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Leveraging a Hypothesis-Generating Transcriptomics Approach to Elucidate Molecular Pathways that Contribute to the Biologic Effects of Quercetin in the Liver
Written By
Nhung Au and Brendan D. Stamper
Submitted: 07 September 2023Reviewed: 11 September 2023Published: 06 October 2023
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Quercetin is a relatively ubiquitous natural product with reported antioxidant, anti-inflammatory, antiviral, and anticarcinogenic properties. Using a bioinformatics approach, differential gene expression analysis was utilized to evaluate quercetin’s potential to protect and promote hepatocellular health through mining of the Gene Expression Omnibus (GEO) and subsequent analysis using the Database for Annotation and Visualization and Integrated Discovery (DAVID). The publicly available microarray datasets GSE4259 and GSE72081 were analyzed to compare the effect of quercetin on two different liver-based model systems to generate a robust set of differentially expressed genes impacted by quercetin exposure. Results from these analyses identified differentially expressed genes related to calcium signaling and signal transduction pathways to be the most significantly altered. A comprehensive literature review following the transcriptome analysis revealed that quercetin-induced gene expression changes in cell membrane receptors (specifically, voltage gated calcium channels NS integrins) share a common direct signaling pathway through extracellular signal-regulated kinase (ERK). Thus, the results from this bioinformatics study identified potential biomarkers related to quercetin’s effects on hepatocellular health. Based on quercetin’s ubiquitous use and good safety profile, future laboratory studies can be directed at validating the observed transcriptional changes on protein expression and the likelihood for hepatoprotection.
Pacific University School of Pharmacy, Hillsboro, OR, United States
Brendan D. Stamper*
Pacific University School of Pharmacy, Hillsboro, OR, United States
*Address all correspondence to: stamperb@pacificu.edu
1. Introduction
Natural products have been used throughout human history for various purposes due to the ability of their chemical constituents to exhibit various pharmacologic activities in humans, many of which have proven to be beneficial for treating and preventing a broad spectrum of diseases [1]. To this day, natural products continue to be an excellent reservoir for lead compound development in drug discovery research. For example, structure-based drug design with a novel nature-based structural skeleton has been used to improve the pharmacodynamic and pharmacokinetic characteristics of a natural product [2]. While there is little doubt, natural products represent a rich diversity of potential drug candidates, challenges such as a lack of well-designed candidate selecting strategies may hinder progress and limit expansion in the field. Despite these challenges, there is continued interest in natural products research from both the public and within the scientific community, which has led to the development of new technologies and approaches to increase the likelihood of a successful line of inquiry into the potential of a given natural product as a future drug candidate [3, 4].
Quercetin (3,3′,4′,5,7-pentahydroxy-flavone) is a polyphenolic compound derived from plant pigments and is a natural product found in many different plant species such as grapes, berries, cherries, apples, citrus fruit, kale, and black tea [5]. In plants, quercetin mainly presents as quercetin glycosides, and exhibits different therapeutic properties, which have been shown to have beneficial effects in treating various disease states with pathophysiologies related to oxidative stress, inflammation, and the immune system [6, 7]. While more work needs to be done to elucidate the precise mechanisms underlying how quercetin exerts its effects on cells, efforts must also be taken to make quercetin more “druggable”. From a pharmacokinetic perspective, quercetin struggles with low bioavailability and extensive metabolism [5]. Both enterohepatic and enteric recycling are thought to contribute to its poor systemic bioavailability, similar to many other quercetin-like polyphenolic structures [8]. Based on the fact that the liver has the potential to be exposed to greater quercetin concentrations compared to other systemic organs, quercetin may possess utility in treating hepatic ailments and promoting hepatocellular health.
