in vivo and in vitro PD models.MethodsFor the in vitro experiments, primary mesencephalic neuron-glia cultures were pretreated with BHBA and stimulated with LPS. [3H]dopamine (DA) uptake, tyrosine hydroxylase-immunoreactive (TH-ir) neurons and morphological analysis were evaluated and analyzed in primary mesencephalic neuron-glia cultures. In vivo, microglial activation and the injury of dopaminergic neurons were induced by LPS intranigral injection, and the effects of BHBA treatment on microglial activation and the survival ratio and function of dopaminergic neurons were investigated. Four our in vitro mechanistic experiment, primary microglial cells were pretreated with BHBA and stimulated with LPS; the cells were then assessed for the responses of pro-inflammatory enzymes and pro-inflammatory cytokines, and the NF-κB signaling pathway was evaluated and analyzed.ResultsWe found that BHBA concentration-dependently attenuated the LPS-induced decrease in [3H]DA uptake and loss of TH-ir neurons in the primary mesencephalic neuron/glia mixed culture. BHBA treatment significantly improved the motor dysfunction of the PD model rats induced by intranigral injection of LPS, and this beneficial effect of BHBA was attributed to the inhibition of microglial overactivation and the protection of dopaminergic neurons in the substantia nigra (SN). Our in vitro mechanistic study revealed that the inhibitory effect of BHBA on microglia was mediated by G-protein-coupled receptor 109A (GPR109A) and involved the NF-κB signaling pathway, causing the inhibition of pro-inflammatory enzyme (iNOS and COX-2) and pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) production.ConclusionsIn conclusion, the present study supports the effectiveness of BHBA in protecting dopaminergic neurons against inflammatory challenge.

BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-κB activation.

β-Hydroxybutyric acid (BHBA) has neuroprotective effects, but the underlying molecular mechanisms are unclear. Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The current study investigates the potential mechanisms whereby BHBA affects the expression of potentially proinflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS). The results showed that BHBA significantly reduced LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6. Blocking of GPR109A by PTX resulted in a loss of this anti-inflammatory effect in BV-2 cells. Western blot analysis showed that BHBA reduced LPS-induced degradation of IκB-α and translocation of NF-κB, while no effect was observed on MAPKs phosphorylation. All results imply that BHBA significantly reduces levels of proinflammatory enzymes and proinflammatory cytokines by inhibition of the NF-κB signaling pathway but not MAPKs pathways, and GPR109A is essential to this function. Overall, these data suggest that BHBA has a potential as neuroprotective drug candidate in neurodegenerative diseases.

Expression, regulation and function of Egr1 during implantation and decidualization in mice

Abstract Early growth response gene 1 (Egr1), a zinc finger transcriptional factor, plays an important role in regulating cell proliferation, differentiation and angiogenesis. Current data have shown that Egr1 is involved in follicular development, ovulation, luteinization and placental angiogenesis. However, the expression, regulation and function of Egr1 in mouse uterus during embryo implantation and decidualization are poorly understood. Here we showed that Egr1 was strongly expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy. Injection of Egr1 siRNA into the mouse uterine horn could obviously reduce the number of implanted embryos and affect the uterine vascular permeability. Further study found that Egr1 played a role through influencing the expression of cyclooxygenase-2 (Cox-2), microsomal prostaglandin E synthase 1 (mPGES-1), vascular endothelial growth factor (Vegf), transformation related protein 53 (Trp53) and matrix metallopeptidase 9 (Mmp9) genes in the process of mouse embryo implantation. Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) might direct the expression of Egr1 in the uterine stromal cells. Under in vivo and in vitro artificial decidualization, Egr1 expression was significantly decreased. Overexpression of Egr1 downregulated the expression of decidual marker decidual/trophoblast PRL-related protein (Dtprp) in the uterine stromal cells, while inhibition of Egr1 upregulated the expression of Dtprp under in vitro decidualization. Estrogen and progesterone could regulate the expression of Egr1 in the ovariectomized mouse uterus and uterine stromal cells. These results suggest that Egr1 may be essential for embryo implantation and decidualization.

GLP-2 suppresses LPS-induced inflammation in macrophages by inhibiting ERK phosphorylation and NF-κB activation.

