Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway.

Neuroinflammation plays a very important role in the pathogenesis of Parkinson's disease (PD). After activation, microglia produce pro-inflammatory mediators that damage surrounding neurons. Consequently, the inhibition of microglial activation might represent a new therapeutic approach of PD. Vanillin has been shown to protect dopaminergic neurons, but the mechanism is still unclear. Herein, we further study the underlying mechanisms in lipopolysaccharide (LPS)-induced PD models. In vivo, we firstly established rat models of PD by unilateral injection of LPS into substantia nigra (SN), and then examined the role of vanillin in motor dysfunction, microglial activation and degeneration of dopaminergic neurons. In vitro, murine microglial BV-2 cells were treated with vanillin prior to the incubation of LPS, and then the inflammatory responses and the related signaling pathways were analyzed. The in vivo results showed that vanillin markedly improved the motor dysfunction, suppressed degeneration of dopaminergic neurons and inhibited microglial over-activation induced by LPS intranigral injection. The in vitro studies demonstrated that vanillin reduces LPS-induced expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), IL-1ß, and IL-6 through regulating ERK1/2, p38 and NF-κB signaling. Collectively, these data indicated that vanillin has a role in protecting dopaminergic neurons via inhibiting inflammatory activation.

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-1ß, 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-1ß, 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.

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

BACKGROUND: Accumulating 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. ß-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. METHODS: For the in vitro experiments, primary mesencephalic neuron-glia cultures were pretreated with BHBA and stimulated with LPS. [(3)H]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. RESULTS: We found that BHBA concentration-dependently attenuated the LPS-induced decrease in [(3)H]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-1ß, and IL-6) production. CONCLUSIONS: In conclusion, the present study supports the effectiveness of BHBA in protecting dopaminergic neurons against inflammatory challenge.

ß-Hydroxybutyric sodium salt inhibition of growth hormone and prolactin secretion via the cAMP/PKA/CREB and AMPK signaling pathways in dairy cow anterior pituitary cells.

ß-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows.

Mycophenolate Mofetil Modulates Differentiation of Th1/Th2 and the Secretion of Cytokines in an Active Crohn's Disease Mouse Model.

Mycophenolate mofetil (MMF) is an alternative immunosuppressive agent that has been reported to be effective and well tolerated for the treatment of refractory inflammatory bowel disease (IBD). The aim of this study was to investigate the therapeutic effect of MMF on intestinal injury and tissue inflammation, which were caused by Crohn's disease (CD). Here, trinitrobenzene sulfonic acid-relapsing (TNBS) colitis was induced in mice; then, we measured the differentiation of Th1/Th2 cells in mouse splenocytes by flow cytometry and the secretion of cytokines in mice with TNBS-induced colitis by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay (RT-PCR/ELISA). The results show that MMF significantly inhibited mRNA expression of pro-inflammatory cytokines IFN-γ, TNF-α, IL-12, IL-6, and IL-1ß in mice with TNBS-induced colitis; however, MMF did not inhibit the expression of IL-10 mRNA. Additionally, ELISA showed that the serum levels of IFN-γ, TNF-α, IL-12, IL-6, and IL-1ß were down-regulated in a TNBS model of colitis. Flow cytometric analysis showed MMF markedly reduced the percentages of Th1 and Th2 splenocytes in the CD mouse model. Mycophenolic acid (MPA) also significantly decreased the percentages of splenic Th1 and Th2 cells in vitro. Furthermore, MMF treatment not only significantly ameliorated diarrhea, and loss of body weight but also abrogated the histopathologic severity and inflammatory response of inflammatory colitis, and increased the survival rate of TNBS-induced colitic mice. These results suggest that treatment with MMF may improve experimental colitis and induce inflammatory response remission of CD by down-regulation of pro-inflammatory cytokines via modulation of the differentiation of Th1/Th2 cells.

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

ß-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-1ß, 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.

Molecular characteristics and evolutionary analysis of field Marek's disease virus prevalent in vaccinated chicken flocks in recent years in China.

