Pinoresinol targets NF-kB alongside STAT3 pathway to attenuate IL-6-induced inflammation

doi:10.21203/rs.3.rs-3900297/v1

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Pinoresinol targets NF-kB alongside STAT3 pathway to attenuate IL-6-induced inflammation

https://doi.org/10.21203/rs.3.rs-3900297/v1

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Dysregulated synthesis of IL-6 plays a critical role in inflammation-induced disease pathophysiology. IL-6 is known to induce NF-κB along with canonical JAK-STAT pathway, indicating the importance of cascade proteins of these two pathways as the targets of anti-inflammatory compounds. Here, we report the mechanism of downregulation of NF-κB and JAK-STAT pathways by pinoresinol, a plant lignan, in IL6-induced macrophages. Bioinformatic analysis revealed Pinoresinol, among 100 dietary polyphenols, as the most potent candidate to interact with the proteins in NF-κB and JAK-STAT pathways. Pinoresinol significantly abrogated IL-6-mediated activation of p65 NF-κB and STAT3 and their nuclear translocation in differentiated THP-1 cells. Remarkably, pinoresinol attenuated IL-6-mediated activation of NF-κB regulators, IKK and IκB-α and the degradation of the latter suggesting its critical role in inhibition of NF-kB pathway. The expressions of downstream genes of the two pathways, e.g. IL-1β, TNF-α, and COX-2 were also inhibited by pinoresinol. The polyphenol treatment also reduced the IL-6-mediated macrophage adhesion and migration, which was supported by the downregulation of VCAM-1, ICAM-1, MCP-1, MMP-9 and MMP-2. Our study highlights the mechanism of anti-inflammatory efficacy of pinoresinol in IL-6-induced differentiated macrophages.

Pinoresinol

IL-6

NF-kB

STAT3

Inflammation

Dietary polyphenol

Adhesion

Migration

Interleukin-6 (IL-6) is a multifaced inflammatory cytokine that is produced at the site of inflammation and executes critical functions in the orchestration of acute-phase responses, immune responses, and hematopoiesis. Although subjected to efficient regulation through transcriptional and post-transcriptional mechanisms, aberrant expression of IL-6 due to continued production or defective control mechanisms, is implicated in the development of several chronic inflammatory and autoimmune disorders [1]. The pathological role of IL-6 is acknowledged in various animal models of disease progression. Inhibition of IL-6 or its receptor (IL-6R) by IL-6-targeting antibodies is known for its protective effect against disease progression [2-4]. IL-6 is widely recognized for its ability to trigger the members of the Janus Kinase (JAK) family namely, JAK1, JAK2 and Tyk2, which, then stimulates the Signal Transducers and Activators of Transcription-3 (STAT3). Upon IL-6 receptor binding, cytoplasmic STAT3 proteins undergo tyrosine phosphorylation which is crucial for its dimerization and nuclear translocation [5]. In addition to STAT3, nuclear factor-kappa B (NF-κB), is another vital transcriptional mediator intricately involved in modulating the activity patterns of numerous genes associated with immune and inflammatory events. A substantial body of research indicates that NF-κB functions as the transcriptional regulator of genes that encode IL-6 [6, 7]. Under normal conditions, the presence of cytoplasmic IκB proteins serves to impede the activity of the p50/p65 heterodimer complex. IκB kinase (IKK) consists of three different proteins: two kinase constituents (IKKα and IKKβ) and one regulatory component (IKKγ). IκB (Ser32/34) is phosphorylated upon IKK activation and phosphorylation, followed by ubiquitin conjugation and proteasome-driven IκB breakdown leading to NF-κB p65/RelA (Ser536) phosphorylation. After activation, NF-κB complex subunits like p65/RelA translocate to the nucleus and form homodimers and heterodimers with other family members to stimulate gene expression regulated by κB-dependent elements. [8-10]. Since NF-κB and STAT3 signaling pathways are regulated by IL-6, their dysregulation has been implicated in inflammatory ailments like multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis and Crohn’s disease [11-16]. Abnormal regulation of IL-6 is also linked to chronic inflammatory and autoimmune disorders, such as atherosclerosis. [17]. Atherosclerosis is a persistent inflammatory condition characterized by the recruitment and aggregation of macrophages in the sub-endothelial layer of blood vessels resulting in plaque development. For migration, infiltration and adhesion of monocytes that differentiate into macrophages, mediators such as Vascular cell adhesion molecule-1 (VCAM-1), Intercellular adhesion molecule-1 (ICAM-1) and Monocyte chemoattractant protein-1 (CCL2 or MCP-1) are important [18-23]. The NF-κB and IL-6/STAT3 cascade are well recognized as the primary inducer of gene expression levels of these molecules [24-27]. The extravasation process of circulating monocytes/macrophages also requires the secretion of matrix metalloproteinases (MMPs), critical for enabling the monocytes/macrophages to cross the vascular barrier and infiltrate the vessel wall, subsequently entering the extracellular matrix. Elevated MMP expression has been associated with atherosclerosis, as well as Crohn’s disease, and inflammatory bowel diseases. [28-32].

