PubMed: 23178521

Title
Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation.
Journal
European journal of pharmacology
Volume
698
Issue
None
Pages
145-53
Date
2013-01-05
Authors
Abdullah L | Ait-Ghezala G | Bachmeier C | Beaulieu-Abdelahad D | Crawford F | Mullan M | Paris D | Reed J | Verma M

Evidence c0527e8527

We observed that anatabine inhibits basal STAT3 phosphorylation levels (Mann– Whitney U=0, Z=-2.882, P=0.004) and reduces the induction of p65 NFkB phosphorylation by TNFa (Mann–Whitney U=0, Z=-2.882, P=0.004) within this 15 min time-frame (Fig. 1)

Evidence 2d50d5ff58

Under these culture conditions, anatabine also inhibited both STAT3 and p65 NFkB phosphorylation induced by TNFa (Fig. 2)

Evidence 517a336e2e

A significant inhibition of STAT3 phosphorylation was observed with 600 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021) and with 800 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021) and a significant inhibition of p65 NFkB phosphorylation was observed with 600 mg/ml of anatabine (Mann–Whitney U=1.0, Z=-2.021, P=0.043) and with 800 mg/ml of anatabine (Mann–Whitney U=0,Z=-2.309, P=0.021)

Evidence 3383e1084d

We observed that anatabine significantly suppressed the stimulation of p65 NFkB and STAT3 phosphorylation by LPS in microglia (Fig. 3)

Evidence b0265753eb

Kruskal–Wallis test revealed that doses of anatabine significantly suppressed both LPS induced STAT3 phosphorylation (H=15.658, df=4, P=0.005) and p65 NFkB phosphorylation (H=14.150, df=4, P=0.007) in human microglia

Evidence 210f8f5bae

A significant inhibition of STAT3 phosphorylation was observed with 10 mg/ml of anatabine (Mann–Whitney U=1, Z=-2.021, P=0.043), with 100 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021), with 300 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021) and with 600 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021)

Evidence 1aeb963ed3

An inhibition of TNFa induced STAT3 (Mann–Whitney U=0, Z=-2.121, P=0.034), and p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-2.121, P=0.034) was observed in these cells following the anatabine treatment (Fig. 4) suggesting that anatabine may also mediate its anti-inflammatory activity independently of nicotinic acetylcholine receptor expression

Evidence 915eca5f23

Anatabine appears to completely antagonize LPS induced STAT3 (Mann–Whitney U=0, Z=-3.077, P=0.002) and p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-3.077, P=0.002) in human mononuclear cells

Evidence 9615f946f1

Similarly, LPS induced p65 NFkB phosphorylation in microglia was significantly inhibited with 10 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021), with 100 mg/ml of anatabine (Mann– Whitney U=0, Z=-2.309, P=0.021), with 300 mg/ml of anatabine (Mann–Whitney U=0, Z=-2.309, P=0.021)

Evidence 58aa9fe30b

Western-blot experiments revealed that LPS significantly stimulated of STAT3 phosphorylation in the spleen (Mann–Whitney U=0, Z=-2.309, P=0.021) (Fig. 8) and kidney (Mann–Whitney U=0, Z=-2.882, P=0.004) (data not shown) whereas anatabine significantly inhibited STAT3 phosphorylation in the spleen (Mann–Whitney U=1, Z=-2.021, P=0.043) and kidney (Mann– Whitney U=5, Z=-2.082, P=0.037) (data not shown)

Evidence 230482eaae

A significant elevation of STAT3 phosphorylation was detected in the brain of Tg APPsw compared to their control littermates (Mann–Whitney U=1, Z=-3.767, p<0.001) and a significant reduction in STAT3 phosphorylation (Mann–Whitney U=8, Z=-2.066, p=0.039) was observed in the brain of Tg APPsw treated with anatabine compared to untreated Tg APPsw littermates (Fig. 10).

Evidence 9678620723

Following 90 days of treatment with anatabine, a significant reduction in brain TNF-a (Mann–Whitney U=6, Z=-2.146, p=0.032) and in IL-6 (Mann–Whitney U=0, Z=-2.887, p=0.004) was observed in Tg APPsw mice compared to Tg APPsw that received regular drinking water (Fig. 9)

Evidence a2043e5896

An increased STAT3 (Mann–Whitney U=0, Z=-2.309, P=0.021) and p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-2.309, p=0.021) was observed in LPS treated microglia (Fig. 3)

Evidence 1b86a023ee

Following a 24 h incubation with LPS, a significant stimulation of STAT3 (Mann– Whitney U=0, Z=-2.882, P=0.004) and p65 NFkB phosphorylation was observed (Mann–Whitney U=1, Z=-2.722, P=0.006)

