Nirmala Tilija Pun

Globular Adiponectin Causes Tolerance to LPS-Induced TNF-α Expression via Autophagy Induction in RAW 264.7 Macrophages: Involvement of SIRT1/FoxO3A Axis

Adiponectin, an adipokine predominantly produced from adipose tissue, exhibited potent anti-inflammatory properties. In particular, it inhibits production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In the present study, we investigated the role of autophagy induction in the suppression of Lipopolysaccharide (LPS) -induced TNF-α expression by globular adiponectin (gAcrp) and its potential mechanisms. Herein, we found that gAcrp treatment increased expression of genes related with autophagy, including Atg5 and microtubule-associated protein light chain (LC3B), induced autophagosome formation and autophagy flux in RAW 264.7 macrophages. Similar results were observed in primary macrophages isolated peritoneum of mice. Interestingly, inhibition of autophagy by pretreatment with Bafilomycin A1 or knocking down of LC3B gene restored suppression of TNF-α expression, tumor necrosis factor receptor- associated factor 6 (TRAF6) expression and p38MAPK phosphorylation by gAcrp, implying a critical role of autophagy induction in the development of tolerance to LPS-induced TNF-α expression by gAcrp. We also found that knocking-down of FoxO3A, a forkhead box O member of transcription factor, blocked gAcrp-induced expression of LC3II and Atg5. Moreover, gene silencing of Silent information regulator 1 (SIRT1) blocked both gAcrp-induced nuclear translocation of FoxO3A and LC3II expression. Finally, pretreatment with ROS inhibitors, prevented gAcrp-induced SIRT1 expression and further generated inhibitory effects on gAcrp-induced autophagy, indicating a role of ROS production in gAcrp-induced SIRT1 expression and subsequent autophagy induction. Taken together, these findings indicate that globular adiponectin suppresses LPS-induced TNF-α expression, at least in part, via autophagy activation. Furthermore, SIRT1-FoxO3A axis plays a crucial role in gAcrp-induced autophagy in macrophages.

Globular Adiponectin Inhibits Lipopolysaccharide-Primed Inflammasomes Activation in Macrophages via Autophagy Induction: The Critical Role of AMPK Signaling

The inflammasome acts as a key platform for the activation of pro-inflammatory cytokines. Adiponectin exhibits potent anti-inflammatory properties. However, the effect of adiponectin on the modulation of the inflammasome has not been explored. Herein, we show that globular adiponectin (gAcrp) suppressed lipopolysaccharide (LPS)-primed inflammasomes activation in murine peritoneal macrophages judged by prevention of interleukin-1β (IL-1β) maturation, caspase-1 activation, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, and pyroptotic cell death. Interestingly, pretreatment with 3-methyl adenine, a pharmacological inhibitor of autophagy, abrogated the suppressive effects of gAcrp on IL-1β secretion and caspase-1 activation, indicating the crucial role of autophagy induction in gAcrp-modulation of the inflammasome activation. In addition, inhibition of 5′Adenosine monophaspahate (AMP)-activated protein kinase (AMPK) signaling abolished suppressive effect of gAcrp on inflammasomes activation. Furthermore, autophagy induction or inhibition of the inflammasome activation by gAcrp was not observed in macrophages deficient in AMPK. Taken together, these results indicate that adiponectin inhibits LPS-primed inflammasomes activation in macrophages via autophagy induction and AMPK signaling-dependent mechanisms.