It is a well-established practice to harness the resourcefulness of natural products in the management of inflammation-associated conditions. The antioxidative, anti-inflammatory, and immunomodulatory properties of plant polyphenols establish them as a viable and non-toxic NSAID alternative. Pinoresinol, a lignan constituent of the Forsythiae fructus plant was shown to possess anti-oxidative and anti-inflammatory properties [33, 34]. It is also found in sesame and flax seeds, and is known to reduce the risk of certain cancers and cardiovascular diseases [35-38]. In primary microglia, pinoresinol suppressed LPS-induced ERK 1/2 phosphorylation levels and nuclear migration of p65 NF-κB. Since LPS stimulates iNOS, COX-2 and TNF-α secretion in microglia via the MAPK and NF-κB regulatory pathways, suppression of these signaling pathways may highlight the efficacy of pinoresinol as a suppressor of inflammation [34]. In Caco-2 cells pinoresinol reduced IL-6, COX-2-derived prostaglandin E2, MCP-1, and NF-κB activity in a dose-dependent manner [39]. However, its precise molecular mechanisms and therapeutic potential in modulating IL-6-induced inflammation remain unexplored. Previously, we reported that IL-6 induced the activation of both NF-κB and JAK-STAT signal transduction pathways in THP-1 macrophages [7]. In this report, we aim to evaluate the influence of pinoresinol on the IL-6-activated NF-κB and JAK-STAT pathways and the subsequent impact on the inflammatory responses in THP-1 macrophage cells.

2.1 Molecular docking:

The protein structures of the key proteins associated with NF-κB and JAK-STAT pathway were downloaded from the PDB database: IKK-α (5EBZ), IKK-β (4KIK), IκB- α (1IKN), p65 (1NFI), p50 (1SVC), p65-p50 heterodimer (1VKX), RelB (3DO7), NIK (4G3D), p52 (3DO7), JAK1 (6BBU), JAK2(6BBV), JAK3 (6DA4), STAT1(1YVL), STAT2 (5OEN), STAT3 (6TLC), STAT4(1BGF), STAT5A(1Y1U), TYK(6DBK). A total of 100 plant polyphenols were selected based on dietary intake consisting of flavonoids, non-flavonoids, and phenolic acid. SMILES of the chosen ligands were acquired from the PubChem database and transformed into conventional .pdbqt setup with the help of Open Babel software [40]. The molecular docking study of the identified ligands and the target proteins were carried out via AutoDock Vina and HADDOCK web server [41, 42]. For docking studies in AutoDock Vina, water molecules present in the target protein were detached, and polar hydrogens in addition to Kollman charges were affixed. The structures were next transformed to .pdbqt setup. The grid box was generated to help site-specific docking by locating binding points and the coordinates for x, y, z were fixed to 40, 40, and 40, respectively. For docking experiments of all 100 polyphenols, similar-sized grid box along with other similar parameters were employed, and the full set-up was performed to acquire the docked conformations. To determine the binding energy between proteins of the NF-κB and JAK-STAT pathway and the polyphenols, the best-fitted configuration with the lowermost root mean square deviation (RMSD) were chosen. The same protein and ligand structures were used to carry out docking studies in HADDOCK web server. Easy interface segment of HADDOCK web server was selected to upload the proteins and ligand structures as molecule-1 and molecule-2, respectively. The active sites of the protein critically involved in various interactions were specified. LigPlot+ was utilized to examine the interactions involved in the docked complexes containing the results from AutoDock and HADDOCK server [43].