Marek's disease is a highly contagious, oncogenic, and immunosuppressive avian viral disease. Surveillance of newly registered Marek's disease virus (MDV) isolates is meaningful for revealing the potential factors involved in increased virulence. Presently, we have focused on the molecular characteristics of all available MDVs from China, including 17 new Henan isolates. Based on Meq, gE, and gI genes, we found that most Chinese isolates contain conserved amino acid point mutations in Meq, such as E(77), A(115), A(139), R(176), and A(217), compared to USA virulent MDVs. However, the 59-aa or 60-aa insertions are only found in a few mild MDVs rather than virulent MDVs in China. Further phylogenetic analysis has demonstrated that a different genotype of MDV has been prevalent in China, and for virulent MDVs, their recent evolution has possibly been geographically restricted. Our study has provided more detailed information regarding the field MDVs circulating in China.

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-1ß, 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.

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-3 in mouse uterus during preimplantation period.

Angiogenesis is necessary for successful implantation and decidualization. This study was to investigate the differential expression of angiopoietin-3 (Ang-3) in mouse uterus during early pregnancy and its regulation by steroid hormones using in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR). There was no detectable Ang-3 mRNA signal on days 1-5 of pregnancy by in situ hybridization. On day 6 of pregnancy, a low level of Ang-3 mRNA signal was seen in the primary decidua. Ang-3 mRNA expression gradually increased on days 7 and 8 of pregnancy along with the development of decidua, and its expression scope was also expanded. The RT-PCR result indicated that Ang-3 mRNA expression was low on days 1-4 of pregnancy. On day 5, as embryo implanted, Ang-3 mRNA was highly expressed in mouse uterus, and the expression gradually increased on days 6-8 of pregnancy, with peak level on day 8 of pregnancy. Similarly, Ang-3 mRNA was also strongly expressed in decidualized cells under artificial decidualization. Compared with the delayed uterus, a high level of Ang-3 mRNA expression was detected in activated implantation uterus by RT-PCR. In the ovariectomized mouse uterus, Ang-3 mRNA expression increased and reached the highest level at 12 hr after injection of estrogen, progesterone, and estrogen plus progesterone, respectively. These results suggest that Ang-3 may play an important role during the process of mouse decidualization. Both estrogen and progesterone can induce the expression of Ang-3 in ovariectomized mouse uterus.

(E)-N'-(2,5-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide.

In the title compound, C(16)H(16)N(2)O(5), the dihedral angle between the two benzene rings is 4.2 (2)° and an intra-molecular O-H⋯O hydrogen bond generates an S(6) ring. In the crystal, mol-ecules are linked into layers lying parallel to the bc plane by O-H⋯O and N-H⋯O hydrogen bonds.

Acute electrical stimulation of nucleus ambiguus enhances immune function in rats.

BACKGROUND: Up to now, many "immunoactive" brain areas have been identified, such as hypothalamic nuclei, brain reward system; but the nucleus ambiguous (Amb), a nucleus nervi vagis of medulla oblongata, was less well studied in neuroimmunomodulation. METHODS: In order to obtain more profound comprehension and more knowledge on Amb, we studied the effect of acute electrical stimulation of Amb on thymus and spleen activity in rat. A stimulator was applied to stimulate the Amb of the anaesthetic rats using the parameter at 100 microgA x 5 ms x 100 Hz every 1 s for 1 min. The levels of TGF-13 and thymosin-beta4 mRNA in thymus, the release of IL-2 and IL-6 at splenocyte in vitro and splenic lymphocyte proliferation were measured at hour 0.5, 1, 2, 3 following the electrical stimulation. RESULTS: The results showed that concanavalin A (Con A)-induced splenic lymphocyte proliferation and the release of IL-2 and IL-6 were all significantly enhanced at 0.5, 1, and 2 h following effective Amb stimulation as compared to in the control group. However, as compared to in the control group, the levels of TGF-beta and thymosin-beta4 mRNA in the thymus were both remarkably reduced at 0.5, 1, and 2 h following effective Amb stimulation. CONCLUSIONS: These findings reveal that the Amb participates in the modulation of animal immune functions.

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.