Through investigations into how quercetin promotes hepatocellular health, researchers have identified a variety of hepatic signaling pathways that are altered following quercetin exposure, such as those associated with antioxidant defense, MAPKs (mitogen-activated protein kinases), inflammation, apoptosis, autophagy, insulin, AMPK (AMP-activated protein kinase), β-Catenin signaling, and antiviral activity [9]. For example, quercetin has been shown to elicit a dose-dependent protective effect against ethanol-induced oxidative stress in human hepatocytes [10]. This protection was found to occur through MAPK-mediated nuclear Nrf2 (nuclear factor erythroid 2-related factor 2) translocation and subsequent induction of heme oxygenase-1 (HO-1). Chronic inflammation also plays a role in various liver disorders such as non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma. Research suggests quercetin is capable of reducing liver inflammation and fibrosis through inhibition of macrophage infiltration and hepatic stellate cell activation [11]. In addition, quercetin’s ability to inhibit NF-kB (nuclear factor-kappa B) signaling may also be a contributing factor to its anti-inflammatory effects [12]. Quercetin’s significant effects on NF-kB signaling, MAPK pathways, and the antioxidant response system have buoyed its popularity as a promising and druggable natural product since these facotrs are associated with important cellular processes such as proliferation, differentiation, stress response, inflammation, and apoptosis.
Over the past thirty years, numerous studies have identified other pathways affected by quercetin exposure, many of which crosstalk with NF-kB, MAPK and Nrf2. In general, quercetin has been shown to modulate PI3K/Akt (phosphatidylinositol 3-kinase/protein kinase B) signaling, mTOR (mammalian target of rapamycin), and AMPK (AMP-activated protein kinase), which in turn influence cellular effects related to autophagy [13, 14], insulin resistance [15], lipophagy [16], and apoptosis [17]. Interestingly, quercetin may also have utility as a chemosensitizer [18]. This study found quercetin capable of reversing multidrug resistance in hepatocellular carcinoma through the FZD7 (Frizzled-7)/β-catenin signaling, a pathway involved in liver development and regeneration. Taken together, the growing body of literature covering quercetin-mediated effects on the liver has shown that quercetin alters numerous hepatic signaling pathways across a diverse range of model systems and over a wide variety of treatment conditions; thus, implicating a complex, multifaceted, and “dirty” mechanism by which quercetin is able to promote hepatocellular health.
In clinical trials, quercetin has shown promise in the treatment and protection of the liver from injury and damage in various disease states. In patients with beta-thalassemia, quercetin was coadministered with deferoxamine and demonstrated an ability to lower alanine aminotransferase (ALT) levels and positively affect liver function [19]. However, in a separate trial looking at patients with non-alcoholic fatty liver disease (NAFLD), quercetin showed no significant effect on hepatic function biomarkers and liver enzymes [20]. Quercetin’s potential for hepatoprotection has also been investigated for the treatment of chronic hepatitis C infection [21]. While no changes in ALT levels were observed in this study, a decrease in viral load for 8 of 30 patients taking quercetin was observed. It is also worth noting that across all of these studies, quercetin displayed a good safety profile [22]. Yet, while some of this clinical data shows promise and supplementary compelling evidence from animal studies is encouraging, progress has been slow and the need for more extensive clinical trials is needed to validate the potential positive effects of quercetin for patients with various liver conditions [23, 24].
