The vagus nerve as a modulator of intestinal inflammation - TI Pharma

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Chapter 4 Figure 2. (A) RAW264.7 cells were treated with LPS for three hours, and STAT3 tyrosine (Y705) and serine (S727) phosphorylation were analyzed by Western-blot. Total STAT3 protein was used as a loading control. (B) JAK2 inhibition by AG490 prevents LPS-induced STAT3 tyrosine (Y705) phosphorylation in a concentration dependent manner. The upper panel shows a representative Western-blot, while the lower panel represents the densitometric data of three different experiments represented in mean ± STD. (C) RAW264.7 cells transfected with the NF-kB gene reporter were pretreated with different concentrations of the JAK2 inhibitor AG490 30 minutes prior to 3 hrs of 10ng/ml LPS stimulation. NF-kB activity was analyzed by luminescence. 70 A B C P-STAT3 (%) P-STAT3 (%) NF-kB activity (%) 100 80 60 40 20 0 100 75 50 25 0 100 75 50 25 0 0 1 10 100 1000 LPS(ng/ml) 0 1 10 25 AG490 ( µM) 0 1 10 25 10ng/ml LPS * * PY 705-STAT3 PS 727-STAT3 PS 727-STAT3 PY 705-STAT3 µM AG-490
Deciphering STAT3 signaling in cholinergic immune-modulation effect was prevented by transfection with STAT3D (Fig.3). This implies that the antiinflammatory effect of nAChR activation is dependent on STAT3 DNA binding and the presence of STAT3 protein, rather than STAT3 phosphorylation. TNF (%) 100 50 0 * * - + - + - + EV 3D 3F LPS (10/ng) * Nicotine (1 µM) Figure 3. Nicotinic inhibition of LPS-induced TNF production is dependent on STAT3 DNA binding, rather than STAT-3 phosphorylation. Peritoneal macrophages were transfected with control plasmid expressing empty vector (EV) STAT3 or the mutant forms expressing STAT3D (3D) or STAT3F (3F). Cell cultures were incubated with nicotine (Nico; 1µM), or vehicle, and stimulated with 10 ng/ml endotoxin for 3 hrs. TNF levels in the supernatants were analyzed by ELISA and values are expressed as the percent of TNF released compared to vehicle. Data are mean s.e.m. of three independent experiments done in duplicate. *, P < 0.05 Nicotine and STAT3 modulate the classical p65RelA/p50NF-kB1 pathway To determine at what molecular level nicotine inhibits the NF- B signaling pathway, we assessed the effect of nicotine on nuclear translocation of p65 subunits in stimulated RAW macrophages. In line with previous data, nicotine reduced LPSinduced NF-kB transactivation in RAW macrophages significantly (Fig.4A). As is shown in figure 4B, LPS stimulation increased translocation of NF-κB p65 to the cell nucleus, which was inhibited by nicotine pretreatment. Next, we analyzed whether the inhibition of STAT3 phosphorylation could modulate NF-kB. STAT3 inhibition with stattic, a well-characterized inhibitor of STAT3 phosphorylation7 , prevented the LPS-induced activation of p65RelA in peritoneal macrophages transfected with the NF-kB luciferase reporter (Fig 4C). The specificity of this inhibition was evaluated by analyzing the different NF-kB proteins using specific DNA probes for each pathway. LPS enhanced the NF-kB pathway by activating both p65 and p50 (Fig. 4C), and stattic prevented this activation. This inhibition of p65/p50 was specific as stattic failed to affect p52, c-Rel, and RelB. 71
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The vagus nerve as a modulator ofin
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The vagus nerve as a modulator of i
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Promotiecommissie Promotores: Prof.
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Table of contents Chapter 1 Introdu
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Introduction and outline The aim of
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Introduction and outline Hence, the
Page 13 and 14:
Introduction and outline 18. van de
Page 15 and 16: Chapter 2 ABSTRACT The cholinergic
Page 17 and 18: Chapter 2 neurotransmitter ACh with
Page 19 and 20: Chapter 2 THE VAGUS NERVE AND CHOLI
Page 21 and 22: Chapter 2 nicotine treatment impair
Page 23 and 24: Chapter 2 Figure 1. Hypothetical sc
Page 25 and 26: Chapter 2 unclear whether this hybr
Page 27 and 28: Chapter 2 reduction of NF-κB activ
Page 29 and 30: Chapter 2 CONCLUDING REMARKS The hy
Page 31 and 32: Chapter 2 34 17. The FO, Boeckxstae
Page 33 and 34: Chapter 2 55. Moser N, Mechawar N,
Page 35 and 36: Chapter 2 93. Shafique S, Dalsing M
Page 37 and 38: Chapter 3 ABSTRACT Acetylcholine re
Page 39 and 40: Chapter 3 by bowel manipulation 12
Page 41 and 42: Chapter 3 for preparation of whole
Page 43 and 44: Chapter 3 Figure 1. Nicotine attenu
Page 45 and 46: Chapter 3 Figure 2. Inhibition of m
Page 47 and 48: Chapter 3 macrophage cell lysates s
Page 49 and 50: Chapter 3 reports 2 . We next incub
Page 51 and 52: Chapter 3 Figure 5. Perioperative e
Page 53 and 54: Chapter 3 Figure 7. Stimulation of
Page 55 and 56: Chapter 3 Activation of the STAT3 c
Page 57 and 58: Chapter 3 REFERENCE LIST 60 1. Trac
Page 59 and 60: 4 CHAPTER Deciphering STAT3 signali
Page 61 and 62: INTRODUCTION Deciphering STAT3 sign
Page 63 and 64: Deciphering STAT3 signaling in chol
Page 65: Deciphering STAT3 signaling in chol
Page 69 and 70: DISCUSSION Deciphering STAT3 signal
Page 71 and 72: REFERENCES Deciphering STAT3 signal
Page 73 and 74: Chapter 5 ABSTRACT Background and A
Page 75 and 76: Chapter 5 METHODS Reagents and anti
Page 77 and 78: Chapter 5 described previously 6 .
Page 79 and 80: Chapter 5 Figure 1 Cholinergic agon
Page 81 and 82: Chapter 5 Figure 3 Cholinergic agon
Page 83 and 84: Chapter 5 we analyzed whether nicot
Page 85 and 86: Chapter 5 Zymosan induced formation
Page 87 and 88: Chapter 5 marker CD11 c (Fig. 7E),
Page 89 and 90: Chapter 5 Our data demonstrate that
Page 91 and 92: Chapter 5 REFERENCE LIST 96 1. Boro
Page 93 and 94: Thesis E.P.M. van der Zanden The va
Page 95 and 96: Summary and conclusions The goal of
Page 97 and 98: Summary and conclusions whether the
Page 99 and 100: Summary and conclusions exposure co
Page 101 and 102: REFERENCE LIST Summary and conclusi
Page 103 and 104: Nederlandse samenvatting
Page 105 and 106: Chapter 7 van STAT3 in de ‘cholin
Page 107 and 108: Chapter 7 De bevinding dat nicotine
Page 109 and 110: Chapter 7 macrofagen aan te tonen (
Page 111 and 112: Chapter 7 18. Yu Z, Zhang W, Kone B
Page 113 and 114: Shizuo Akira Department of Host Def
Page 115 and 116: List of publications
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Dankwoord
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om de ene keer olijfolie te drinken
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Curriculum Vitae Curriculum Vitae E
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The vagus nerve as a modulator of intestinal inflammation - TI Pharma

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