2.2 MD simulation setup for the complexes:

The AMBER 20 Molecular Dynamics Package was used to simulate the docked p65 NF-κB-pinoresinol and STAT3-pinoresinol complexes [44]. The Amber LEaP module produced the topology and coordinate files necessary for MD simulation [45]. The protein was described using the ff99SB force field [46] and the generic Amber force field (GAFF) parameters for the ligand-protein complexes. The docked complex systems were solvated in a cubic periodic box using the explicit TIP3P (transferable intermolecular potential with 3 points) water model [47]. The requisite quantity of counter ions was added to the complicated systems to neutralize them, and the strong van der Waals was eliminated through energy reduction. The energy minimization dynamics are a part of the standard protocol for the Molecular Dynamics Simulations technique. We employed energy-minimized systems as a starting point for the succeeding MD stages. The above-mentioned constructed complex was subsequently put through an MD minimization run, which involves two steps of minimization: steepest descent and conjugate gradient. Following a nanosecond of NVT equilibration with the time step and NPT equilibration, the complicated structures were reduced using the steepest descent method for the picosecond. Structures were improved until the final RMS energy gradient was smaller than 0.1 Kcal/mol. Except as otherwise noted, the reduction was finished at each stage. The protein systems were equilibrated for one nanosecond under NPT conditions, which involve 300 K and one atm of pressure. The temperature, energy, and pressure graphs were plotted and examined to make sure the systems had successfully equilibrated. Subsequently, a 100 ns MD production run was conducted for the equilibrated structures of both systems using the Particle Mesh Ewald (PME) algorithm with a time step of 2 fs [48, 49]. During the simulation, nonbonding interactions (van der Waals and short-range electrostatic interactions) were handled with a cutoff of 8 Å, whereas the PME approach was used to tackle long-range electrostatic interactions. The SHAKE method was used to limit every bond in the systems [50]. The pressure and temperature (0.5 ps of heat bath and 0.2 ps of pressure relaxation) were maintained constant throughout the simulation process by the Berendsen weak coupling algorithm [51], and all the bonds that were present in the systems were constrained using the SHAKE algorithm [50].

2.3 Binding free energy calculations:

The binding free energies (BFE) of the p65 NF-κB-pinoresinol and STAT3-pinoresinol complexes in the presence of the pinoresinol molecule were calculated using the FastDRH (http://cadd.zju.edu.cn/fastdrh/) web server and Generalized-Born surface area continuum solvation (MM/GBSA) methods [52]. The total binding free energies (BFE) of the NF-κB-Pinoresinol and STAT3-Pinoresinol complex as well as the other derived components (VDW, ELE, GB, and SA) that contribute to the total BFE of the two complexes, were calculated.

2.4 Antibodies and reagents:

Pinoresinol, Phorbol-12-myristate- 13-acetate (PMA) and Type 1 Collagen were procured from Sigma-Aldrich, USA. Alexa Fluor 594 and IL-6 (human recombinant) protein were bought from Invitrogen, USA. RPMI-1640 medium was procured from Himedia, India. NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, ProLong™ Gold Antifade Mountant, Halt™ Protease Inhibitor Cocktail, Halt™ Phosphatase Inhibitor Cocktail, and Verso cDNA synthesis kit were procured from Thermo Fisher Scientific, USA. Taq polymerase was obtained from BioBharati Life Science, India. Fetal bovine serum and penicillin/streptomycin were acquired from Life Technologies, Gibco, USA. The antibodies, mouse anti-TNF-α was obtained from Cloud-Clone Corp. and rabbit anti-β actin, mouse anti-STAT3, rabbit anti phospho STAT3 (Tyr705), mouse anti-NF-κB p65, rabbit anti-phospho NF-κB p65 (Ser536), rabbit anti-IKK, rabbit anti-phospho IKK (Ser176), rabbit anti-Ikβ-α, rabbit anti-phospho Ikβ- α (Ser32), rabbit anti-COX-2, rabbit anti-IL-1β, rabbit anti-MCP-1, rabbit anti-MMP-2, rabbit anti-MMP-9, rabbit anti-GAPDH, and rabbit anti-PARP were all procured from Cell Signaling Technology, USA.

2.5 Cell culture and treatment:

THP-1 cell line derived from human monocytes was procured from the American Type Culture Collection (ATCC, USA). Cells were maintained in RPMI-1640 enriched with 10% FBS as well as penicillin/streptomycin at 37⁰C in 5% CO₂. The culture was passaged in fresh medium every 2-3 days. For gene and protein expression studies, around 1.5 X 10⁶-2.5 X 10⁶THP-1 monocytes were differentiated to THP-1 macrophages by treatment with 5 ng/ml PMA for a period of 48 hours. The differentiated cells underwent pre-treatment with Pinoresinol at two dosages, 50 μM and 100 μM, for a duration of 4 hours in 1% FBS-containing RPMI media (without antibiotics) and then induced with IL-6 (50 ng/ml) for another 2 hours. Cells were subsequently harvested for protein and gene expression.