Evidence 128ab8ebec

Anatabine at 200 mg/ ml significantly lowered LPS induced IL-1b levels in whole blood (Mann–Whitney U=0.0, Z=-2.3, P=0.02)

Evidence 7a7344e0de

Our results showed that intraperitoneal injection of LPS (1 mg/kg) caused a significant elevation of plasma IL-1b (Mann–Whitney U=3, Z=-2.988, P=0.003), IL-6 (Mann–Whitney U=0, Z=-3.366, P=0.001) and TNFa (Mann– Whitney U=0, Z=-3.508, P<0.001) 4 h after the LPS challenge whereas in mice co-treated with an intraperitoneal injection of anatabine (2 mg/kg) and LPS, a significant reduction in plasma IL-1b (Mann–Whitney U=7, Z=-2.645, P=0.008) and TNFa (Mann–Whitney U=4, Z=-2.941, P=0.003) levels was observed (Fig. 6)

Evidence 036e29d5ba

An elevation of IL-6 (Mann–Whitney U=0, Z=-3.487, P<0.001), IL1b (Mann– Whitney U=0, Z=-3.24, P=0.001) and TNFa (Mann–Whitney U=0, Z=-3.361, P=0.001) was observed in the spleen of LPS challenged mice (Fig. 7)

Evidence a10ff37bb9

Similarly, IL-1b (Mann–Whitney U=0, Z=-3.098, P=0.002), IL-6 (Mann–Whitney U=0, Z=-3.363, P<0.001) and TNF-a levels (Mann–Whitney U=0, Z=-3.361, P=0.001) were elevated in the kidney following the LPS challenge (data not shown)

Evidence 4754f545b6

Nicotine has been shown to modulate inflammation by affecting STAT3 phosphorylation (Chatterjee et al., 2009; Hosur and Loring, 2011) and by opposing NFkB activation (Leite et al., 2010; Zhou et al., 2010)

Evidence baa1858960

As a positive control, we used stattic, a known inhibitor of STAT3 dimerization and phosphorylation

Evidence dc47eb9591

We found that stattic inhibited STAT3 phosphorylation (Mann–Whitney U=0, Z=-2.324, P=0.02) and also suppressed p65 NFkB phosphorylation (Mann–Whitney U¼0, Z¼2.324, P¼0.02) mimicking the effect of anatabine in SHSY5Y cells (Fig. 1)

Evidence 93fb063694

We next tested the impact of anatabine on STAT3 and p65 NFkB phosphorylation induced by a 24 h treatment with LPS on human microglial cells, a cell type known to express alpha7-nicotinic acetylcholine receptor subtype (Suzuki et al., 2006)

Evidence 316707286c

Anatabine significantly inhibited LPS induced IL-6 (Mann–Whitney U=4, Z=-3.303, P=0.001), TNF-a (Mann–Whitney U=0, Z=-3.361, P=0.001) and IL-1b levels in the spleen (Mann–Whitney U=9, Z=-1.981, P=0.048) (Fig. 7)

Evidence 63f15d5cf0

A significant reduction in TNF-a (Mann–Whitney U=12, Z=-2.309, P=0.021) but no significant reduction in IL-6 levels (Mann–Whitney U=25, Z=-1.223, P=0.221) or IL-1b (Mann–Whitney U=8, Z=-1.857,P=0.063) were observed in the kidney of LPS and anatabine cotreated animals (data not shown)

Evidence 0a0dca111b

Plasma IL-6 levels show a trend for a reduction but were not significantly affected (Mann–Whitney U=19, Z=-1.368, P=0.171) by the anatabine treatment (Fig. 6)

Evidence 377f948ef7

Higher doses of anatabine resulted in a complete suppression of IL-1b levels in LPS challenged human blood (Mann–Whitney U=0.0, Z=-2.5, P=0.01)

Evidence 405027a3d4

Following this 15 mins of stimulation with TNFa, an increased p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-2.309, P=0.021) without a noticeable increase in STAT3 phosphorylation (Mann–Whitney U=7, Z=-0.289, P=0.773) was observed compared to the control conditions (Fig. 1)

Evidence 82889eb872

An increased in both STAT3 (Mann–Whitney U=0, Z=-2.309, P=0.021) and p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-2.309, P=0.021) was observed following 24 h of treatment with TNFa

Evidence 99c7cae9a4

TNFa significantly stimulated STAT3 (Mann–Whitney U=0, Z=-2.309, P=0.021) and p65 NFkB phosphorylation (Mann–Whitney U=0, Z=-2.309, P=0.021)

Evidence 679f3a1eef

An elevation of brain IL-6 (Mann–Whitney U=0, Z=-3, p=0.003) and TNF-a (Mann–Whitney U=0, Z=-2.887, p=0.004) was observed in Tg APPsw mice compared to their wild-type littermates (Fig. 9)

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