Background/Aims: GLP-2 has been shown to exert anti-inflammatory effects, but the underlying molecular mechanisms remained undefined. As macrophages are important in the development and maintenance of inflammation, we investigated whether exogenous GLP-2 modulates the expression of pro-inflammatory proteins in LPS stimulated murine peritoneal macrophages. Methods: Macrophages were pretreated with various concentrations of GLP-2 for 1 h and then stimulated with LPS. The effects on pro-inflammatory enzymes (iNOS and COX-2), and pro-inflammatory cytokines (TNF-a, IL-1ß and IL-6) were analysed by Western blotting, ELISA and qRT-PCR. We also examined whether NF-κB or MAPK signaling was involved in the effects of GLP-2. Results: In macrophages, GLP-2 blunted the effect of LPS on protein and mRNA expression levels of iNOS, COX-2, TNF-a, IL-1ß and IL-6. Pre-incubation of macrophages with GLP-2 also blunted LPS-induced IκB-a degradation, IκB-a phosphorylation and NF-κB translocation. In the presence of GLP-2, the effect of LPS treatment on ERK phosphorylation was also profoundly blunted. GLP-2 did, however, not significantly modify the effects of LPS on p38 and JNK activities. Conclusions: These findings demonstrate that in LPS primed macrophages, GLP-2 reduced pro-inflammatory enzymes and cytokine production via mechanisms involving the suppression of NF-κB activity and ERK phosphorylation.

IL-21 Modulates Release of Proinflammatory Cytokines in LPS-Stimulated Macrophages through Distinct Signaling Pathways

The aim of this study was to investigate the anti-inflammatory effect of IL-21 on LPS-induced mouse peritoneal macrophages. The results showed that IL-21 significantly inhibited LPS-induced mRNA expression of IL-1β, TNF-α, and IL-6 in macrophages, but not of IFN-γ, IL-10, CCL5, or CXCL2. ELISA analysis showed that IL-21 also suppressed LPS-induced production of TNF-α and IL-6 in culture supernatants. Western blot analysis showed that IL-21 clearly inhibited ERK and IκBα phosphorylation and NF-κB translocation in LPS-stimulated macrophages, but it increased STAT3 phosphorylation. Flow cytometric and Western blot analysis showed that IL-21 decreased M1 macrophages surface markers expression of CD86, iNOS, and TLR4 in LPS-stimulated cells. All results suggested that IL-21 decreases IL-6 and TNF-α production via inhibiting the phosphorylation of ERK and translocation of NF-κB and promotes a shift from the M1 to M2 macrophage phenotype by decreasing the expression of CD86, iNOS, and TLR4 and by increasing STAT3 phosphorylation in LPS-stimulated cells.

Licochalcone A Prevents the Loss of Dopaminergic Neurons by Inhibiting Microglial Activation in Lipopolysaccharide (LPS)-Induced Parkinson’s Disease Models

The neuroprotective effects of Licochalcone A (Lico.A), a flavonoid isolated from the herb licorice, in Parkinson’s disease (PD) have not been elucidated. The prominent pathological feature of PD is the loss of dopaminergic neurons. The crucial role of neuroinflammation induced by activated microglia in dopaminergic neurodegeneration has been validated. In this study, we explore the therapeutic effects of Lico.A in lipopolysaccharide (LPS)-induced PD models in vivo and in vitro. We find that Lico.A significantly inhibits LPS-stimulated production of pro-inflammatory mediators and microglial activation by blocking the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and nuclear factor κB (NF-κB) p65 in BV-2 cells. In addition, through cultured primary mesencephalic neuron-glia cell experiments, we illustrate that Lico.A attenuates the decrease in [3H] dopamine (DA) uptake and the loss of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in LPS-induced PD models in vitro. Furthermore, LPS intoxication in rats results in microglial activation, dopaminergic neurodegeneration and significant behavioral deficits in vivo. Lico.A treatment prevents microglial activation and reduction of dopaminergic neuron and ameliorates PD-like behavioral impairments. Thus, these results demonstrate for the first time that the neuroprotective effects of Lico.A are associated with microglia and anti-inflammatory effects in PD models.

GLP-2 Attenuates LPS-Induced Inflammation in BV-2 Cells by Inhibiting ERK1/2, JNK1/2 and NF-κB Signaling Pathways

The pathogenesis of Parkinson’s disease (PD) often involves the over-activation of microglia. Over-activated microglia could produce several inflammatory mediators, which trigger excessive inflammation and ultimately cause dopaminergic neuron damage. Anti-inflammatory effects of glucagon-like peptide-2 (GLP-2) in the periphery have been shown. Nonetheless, it has not been illustrated in the brain. Thus, in this study, we aimed to understand the role of GLP-2 in microglia activation and to elucidate the underlying mechanisms. BV-2 cells were pretreated with GLP-2 and then stimulated by lipopolysaccharide (LPS). Cells were assessed for the responses of pro-inflammatory enzymes (iNOS and COX-2) and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α); the related signaling pathways were evaluated by Western blotting. The rescue effect of GLP-2 on microglia-mediated neurotoxicity was also examined. The results showed that GLP-2 significantly reduced LPS-induced production of inducible nitric oxide synthase (iNOS), cyclooxygenase-s (COX-2), IL-1β, IL-6 and TNF-α. Blocking of Gαs by NF449 resulted in a loss of this anti-inflammatory effect in BV-2 cells. Analyses in signaling pathways demonstrated that GLP-2 reduced LPS-induced phosphorylation of ERK1/2, JNK1/2 and p65, while no effect was observed on p38 phosphorylation. In addition, GLP-2 could suppress microglia-mediated neurotoxicity. All results imply that GLP-2 inhibits LPS-induced microglia activation by collectively regulating ERK1/2, JNK1/2 and p65.