2.6 Cell viability assay:

Around 10,000 THP-1 cells in a 96-well plate were differentiated as described above. After 24 hours of rest, cells were subjected to either IL-6 (50ng/ml) alone or with a range of pinoresinol concentrations (12.5-200 μM). Following treatment, the cells were incubated for 24 hours. Upon adding 15μl of 5 mg/ml MTT to each well after 24 hours, absorbance was measured at 590 nm.

2.7 Semi-quantitative PCR:

Total cellular RNA was extracted using TriZol reagent and reverse transcribed with Verso cDNA synthesis kit (Thermo Scientific, USA) following manufacturer’s protocol. Semi-quantitative PCR reactions were performed using the cDNA samples as templates with gene-specific primers (Supplementary Table 3).

2.8 Cell fractionation studies:

3 X 10⁶ THP-1 monocytes were seeded and subsequently differentiated by PMA treatment (5ng/ml). Cells were subjected to pinoresinol (50μM and 100μM) treatment for 4 hours prior to incubation with IL-6 (50ng/ml) for 2 hours. Cells were harvested, washed with ice-cold 1XPBS and subjected to centrifugation at 500 x g (5 minutes). NE-PER™ Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific), was used to prepare the nuclear and cytoplasmic fractions following manufacturer’s protocol.

2.9 Western blotting:

Cells underwent treatment, as previously described, and harvested with RIPA buffer containing phosphatase and protease inhibitors for western blot studies. SDS-PAGE was carried out to resolve the proteins from whole-cell lysate samples, which were then transferred to PVDF membranes. After washing, the membranes were subjected to primary antibody incubation at 4^oC overnight, followed by incubation with secondary antibody (HRP-linked) for an hour at room temperature. The blots were then exposed to a chemiluminescence substrate (Clarity Western ECL Substrate, Bio-Rad). The images were captured with the help of the ChemiDocXRS + system (Bio-Rad, USA). The protein bands were assessed and quantified using ImageJ software [53].

2.10. Adhesion Assay:

A 96-well plate was coated with 75 μl of 40 μg/ml type 1 collagen, sealed, and incubated overnight at 4ºC. The following day, 0.5 x 10⁶ cells were loaded onto a 6-well cell culture plate. Cells were treated with pinoresinol and IL-6 as described earlier. The collagen-coated plate from 4ºC incubation was kept at room temperature for 2 hours, excess collagen was removed and then placed in CO₂ incubator. Post-treatment, cells were transferred to microfuge tubes, centrifuged (2000 rpm, 5 mins) and the cell pellet was resuspended in incomplete RPMI medium. The collagen-coated wells were rinsed with incomplete RPMI medium, and 100 μl of cell preparations from every sample were dispensed into the wells, followed by 1-hour incubation for adhesion. Post incubation, medium was withdrawn, adhered cells were fixed by 5% glutaraldehyde treatment for 20-30 min at room temperature. After couple of washes, cell were incubated with 2% crystal violet for 5 minutes followed by imaging 10X objective. Cells were then solubilized with 10% acetic acid and absorbance were measured at 580 nm.

2.11 Migration Assay:

Differentiated THP-1 macrophages (0.25 x 10⁶/well) were seeded in the lower chamber of a 24-well plate. Cells were pretreated with two pinoresinol concentrations (50 μM and 100 μM) followed by 2-hour stimulation with IL-6 (50 ng/ml) as described earlier. Post-treatment, the cells were washed twice with 1X DPBS and incomplete media was added. Transwell inserts with 5μm pore size (Corning, USA) loaded with THP-1 monocytes (0.2-2 x 10⁶/well) were placed in upper chamber in a serum-deficient medium. Migrated monocytic cells beneath the insert were fixed at room temperature using 3.7% formaldehyde in PBS. After washing, cells were permeabilized with 100% methanol for 20 minutes and stained with Giemsa for 15 minutes. The stain was removed, followed by washing with PBS, and observed under a 10X objective on a bright-field microscope.