One popular and common method for assessing how a compound might affect biology is transcriptome analysis. Microarray technology has existed for approximately forty years and has provided researchers and clinicians an opportunity to better understand transcriptomic changes at a global level [25]. Observed gene expression profile changes that can then be applied to curated biochemical pathways to posit the possible impact on human health and disease. With an ability to analyze the expression levels of thousands of different loci simultaneously, whole transcriptome microarrays offer an attractive high throughput method for researchers to assess a complete picture of gene expression in response to a variety of conditions, such as xenobiotic exposure and disease status. In the early 2000s, the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI) established the Gene Expression Omnibus (GEO) [26] and Array Express [27], respectively to house the plethora of functional genomics data that has been generated over the past half century. A positive component of these two repositories is that the data is publicly available and follows a standardized format for inclusion known as minimum information about a microarray experiment (MIAME) [28]. Free and unfettered access allows objective investigators, unaffiliated with the original researchers who deposited the data, to probe and analyze these datasets independently. In many cases, the original depositors of a microarray experiment are querying their data to ask specific questions. This allows the unaffiliated researcher to compare data against other deposited datasets to leverage multiple studies across multiple species utilizing multiple platforms to elucidate biochemical pathways, identify robust biomarkers, and cultivate potential drug targets. Despite quercetin’s relatively ubiquitous use as a natural product, there are surprisingly few microarray studies on its transcriptomic effects in the liver and liver-based models. The following work showcases how bioinformatic data repositories and platforms can be used as tools for hypothesis-generation in the laboratory by using quercetin-induced hepatocellular effects as an exemplar. Two microarray studies (one from Canada and the other from the Netherlands), which investigated the effects of quercetin on two different liver models, were leveraged to identify biomarkers and pathways in the liver that are impacted following quercetin exposure.
The Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) is an international public repository that archives and freely distributes microarray, next-generation sequencing, and other forms of high throughput functional genomics data. In this study, data from two separate studies investigating the effects of quercetin exposure were mined from GEO; specifically, GSE4259 [29], a study in C57BL/6 mice and GSE72081 [30], a study using primary mouse hepatocytes isolated from C57BL/6 mice. Raw gene expression data was downloaded from GEO and imported into Microsoft Excel for single gene analysis. Transcripts from quercetin-treated samples were compared against their respective controls to generate log2 fold changes, as well as non-log transformed fold changes and p-values (student T-test). The data was then filtered using a threshold (p < 0.05) to identify all transcripts with significantly altered expression. Transcripts from quercetin-treated samples demonstrating a significant change in expression compared to their respective controls were compiled and designated as differentially expressed gene sets.
2.2 Bioinformatic pathway analysis
Differentially expressed gene sets were then submitted for analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) platform. DAVID is a web-accessible tool that provides a comprehensive set of functional annotation tools for investigators to understand biological meaning behind large gene lists. In this study, DAVID was used to conduct pathway analysis in order to identify Kyoto Encyclopedia of Genes and Genomes pathways (KEGG; https://www.genome.jp/kegg/pathway.html) impacted by the GEO-identified differentially expressed gene sets. Only pathways that met a significance cutoff of (p-value <0.05) using DAVID were reported. Of note, individual genes identified across multiple pathways were grouped together and dubbed the core gene battery.
3. Hepatic transcriptome changes associated with quercetin exposure
3.1 Conceptualization of a core gene battery
Natural products serve as a great resource for identifying potential drug candidates, and while many possess attractive pharmacodynamic activities, many are also limited by their pharmacokinetics. Quercetin is no exception to this. Like a double-edged sword, quercetin has attractive pharmacodynamic properties such as antioxidant, anti-inflammatory, and immune-supportive properties; however, quercetin also has poor bioavailability due to extensive metabolism, enterohepatic recirculation, and enteric recycling. Despite the challenges associated with delivery and retention of quercetin in the human body, a growing body of literature continues to suggest that quercetin is capable of offering positive health effects, including hepatoprotection.
In this study, two independent microarray experiments were leveraged to investigate the effects of quercetin on two different liver models in hopes of identifying robust biomarkers and mechanisms associated with quercetin exposure. One study treated male C57BL/6 mice with 7 mg quercetin per mouse for 24 h (GSE4259) [29], whereas the other utilized primary mouse hepatocytes treated with 200uM quercetin for 24 h (GSE72081) [30]. Filtering the gene sets from these two studies based on significance (p < 0.05) resulted in the identification of over 1000 transcripts differentially regulated by quercetin treatment in each study (Figure 1). For the in vivo study, the 1244 differentially expressed genes were associated with 10 KEGG pathways, whereas 14 KEGG pathways were found to be enriched from the set of 1451 differentially expressed genes from the in vitro study (Table 1).