Role of p62 in the suppression of inflammatory cytokine production by adiponectin in macrophages: Involvement of autophagy and p21/Nrf2 axis

Adiponectin possesses potent anti-inflammatory properties. p62, an adaptor protein composed of multi-functional domain, is known to play a role in controlling inflammatory responses. In the present study, we examined the role of p62 in suppressing inflammatory cytokines produced by globular adiponectin (gAcrp) and the potential underlying mechanisms in macrophages. We demonstrated that gAcrp significantly increased p62 expression. Knockdown of p62 abrogated the suppressive effects of gAcrp on LPS-stimulated TNF-α and IL-1β expression and TRAF6/p38 MAPK pathway, indicating that p62 signaling is critical for suppressing inflammatory cytokines production by gAcrp. We next examined the role of p62 in gAcrp-induced autophagy activation, because autophagy has been shown to play a pivotal role in suppressing TNF-α. Herein, we observed that gene silencing of p62 prevented gAcrp-induced increases in autophagy-related genes and autophagosome formation. In addition, we found that Nrf2 knockdown prevented gAcrp-induced p62 expression, and p21 knockdown prevented Nrf2 induction, suggesting the role of p21/Nrf2 axis in gAcrp-induced p62 expression. Taken together, these findings imply that p62 signaling plays a crucial role in suppressing inflammatory cytokine production by globular adiponectin in macrophages, at least in part, through autophagy induction. Furthermore, the p21/Nrf2 signaling cascade contributes to p62 induction by globular adiponectin.

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.

Design, synthesis, and structure-activity relationship study of halogen containing 2-benzylidene-1-indanone derivatives for inhibition of LPS-stimulated ROS production in RAW 264.7 macrophages

As a continuous effort to discover new potential anti-inflammatory agents, we systematically designed and synthesized sixty-one 2-benzylidene-1-indanone derivatives with structural modification of chalcone, and evaluated their inhibitory activity on LPS-stimulated ROS production in RAW 264.7 macrophages. Systematic structure-activity relationship study revealed that hydroxyl group in C-5, C-6, or C-7 position of indanone moiety, and ortho-, meta-, or para-fluorine, trifluoromethyl, trifluoromethoxy, and bromine functionalities in phenyl ring are important for inhibition of ROS production in LPS-stimulated RAW 264.7 macrophages. Among all the tested compounds, 6-hydroxy-2-(2-(trifluoromethoxy) benzylidene)-2,3-dihydro-1H-inden-1-one (compound 44) showed the strongest inhibitory activity of ROS production. Further studies on the mode of action revealed that compound 44 potently suppressed LPS-stimulated ROS production via modulation of NADPH oxidase. The findings of this work could be useful to design 2-benzylidene-indanone based lead compounds as novel anti-inflammatory agents.

ZFP36L1 and AUF1 Induction Contribute to the Suppression of Inflammatory Mediators Expression by Globular Adiponectin via Autophagy Induction in Macrophages

Adiponectin, a hormone predominantly originated from adipose tissue, has exhibited potent anti-inflammatory properties. Accumulating evidence suggests that autophagy induction plays a crucial role in anti-inflammatory responses by adiponectin. However, underlying molecular mechanisms are still largely unknown. Association of Bcl-2 with Beclin-1, an autophagy activating protein, prevents autophagy induction. We have previously shown that adiponectin-induced autophagy activation is mediated through inhibition of interaction between Bcl-2 and Beclin-1. In the present study, we examined the molecular mechanisms by which adiponectin modulates association of Bcl-2 and Beclin-1 in macrophages. Herein, we demonstrated that globular adiponectin (gAcrp) induced increase in the expression of AUF1 and ZFP36L1, which act as mRNA destabilizing proteins, both in RAW 264.7 macrophages and primary peritoneal macrophages. In addition, gene silencing of AUF1 and ZFP36L1 caused restoration of decrease in Bcl-2 expression and Bcl-2 mRNA half-life by gAcrp, indicating crucial roles of AUF1 and ZFP36L1 induction in Bcl-2 mRNA destabilization by gAcrp. Moreover, knock-down of AUF1 and ZFP36L1 enhanced interaction of Bcl-2 with Beclin-1, and subsequently prevented gAcrp-induced autophagy activation, suggesting that AUF1 and ZFP36L1 induction mediates gAcrp-induced autophagy activation via Bcl-2 mRNA destabilization. Furthermore, suppressive effects of gAcrp on LPS-stimulated inflammatory mediators expression were prevented by gene silencing of AUF1 and ZFP36L1 in macrophages. Taken together, these results suggest that AUF1 and ZFP36L1 induction critically contributes to autophagy induction by gAcrp and are promising targets for anti-inflammatory responses by gAcrp.