2.12 Statistical analysis:

Data were collected from mean ± SEM from a minimum of three distinct experiments. ImageJ software was used for densitometric analysis of bands acquired from Western blotting and PCR experiments. One-way ANOVA were performed to compare the distinct sets of data. We considered statistical significance at a P-value equal to or below 0.05.

3.1 Molecular docking analysis and selection of lead polyphenolic compound:

We investigated the binding affinity and interactions between 100 common dietary polyphenols and various proteins of the JAK-STAT and NF-κB signaling cascades using AutoDock Vina and HADDOCK web server. Out of 100 polyphenols, pinoresinol exhibited the highest binding affinity towards the proteins selected from NF-κB and JAK-STAT pathways in both the servers (Supplementary Table 1). The binding affinities of pinoresinol against 9 target proteins from each of NF-κB and JAK-STAT pathways using two servers are enumerated in Supplementary Table 2. The binding affinities indicated the potential of pinoresinol as an inhibitor of both these critical signaling pathways. The interaction profile of pinoresinol with p65 NF-κB and STAT3, the most important members from NF-κB and JAK-STAT pathways are shown in Fig. 1A. The data suggests strong binding of the polyphenol with these two target proteins.

3.2 RMSD Analysis:

The Cα atom RMSD analysis of the complexes (p65 NF-κB and STAT3 in the presence of the pinoresinol molecule) were determined to assess the stability. The RMSD plots for the complex structures (p65 NF-κB-pinoresinol and STAT3-pinoresinol) were depicted in Fig. 1B (a). In the p65 NF-κB -pinoresinol complex, the RMSD value fluctuated at the beginning until 56 ns as a simulation time function, then converged at around 2.8 Å. On the contrary, the STAT3-pinoresinol complex showed minimal oscillations until 40 ns of the simulation period and converged at around 2 Å. Therefore, in the presence of the pinoresinol, the overall structure of the STAT3 complex was found to exhibit more stable conformational dynamics.

3.3 Binding free energy analysis:

Binding free energy is crucial for understanding structural stability, and predicting interacting hotspots and protein-ligand affinities. We calculated the total binding free energy for both the complexes (p65 NF-κB-pinoresinol and STAT3-pinoresinol). The total ΔG_binding for the p65 NF-κB-pinoresinol complex was found to be -13.6 kcal/mol, while for the STAT3-pinoresinol complex it was -15.21 kcal/mol. We noted a slightly greater binding free energy in the STAT3-pinoresinol complex compared to the p65 NF-κB-pinoresinol complex. The remaining energy parameters, including van der Waals (vdW), electrostatic, surface accessible (SA), etc. are also presented in Fig. 1B (b).

3.4 Pinoresinol mitigates IL-6-induced activation of p65 NF-κB and STAT3:

The dynamic interplay between NF-κB and STAT3 activation by IL-6 exerts a critical influence on the pathogenesis of several inflammatory conditions [7, 54]. Hence, we aimed to study the modulatory effect of pinoresinol on IL-6-mediated activation of p65 NF-κB and STAT3. Pinoresinol treatment up to 100 μM showed no cytotoxicity against THP-1 cells and hence, 50 and 100 μM of pinoresinol treatments were used in subsequent studies (Supplementary Fig. 1). Treatment with IL-6 (50ng/ml) substantially activated p65 NF-κB and STAT3 through increased phosphorylation at Ser⁵³⁶ and Tyr⁷⁰⁵residues, respectively in THP-1 macrophages. Pre-treatments with 50 and 100 μM pinoresinol significantly downregulated their activation in a dose-responsive manner (Fig. 2).

3.5 Pinoresinol inhibits nuclear translocation of IL-6 Induced p-p65 NF-κB and p-STAT3:

To understand the inhibitory effect of pinoresinol on IL-6-activated p65 NF-κB and STAT3, we studied the nuclear translocation of these transcription regulators. PMA-differentiated THP-1 cells were pretreated with 50 and 100 μM followed by IL-6 treatment. Western blot analysis of the nuclear and cytoplasmic fractions showed activation and elevated nuclear migration of both p-p65 NF-κB and p-STAT3 following IL-6 treatment. In contrast, pinoresinol treatment impeded the nuclear translocation of both these transcription factors in a dose-dependent fashion [Fig. 3A (a)]. The fold changes in expressions were evaluated by measuring the band intensities [Fig. 3A (b) and (c)]. The observation was supported by an immunofluorescence study where pinoresinol treatment reduced the IL-6-mediated nuclear accumulation of p-p65 NF-κB and p-STAT3 (Fig. 3B).

