Comparative in silico, in vitro and ex vivo anti-inflammatory activity of quercetin

Abstract

Inflammation is characterized by activation of the immune and nonimmune cells that act by removing the stress stimuli such as pathogens, toxins and so on. It can be categorized in two types namely, acute and chronic inflammation that depends on the extent of the injury caused due to inflammation. Basically, acute inflammatory responses regulate various cellular and molecular events and the interaction of different types of immune cells, that helps to minimize the injury. These events may lead to recovery of tissue homeostasis during acute inflammation. However, during chronic inflammation as in neurodegenerative diseases, a variety of key players orchestrate the process to amplify the magnitude of inflammation.¹

Mitogen-activated protein kinases (MAPKs) are one of the widely studied kinase family that composed of three well known subfamily: p38 MAPKs, extracellular signal-regulated protein kinases (ERKs) and c-Jun N-terminal kinases (JNKs). The p38MAPKs are encoded by p38α, p38β, p38γ, and p38δ genes. It has been shown that the p38MAPK pathway is critically involved in the regulation of inflammation via activation of TLR4 receptor. MAPK-activated protein kinase 2 (MK2) is one of the key substrates downstream to p38α/p38β, which on phosphorylation regulate the production and signaling of pro-inflammatory cytokines.^{2, 3}

The inhibition of MK2 by tool compound, PF-3644022 has shown reduction of inflammatory biomarkers in various in vitro and in vivo models.⁴ So, the present study is designed to investigate and compare the anti-inflammatory effects of quercetin⁵ with MK2 inhibitor, PF-3644022 in LPS induced SH-SY5Y cell line in vitro and rat whole blood ex vivo.

The computational docking study (binding affinity) data was obtained through the Auto Dock Vina software and the interaction among the protein-ligand inhibition was studied through Biovia (discovery studio) and Ligplot+ software (Figure 1A-D). The structure-based binding site identification study method was performed to compare the binding affinities among PF-3644022 and quercetin against MK2 protein. The binding affinity of PF-3644022 with MK2 was found to be −8.4 Kcal/mol. The interaction between PF-3644022 and MK2 is divided into two types. At first, it formed one hydrophilic bond having bond length 3.17 Å against Arg 149 (A chain) of MK2 protein. Second, the hydrophobic bonds were formed with Glu 165, Ser 169, Asn 200, Lys 168, Ile 202, Tyr 194, Ile 166, and pro 199 in MK2 protein. The binding affinity of quercetin was found to be −8.1 Kcal/mol. The interaction between quercetin and MK2 showed that there were four hydrophilic bonds with showing different bond length against MK2 protein amino acid sequence like Asp 207 (2.70 Å), Glu 139(2.70 Å), Leu 141(2.80 Å), and Glu 145(2.91 Å). This binding also showed hydrophobic interaction against MK2 protein amino acid such as Gly 73, Leu 72, Glu 190, Asn 191, Val 78, Thr 206, Met 138, Ala 91, Cys 140, and Leu 70, expressed on the A chain of target protein. PF-3644022 showed higher binding affinity to MK2 protein than quercetin. Indeed, the Lipinski rule phenomena and GIT absorption data reflects a decent inhibition against MK2 protein molecules as similar to the internal ligand, CD21 (Figure 1E,F).

The homogeneous time-resolved fluorescence (HTRF) assay was performed to evaluate the effect of PF-3644022 and quercetin for inhibition of MK2 kinase. The PF-3644022 and quercetin were able to inhibit the MK2 enzyme activity dose dependently (Figure 1G). The maximum inhibition was seen at higher doses of PF-3644022 and quercetin.

The rat whole blood collected with EDTA was pretreated with different concentrations of compounds PF-3644022 and quercetin ranging from 10 μM to 1 nM and incubated for 15 min at 37, then further treated with 1 μg/mL of LPS and incubated for 6 h at 37 The plasma was collected to check the level of pro-inflammatory cytokines, TNF-α (Figure 1H), and interleukin (IL)-6 (Figure 1I). It was observed that the cytokine release was significantly upregulated in LPS treated group (p < .001) when compared to control and significantly downregulated in the groups pretreated with 10 μM, 1 μM, and 100 nM of PF-3644022 and quercetin in a dose-dependent manner compared to LPS treated group.

In addition, SH-SY5Y cells were pretreated with PF-3644022 and quercetin (10 μM–1 nM) for 1 h and then induced with LPS (1 μg/mL) for 24 h in cell incubator. The cell supernatants were collected and the level of TNF-α and IL-6 were evaluated. It was observed that the level of these cytokines was decreased in a dose-dependent manner in the groups pretreated with PF-3644022 and quercetin compared to the LPS treated group. The level of these cytokines release were significantly upregulated in LPS treated group (###p < .001) compared to control; while both were significantly downregulated in the groups pretreated with PF-3644022 and quercetin (***p < .001 and *p < .05, Figure 1J,K) in a dose-dependent fashion.

In our study, we used PF-3644022 as standard MK2 inhibitor and compared it with quercetin. Since quercetin is known for its excellent anti-inflammatory property [5]; therefore we evaluated MK2 binding affinity of both PF3644022 and quercetin through in silico and in vitro approach, and the anti-inflammatory effect in vitro by using SH-SY5Y cells and ex vivo by using rat whole blood. The results of docking clearly indicated that the binding affinity of quercetin (−8.1 kcal/mol) on MK2 is comparable to PF-3644022 (−8.4 kcal/mol). This data provided preliminary evidence that quercetin may alter inflammation via MK2 binding. Furthermore, PF-3644022 and quercetin inhibited MK2 kinase in HTRF assay and also showed a significant reduction in the release of LPS-induced pro-inflammatory cytokines dose dependently.

Based on the comparable binding affinity and enzyme inhibition data of quercetin and PF-3644022 for MK2 assay, it is concluded that quercetin may exert the anti-inflammatory activity in rat whole blood as well as neuronal cell line through MK2 inhibition. However, further research is needed to reveal the exact pathway of MK2 inhibition by quercetin.

Shruti S. Choubey: Conceptualization (equal); data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); writing—original draft (equal). Avtar S. Gautam: Data curation (equal); formal analysis (equal); methodology (equal); writing—original draft (equal). Lasure Vaibhav: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); writing—original draft (equal). Shikha Asthana: Data curation (equal); formal analysis (equal); visualization (equal). Anjuman Nanda: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); visualization (equal); writing—original draft (equal). Mangaldeep Dey: Data curation (equal); formal analysis (equal); investigation (equal); Resources (equal); visualization (equal). Rakesh K. Singh: Conceptualization (lead); project administration (lead); resources (lead); supervision (lead); visualization (lead); writing—review and editing (lead). All authors have read and approved the final manuscript.

The authors declare no conflict of interest.

All the procedures and protocols were approved by Institutional Animal Ethics Committee (IAEC) of NIPER-Raebareli, Lucknow, India, (protocol number NIPER/RBL/IAEC/65/August 2021) and used in the experiment as per the provisions laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India.