Short-Chain Fatty Acids Inhibit Growth Hormone and Prolactin Gene Transcription via cAMP/PKA/CREB Signaling Pathway in Dairy Cow Anterior Pituitary Cells

Short-chain fatty acids (SCFAs) play a key role in altering carbohydrate and lipid metabolism, influence endocrine pancreas activity, and as a precursor of ruminant milk fat. However, the effect and detailed mechanisms by which SCFAs mediate bovine growth hormone (GH) and prolactin (PRL) gene transcription remain unclear. In this study, we detected the effects of SCFAs (acetate, propionate, and butyrate) on the activity of the cAMP/PKA/CREB signaling pathway, GH, PRL, and Pit-1 gene transcription in dairy cow anterior pituitary cells (DCAPCs). The results showed that SCFAs decreased intracellular cAMP levels and a subsequent reduction in PKA activity. Inhibition of PKA activity decreased CREB phosphorylation, thereby inhibiting GH and PRL gene transcription. Furthermore, PTX blocked SCFAs- inhibited cAMP/PKA/CREB signaling pathway. These data showed that the inhibition of GH and PRL gene transcription induced by SCFAs is mediated by Gi activation and that propionate is more potent than acetate and butyrate in inhibiting GH and PRL gene transcription. In conclusion, this study identifies a biochemical mechanism for the regulation of SCFAs on bovine GH and PRL gene transcription in DCAPCs, which may serve as one of the factors that regulate pituitary function in accordance with dietary intake.

Differential Expression and Regulation of Angiopoietin‐2 in Mouse Uterus During Preimplantation Period

Angiogenesis is crucial to successful implantation and decidualization, however, as an important angiogenic growth factor, the effect of Ang‐2 in the process of implantation and decidualization is still unknown. This study is to investigate the differential expression of Ang‐2 in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and RT‐PCR. There is no detectable Ang‐2 mRNA signal on days 1–5 of pregnancy by in situ hybridization. On days 6–8, Ang‐2 mRNA is mainly expressed in the primary decidua of mesometrial side, and the expression gradually increases. By RT‐PCR, a significantly higher level of Ang‐2 expression is observed on day 8 of pregnancy, although Ang‐2 expression can be found through days 1–8. Similarly, Ang‐2 is highly expressed in decidualized cells under artificial decidualization. In the ovariectomized mouse uterus, Ang‐2 expression gradually increases after estrogen injection and with peak levels at 12 hr, while progesterone injection can cause a decline in uterine Ang‐2 mRNA level, which reaches a nadir at 12 hr. These results suggest that Ang‐2 may play a key role in the process of mouse decidualization. Estrogen can induce the expression of Ang‐2 while progesterone can inhibit its expression in the ovariectomized mouse uterus. Anat Rec, 2012.

Sodium Butyrate Inhibits Inflammation and Maintains Epithelium Barrier Integrity in a TNBS-induced Inflammatory Bowel Disease Mice Model

G Protein Coupled Receptor 109A (GPR109A), which belongs to the G protein coupled receptor family, can be activated by niacin, butyrate, and β-hydroxybutyric acid. Here, we assessed the anti-inflammatory activity of sodium butyrate (SB) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mice, an experimental model that resembles Crohns disease, and explored the potential mechanism of SB in inflammatory bowel disease (IBD). In vivo, experimental GPR109a−/− and wild-type (WT) mice were administered SB (5 g/L) in their drinking water for 6 weeks. The mice were then administered TNBS via rectal perfusion to imitate colitis. In vitro, RAW246.7 macrophages, Caco-2 cells, and primary peritoneal macrophages were used to investigate the protective roles of SB on lipopolysaccharide (LPS)-induced inflammatory response and epithelium barrier dysfunction. In vivo, SB significantly ameliorated the inflammatory response and intestinal epithelium barrier dysfunction in TNBS-induced WT mice, but failed to provide a protective effect in TNBS-induced GPR109a−/− mice. In vitro, pre-treatment with SB dramatically inhibited the expression of TNF-α and IL-6 in LPS-induced RAW246.7 macrophages. SB inhibited the LPS-induced phosphorylation of the NF-κB p65 and AKT signaling pathways, but failed to inhibit the phosphorylation of the MAPK signaling pathway. Our data indicated that SB ameliorated the TNBS-induced inflammatory response and intestinal epithelium barrier dysfunction through activating GPR109A and inhibiting the AKT and NF-κB p65 signaling pathways. These findings therefore extend the understanding of GPR109A receptor function and provide a new theoretical basis for treatment of IBD.