3.6 Pinoresinol attenuates IL-6-mediated activation of NF-κB regulators, IKK and IκB-α:

IKK (IκB kinase) and IκB-α complex regulates the phosphorylation and cytoplasmic-to-nuclear migration of the NF-κB complex, which exerts important regulatory control over NF-κB-mediated transcriptional activity [55, 56]. Therefore, we investigated the effect of pinoresinol on the IL-6-stimulated phosphorylation of IKK and IκB-α in THP-1 cells. In the cytosolic fraction of the cell lysate, stimulation of IL-6 resulted in an elevation of 3.24-fold in phosphorylated IKK (p-IKK) levels, which was significantly attenuated when the cells underwent treatment beforehand with pinoresinol at concentrations of 50 μM and 100 μM. It was also observed that IL-6 induction led to degradation of IκB-α in the cytosolic fraction, which was significantly inhibited by pinoresinol at both the concentrations. Similarly, the IL-6 stimulated upregulation (1.75-fold) in the phosphorylated IκB-α (p- IκB-α) level was also effectively impeded by pinoresinol with respect to both concentrations. (Fig. 4).

3.7 Pinoresinol inhibits IL-6-induced upregulation of TNF-α, IL-1β and COX-2 levels:

As NF-κB and JAK-STAT pathways are recognized for their role in promoting the transcription of several pro-inflammatory cytokines and enzyme families that catalyze synthesis of inflammatory mediators, we investigated the impact of pinoresinol on pro-inflammatory cytokines stimulated by IL-6. Expression levels of TNF-α, IL-1β, and COX-2 in THP-1 macrophages were studied subsequently. RT-PCR studies demonstrated that IL-6-induced expressions of these mRNAs were significantly inhibited by pinoresinol at both the experimental concentrations (Fig. 5A). The fold changes in expressions were evaluated by measuring the band intensities [Fig. 5 (B-D)]. Similarly, elevated protein levels of TNF- α, IL-1β and COX-2 following IL-6 treatment, were significantly downregulated by pinoresinol treatment in a dose-dependent manner (Fig. 5E). The fold changes in expressions were evaluated by measuring the band intensities [Fig. 5 (F-H)].

3.8 Pinoresinol inhibits IL-6-induced adhesion and migration in THP-1 monocyte:

Since adhesion and migration properties of circulatory monocytes act as a major factor in the aetiology of chronic inflammatory ailments, the effect of pinoresinol to modulate these properties in IL-6-induced THP-1 monocytes was studied. After treatment with pinoresinol (50 and 100 μM) followed by IL-6 (50ng/ml) for 2 hours, cells were allowed to adhere to wells coated with type I collagen. Our results revealed that pinoresinol significantly suppressed the IL-6-induced adhesion of THP-1 monocytes in a dose-dependent manner (Fig. 6A). Furthermore, expressions of ICAM-1, VCAM-1, and MCP-1 are implicated in adhesion and migratory properties of monocytes [57]. We studied the expressions of ICAM-1, and VCAM-1 in IL-6-induced THP-1 cells using RT-PCR to understand their impact on pinoresinol-mediated modulation of adhesion. Pinoresinol treatment led to a dose-dependent decrease in the expression of these genes (Fig. 6C). Additionally, transwell migration assay demonstrated that pinoresinol significantly inhibited monocyte migration towards IL-6-induced differentiated THP-1 macrophages at both concentrations, with 100 μM treatment showed the most prominent inhibition (Fig. 7A). Moreover, gene and protein expression studies of MCP-1 validated the potency of pinoresinol in suppressing the IL-6-induced upregulation at both concentrations (Fig. 7C and D). In-vitro and mouse model studies indicate that elevated matrix metalloproteinases (MMPs) are involved in multiple inflammatory diseases, indicating their multifaceted roles in inflammation, injury etc. [58]. Given their importance in the process of monocyte infiltration, and considering our findings indicating the potential of pinoresinol in downregulating migration properties of monocytes, we investigated the role of pinoresinol in regulating IL-6-induced MMP-2 and MMP-9 expression in THP-1 macrophages. Protein expression studies showed that MMP-2 and MMP-9 increased by 2.12 and 1.6 folds, respectively, in response to IL-6. Pretreatment with pinoresinol downregulated their expression significantly both at 50 and 100 μM. This highlights the efficacy of pinoresinol in suppressing the levels of MMP-2 and MMP-9 expression induced by IL-6 ( Fig. 7D). Our results imply that the mechanism by which pinoresinol functions potentially involves the inhibition of p65 NF-κB and STAT3 activation within THP-1 cells, leading to subsequent downregulation of MCP-1, ICAM-1, VCAM-1, along with MMP-2 and MMP-9.