Establishment and characterization of dairy cow growth hormone secreting anterior pituitary cell model.

A dairy cow anterior pituitary cell (DCAPC) model was established in vitro for the study of growth hormone (GH) synthesis and secretion in the anterior pituitary gland of the dairy cow. Pituitary glands were obtained from Holstein dairy cows' heads cut by electric saw, and the posterior pituitary glands were removed to obtain integrated anterior pituitary glands. Immunohistochemistry assay of GH in the anterior pituitary glands showed that most somatotrophs were located within the lateral wings of the anterior pituitary. Tissues of the lateral wings of the anterior pituitary were dispersed and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The DCAPCs displayed a monolayer, cobblestone, epithelial-like morphology which are the typical characteristics of the anterior pituitary cells. The DCAPCs were subcultured continuously over ten passages. GH immunoreactivity was present in DCAPCs at passage 10. The transcription of the bovine GH mRNA in DCAPCs at passage 10 was decreased to below 50% compared with the lateral wings of the anterior pituitary tissues. Thus, our DCAPCs model is effective for the in vitro examination of GH synthesis and secretion in the dairy cow anterior pituitary gland. The effects of transforming growth factor beta 1 (TGF-ß1) and interferon-γ (IFN-γ) on the expression of GH mRNA in DCAPCs at passage 3 were also investigated. There were no obvious changes in transcription of the GH gene after treatment with TGF-ß1 for 24 h, while IFN-γ increased transcription of the GH gene in a dose-dependent manner.

Virus-encoded miR-155 ortholog is an important potential regulator but not essential for the development of lymphomas induced by very virulent Marek's disease virus.

The microRNA (miRNA) mdv1-miR-M4, a functional miR-155 ortholog encoded by oncogenic Marek's disease virus (MDV), has previously been suggested to be involved in MDV pathogenesis. Using the technique of bacterial artificial chromosome mutagenesis, we have presently evaluated the potential role of mdv1-miR-M4 in the oncogenesis of the very virulent (vv) MDV strain GX0101. Unexpectedly, deletions of the Meq-cluster or mdv1-miR-M4 alone from the viral genome strongly decreased rather than abolished its oncogenicity. Compared to GX0101, mortalities of mutants GXΔmiR-M4 and GXΔMeq-miRs were reduced from 100% to 18% and 4%, coupled with the gross tumor incidence reduction from 28% to 22% and 8%, respectively. Our data suggests that the mdv1-miR-M4 is possibly an important regulator in the development of Marek's disease (MD) lymphomas but is not essential for the oncogenicity of vvMDV. In addition, some of the other Meq-clustered miRNAs may also play potentially critical roles in vvMDV induction of lymphomas.

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.

Expression of interleukin 6 in brain and colon of rats with TNBS-induced colitis.

AIM: To characterise expression of interleukin 6 (IL-6), a potent proinflammatory cytokine, in the occurrence and development of inflammatory bowel disease (IBD) and investigate its effect on neuroimmunomodulation and immune homeostasis regulation. METHODS: In this study, rats with colitis induced by trinitrobenzene sulfonic acid (TNBS) were sacrificed on days 3, 7, 14, 21 and 28 after induction. In the controls, the TNBS was just replaced by equivalent amount of phosphate buffered solution (PBS, 0.01 mol/L). IL-6 mRNA expression in brain and colon tissues in each phase was evaluated by real-time reverse transcription-polymerase chain reaction, and cellular localisation and protein level of IL-6 was determined by immunohistochemistry. RESULTS: At day 7, mRNA expression of IL-6 was significantly higher in the colon and brain of IBD rats than that of the controls. The protein level was also significantly higher in colon, hypothalamus and cerebral cortex of IBD rats compared with the controls. So there are similar temporal trends in IL-6 mRNA expression and protein levels in all positions with a persistent increase to a peak at day 7, followed by a decline and gradual return to normal levels. CONCLUSION: These results revealed that changes in IL-6 expression in brain and colon tissues occur in different phases of IBD. Therefore, we propose that the nerve centre regulates and controls the occurrence and development of IBD via IL-6.