Adiponectin inhibits inflammatory cytokines production by Beclin‐1 phosphorylation and B‐cell lymphoma 2 mRNA destabilization: role for autophagy induction

Adiponectin potently suppresses inflammatory mediator production. Autophagy is known to play a critical role in the modulation of inflammatory responses by adiponectin. However, the underlying mechanisms are not clearly understood. Interaction between Beclin‐1 and B‐cell lymphoma 2 (Bcl‐2) is a critical event in autophagy induction. We examined the effects of globular adiponectin (gAcrp) on the Beclin‐1/Bcl‐2 association and its underlying mechanisms.

Critical role of tristetraprolin and AU-binding factor 1 in the suppression of cancer cell growth by globular adiponectin

Adiponectin exhibits potent antitumor activities. Herein, we examined the molecular mechanisms underlying suppression of tumor growth by globular adiponectin (gAcrp). We demonstrated that gAcrp suppressed B‐cell lymphoma 2 (Bcl‐2) expression, an anti‐apoptotic gene, by inducing its mRNA destabilization, which was accompanied with a decrease in cell viability and increased caspase‐3 activity in hepatic cancer cells. In addition, gAcrp increased expression of tristetraprolin (TTP) and AU‐rich element RNA‐binding protein 1 (AUF1), which are mRNA stability regulatory proteins. Moreover, gAcrp‐induced suppression of Bcl‐2 expression was abrogated by knockdown of TTP or AUF1. These data indicate that gAcrp induces apoptosis of hepatic cancer cells by TTP‐ and AUF1‐mediated Bcl‐2 mRNA destabilization, and further suggest that TTP and AUF1 are novel targets mediating the antitumor activity of adiponectin.

YJI-7 Suppresses ROS Production and Expression of Inflammatory Mediators via Modulation of p38MAPK and JNK Signaling in RAW 264.7 Macrophages

Chalcone, (2E)-1,3-Diphenylprop-2-en-1-one, and its synthetic derivatives are known to possess anti-oxidative and anti-inflammatory properties. In the present study, we prepared a novel synthetic chalcone compound, (E)-1-(4-hydroxyphenyl)-3-(2-(trifluoromethoxy)phenyl)prop-2-en-1-one name (YJI-7), and investigated its inhibitory effects on endotoxin-stimulated production of reactive oxygen species (ROS) and expression of inflammatory mediators in macrophages. We demonstrated that treatment of RAW 264.7 macrophages with YJI-7 significantly suppressed lipopolysaccharide (LPS)-stimulated ROS production. We also found that YJI-7 substantially decreased NADPH oxidase activity stimulated by LPS, indicating that YJI-7 regulates ROS production via modulation of NADPH oxidase in macrophages. Furthermore, YJI-7 strongly inhibited the expression of a number of inflammatory mediators in a gene-selective manner, suggesting that YJI-7 possesses potent anti-inflammatory properties, as well as anti-oxidative activity. In continuing experiments to investigate the mechanisms that could underlie such biological effects, we revealed that YJI-7 suppressed phosphorylation of p38MAPK and JNK stimulated by LPS, whereas no significant effect on ERK was observed. Furthermore, LPS-stimulated production of ROS, activation of NADPH oxidase and expression of inflammatory mediators were markedly suppressed by treatment with selective inhibitor of p38MAPK (SB203580) and JNK (SP600125). Taken together, these results demonstrated that YJI-7, a novel synthetic chalcone derivative, suppressed LPS-stimulated ROS production via modulation of NADPH oxidase and diminished expression of inflammatory mediators, at least in part, via down-regulation of p38MAPK and JNK signaling in macrophages.