There are extensive literature that indicate a connection between the aberrant synthesis of IL-6 and various inflammatory conditions [59, 60]. While the classical IL-6 signaling pathway typically activates STAT3, recent studies, including those from our laboratory, underscore the concurrent activation of NF-κB during IL-6-mediated inflammation [7]. The intricate coordination between STAT3 and NF-κB signaling pathways across autoimmune diseases, cytokine storm syndromes and cancer along with their synergistic activation within the IL-6 Amp circuit highlight that their compelling candidacy as therapeutic targets [59, 61-65]. Polyphenols are well recognized for their beneficial role in the prevention and progress of chronic diseases due to their immunomodulatory and anti-oxidative properties, which demonstrate their effectiveness as therapeutic agents against a variety of acute and chronic inflammatory disorders [66-68]. The present study employed bioinformatic analysis to screen 100 common dietary polyphenols, revealing pinoresinol as the most potent candidate targeting proteins in both JAK-STAT and NF-κB signaling cascades. Subsequently, it unfolds the mechanism of pinoresinol-mediated attenuation to impede IL-6-mediated inflammation in differentiated THP-1 macrophages.

The rationale for identifying natural compounds with pharmacological activity, specifically targeting proteins of interest, has been significantly advanced by recent computational approaches. Integrative in-silico strategies have considerable potential in identifying novel therapeutic targets for addressing chronic diseases. In this context, the binding affinities of 100 polyphenols with key proteins in the NF-κB and JAK-STAT pathways were assessed using AutoDock Vina and HADDOCK servers. Notably, pinoresinol emerged with the highest binding affinities in both analyses. We further utilized molecular dynamics simulations and free energy calculations to determine the stability of docked p65 NF-κB-pinoresinol and STAT3-pinoresinol complexes. Our results indicate that pinoresinol could be a promising focus in further investigation in vitro of the potential for downregulating these two pathways when activated.

NF-κB and STAT3 coordinate the expression of multiple downstream genes associated with cell proliferation, survival, stress responses, and immune functions. Activation of NF-κB and STAT3 induced by IL-6 is crucial in coordinating inflammatory responses, ultimately driving the progression of inflammatory disorders through the enhancement of the inflammatory milieu [59, 62]. We found pinoresinol effectively reduced IL-6-induced phosphorylation of p65 NF-κB and STAT3 in differentiated THP-1 macrophages in a dose-dependent manner. However, treatment with 100 μM pinoresinol showed remarkable downregulation of NF-κB phosphorylation, suggesting its effectiveness as a target of the polyphenol. Following activation and phosphorylation, p65 NF-κB and STAT3 translocate to the nucleus to initiate the downstream gene transcription. Our study suggested that pinoresinol markedly suppressed the IL-6-stimulated nuclear translocation of p-p65 NF-κB and p-STAT3.

The NF-κB pathway has been widely recognized as a quintessential proinflammatory signalling pathway, primarily due to its involvement in the expression of proinflammatory genes. Upon activation, NF-κB binds to specific DNA sequences termed κB elements in target genes, orchestrating the transcription of over 500 genes involved in inflammation, immunoregulation, carcinogenesis, apoptosis, etc. [69, 70]. The multi-subunit IκB kinase (IKK) and IκB present in the cytoplasm are the two key proteins that initiate NF-κB activation and translocation. IKK is predominantly responsible for the inducible phosphorylation and ubiquitination of IκB, releasing NF-κB subunits into the nucleus [71]. We aimed to uncover the modulatory effect of pinoresinol on these two cytoplasmic regulators. In the cytoplasmic fraction, pinoresinol treatment resulted in significantly decreased phosphorylation levels of IKK. Pinoresinol also effectively inhibited the phosphorylation IκBα as well as its degradation. Thus, pinoresinol-mediated regulation of IKK and IκBα eventually contributed to the inhibition of p65 NF-κB activation and nuclear translocation.

Subsequently, the study also investigated the efficacy of pinoresinol on TNF-α, IL-1β, and COX-2, recognizing the crucial involvement of NF-κB and STAT3 in their regulation [7, 72, 73]. Pinoresinol significantly reduced the IL-6-induced expression levels of these mediators at both experimental concentrations.

