Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-\u03b1 mediated NF-\u03baB and HDAC-3 nuclear translocation

Venkatesh Pooladanda,Nagendra Babu Bathini,Bijay Pattnaik,Sowjanya Thatikonda,Shashi Bala Singh,Chandraiah Godugu,Swarna Bale,Dilep Kumar Sigalapalli

doi:10.1038/s41419-018-1247-9

Abstract

Acute respiratory distress syndrome (ARDS) is characterized by an excessive acute inflammatory response in lung parenchyma, which ultimately leads to refractory hypoxemia. One of the earliest abnormalities seen in lung injury is the elevated levels of inflammatory cytokines, among them, the soluble tumor necrosis factor (TNF-α) has a key role, which exerts cytotoxicity in epithelial and endothelial cells thus exacerbates edema. The bacterial lipopolysaccharide (LPS) was used both in vitro (RAW 264.7, THP-1, MLE-12, A549, and BEAS-2B) and in vivo (C57BL/6 mice), as it activates a plethora of overlapping inflammatory signaling pathways involved in ARDS. Nimbolide is a chemical constituent of Azadirachta indica, which contains multiple biological properties, while its role in ARDS is elusive. Herein, we have investigated the protective effects of nimbolide in abrogating the complications associated with ARDS. We showed that nimbolide markedly suppressed the nitrosative-oxidative stress, inflammatory cytokines, and chemokines expression by suppressing iNOS, myeloperoxidase, and nitrotyrosine expression. Moreover, nimbolide mitigated the migration of neutrophils and mast cells whilst normalizing the LPS-induced hypothermia. Also, nimbolide modulated the expression of epigenetic regulators with multiple HDAC inhibitory activity by suppressing the nuclear translocation of NF-κB and HDAC-3. We extended our studies using molecular docking studies, which demonstrated a strong interaction between nimbolide and TNF-α. Additionally, we showed that treatment with nimbolide increased GSH, Nrf-2, SOD-1, and HO-1 protein expression; concomitantly abrogated the LPS-triggered TNF-α, p38 MAPK, mTOR, and GSK-3β protein expression. Collectively, these results indicate that TNF-α-regulated NF-κB and HDAC-3 crosstalk was ameliorated by nimbolide with promising anti-nitrosative, antioxidant, and anti-inflammatory properties in LPS-induced ARDS.

Highlights

Acute respiratory distress syndrome (ARDS) is a lifethreatening disease caused by shock, sepsis, and pneumonia, which eventually culminates into multiple organ failure[1]
LPS from Escherichia coli (055: B5), and phorbol 12-myristate 13-acetate (PMA), 3-(4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT), dimethyl sulfoxide (DMSO), Griess reagent, 2',7'–dichlorofluorescin diacetate (DCFDA), 4ʹ, 6ʹ-diamidino-2-phenylindole (DAPI), ethylenediaminetetraacetic acid (EDTA), hematoxylin, eosin, toulidine blue (TB), Ehrlich reagent, Giemsa stain, reduced glutathione (GSH), bovine serum albumin (BSA), sodium dodecyl sulphate (SDS), glacial acetic acid, sodium nitrite and bicinchoninic acid (BCA) reagent were purchased from Sigma-Aldrich, USA
As nitrite is the final product of NO, these levels were found to be elevated in LPS stimulated group as compared to normal control (NC), these levels were significantly reduced by nimbolide in both RAW 264.7 (Fig. 1a) and differentiated THP-1 cells (Fig. 1b)

Summary

Introduction

Acute respiratory distress syndrome (ARDS) is a lifethreatening disease caused by shock, sepsis, and pneumonia, which eventually culminates into multiple organ failure[1]. There is a certain scope to treat infectious lung diseases for reducing the mortality rate[3]. Pooladanda et al Cell Death and Disease (2019)10:81 receptor-4 (TLR-4) and the co-receptor cluster of differentiation 14 (CD14) results in lung parenchymal damage, neutrophil accumulation in the interstitial and alveolar compartments, elevated vascular permeability, provocation of pulmonary edema and fibrin deposition[4,5]. LPS stimulation initiates multiple molecular intracellular signaling events, including classical nuclear factor-κB (NFκB) activation, thereby promoting translocation into the nucleus, release inflammatory cytokines principally interleukins (IL-1β, IL-2, and IL-6) and chemokines (macrophage inflammatory proteins, MIP-1α/β)[6]. TLR-4 activation enhances the tumor necrosis factor-α (TNF-α) production, which is a pleiotropic cytokine of the TNF superfamily involves in the pathogenesis of various inflammatory diseases by inducing the oxidative stress, while depleting antioxidant levels[7]. Either suppressing the TNF-α secretion or obstruction its biological actions by pharmacological modulators might have eminent therapeutic potential in treating various inflammatory lung diseases[8]

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Results

Conclusion