Inflammation plays a major role in the pathophysiology of cardiovascular disorders e.g. atherosclerosis. Adhesion and migration of circulatory monocytes from blood to the sub-intimal layer of the artery marks the beginning of atherosclerotic plaque development. The enhanced cellular infiltration of monocytes occurs via the action of cell adhesion molecules and chemotactic factors. As the plaque matures, these monocytes convert to tissue macrophages, sustaining local inflammation and transforming into foam cells [74, 75]. Considering the context, it was crucial for us to ascertain whether pinoresinol could effectively attenuate the adhesion and migration of THP-1 monocytes. Our data showed that pre-treatment with pinoresinol for 4 hours led to a substantial decline in the adhesion and migration and rates of THP-1 monocytes induced by IL-6. ICAM-1 and VCAM-1 play major roles in monocyte adhesion and more significantly, their expression can be regulated by NF-κB and JAK-STAT signaling pathways [24, 76-78]. Pinoresinol significantly attenuated the expressions ICAM-1 and VCAM-1 in IL-6-induced THP-1 macrophages in a dose-dependent manner. Monocyte chemoattractant protein-1 (MCP-1), a key chemokine, significantly governs the migration and infiltration of monocytes/macrophages, and its levels closely correlate with the extent of atherosclerosis. The expression of MCP-1 is markedly increased in atherosclerotic plaques in response to various stimuli, including cytokines, growth factors, oxLDL, etc. [79]. Pinoresinol likewise downregulated expression of MCP-1 suggesting its critical role in regulation of migration. In addition, matrix metalloproteinases (MMPs) are major contributors to monocyte migration and are acknowledged for their increasingly recognizable role in chronic inflammatory disease pathologies. Especially, MMP-2 and MMP-9 are associated with tissue remodeling and elicitation of inflammatory response in the context of cardiovascular disorders [80, 81]. Our study revealed that pinoresinol effectively diminished the IL-6-triggered elevated expression of MMP-2 and MMP-9. To our knowledge, this is the first report describing pinoresinol's downregulatory effect on IL-6-induced adhesion and migration of monocytic cells. Remarkably, MMP-2 and MMP-9 are also located downstream of the NF-κB and JAK-STAT pathways [82, 83], [82, 83], implying that the reduction of MMP-2 and MMP-9 could result from the inhibition of IL-6-activated NF-κB and STAT3 by pinoresinol. Several studies have demonstrated that MMP inhibitors modulate the migration of inflammatory cells by reducing the expressions of ICAM-1 and VCAM-1 [83, 84]. Thus, it implies that MMPs play a role in the elevation of VCAM-1 and ICAM-1 expression, leading to an eventual increase in monocyte-endothelial adhesion. Therefore, the observed decrease in migration and adhesion of THP-1 monocytes could be attributed to the suppression of ICAM-1 and VCAM-1 by a substantial reduction of MMP-2 and MMP-9 expression.

In summary, this study emphasizes the potential of pinoresinol to inhibit the activation and nuclear translocation of p65 NF-κB and STAT3 in the IL-6-stimulated monocyte/macrophage system and also its downstream genes. Additionally, it effectively demonstrates the efficacy of pinoresinol to impede the migration and adhesion of IL-6-induced THP-1 monocytes by downregulating the expression of critical genes involved in these two processes. The action of pinoresinol on IL-6-induced macrophages is represented as an illustration in Fig. 8. These observations shed light on the anti-inflammatory efficacy of pinoresinol and suggest its potential role in the prevention of IL-6-associated inflammatory conditions.

Data Availability: The manuscript includes all relevant data, which will be available from the corresponding author upon request.

Funding: This study was supported by the Science and Engineering Research Board, Govt. of India (CRG/2021/008212).

Compliance with ethical standards:

Conflict of interest: The authors declare that they have no conflict of interest.

Ethical approval: This study did not require any institutional ethical approval as it involves experiments with commercially available cell lines.

Author contributions:

A.D., M.V.S. and R.M. designed the experiments. A.D., D.D., R.C., B.J.B., M.S.M., and P.S. collected and analysed the data. A.D., D.D. and R.M. wrote the manuscript. R.M. was responsible for acquiring the fund. All authors reviewed and approved the final version of the manuscript.

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No competing interests reported.

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Pinoresinol targets NF-kB alongside STAT3 pathway to attenuate IL-6-induced inflammation

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