Figure 1.
Workflow for the identification of differentially expressed genes and enriched biologic pathways across two quercetin studies.
Arrhythmogenic right ventricular cardiomyopathy (ARVC)
Phosphatidylinositol signaling system
Primary bile acid biosynthesis
Amino sugar and nucleotide sugar metabolism
Calcium signaling pathway
Cardiac muscle contraction
NOD-like receptor signaling pathway
Vasopressin-regulated water reabsorption
Insulin signaling pathway
RNA degradation
Protein processing in endoplasmic reticulum
Type II diabetes mellitus
Retrograde endocannabinoid signaling
Lysine degradation
MAPK signaling pathway
Adipocytokine signaling pathway
Adherents junction
Prolactin signaling pathway
cAMP signaling pathway
TNF signaling pathway
Table 1.
Enriched pathways (p-value <0.05) associated with quercetin exposure from two independent studies.
Interestingly, pathway analysis using DAVID identified no overlapping pathways between the two studies (Table 1). In an attempt to uncover commonalities between the two quercetin studies, a unique approach of identifying genes found in multiple enriched KEGG pathways was employed. Thus, individual genes identified across multiple pathways were grouped together and dubbed the core gene battery (Table 2). Similar to the enriched pathways identified by DAVID, no genes within the core gene batteries were shared between the two quercetin studies. There are certainly limitations based on the experimental design of this bioinformatic study that could explain why identical transcripts were not found between the two independent studies. First, retrospective bioinformatic surveys like this are always limited by the data that is accessible. In this case, only two studies investigating the effects of quercetin on hepatic murine gene expression were available (one using C57BL/6 mice that was published in 2006 and the other using primary mouse hepatocytes from C57BL/6 mice that was published in 2016). This limited data posed two significant challenges. First, the two independent studies were separated by 10 years, and significant improvements in how gene expression is quantified were made during this time frame. Second, while the same mouse strain was used in both studies, one performed whole liver analysis, while the other assayed isolated primary hepatocytes. Differences in how the tissue was prepared for analysis can impact effects and lead to divergent results. However, despite the fact that there was no overlap in biochemical pathways or specific genes between the two studies, the identification of genes found in multiple enriched pathways through DAVID revealed that many of the genes within the core gene batteries of each study encoded for related isoforms (e.g., transforming growth factor-beta) or different subunits of the same protein (e.g., voltage-gated calcium channels) (Table 2).
Core gene batteries associated with quercetin exposure from two independent studies.
From an objective big-picture perspective, the results from this pilot bioinformatics study suggest that quercetin alters the transcription of membrane proteins related to calcium signaling and specific signal transduction pathways that appear to consistently funnel through the extracellular signal-regulated kinase (ERK) (Figure 2). The three membrane proteins and one cytokine that were identified from the comparative microarray study are voltage-gated calcium channels (VGCCs), sodium-calcium exchange protein (NCX; SLC8A1), integrins, and the cytokine, transforming growth factor beta 2 (TGFB2), whose general roles are all described in what follows.
Figure 2.
Signaling pathways that link membrane proteins, calcium signaling, and ERK with genes identified through DAVID analysis. Upregulated transcripts that are part of the core gene battery are boxed in green whereas downregulated transcripts that are part of the core gene battery are boxed in red.
3.2 Transcripts within the core gene battery and their convergence on ERK
Calcium signaling plays a crucial role in liver function and survival, and is regulated by changes in the intracellular concentration of calcium, which is highly organized by time and space. VGCCs play a major role in the regulation of intracellular calcium, which in turn indirectly activates mitogen-activated protein kinase (MAPK) signal transduction pathways (e.g., ERK 1/2) [31]. VGCCs are composed of multiple subunits (alpha1, alpha2delta, beta, and gamma), where ion conduction occurs through the alpha1 subunit, whereas other subunits play modulatory roles [32, 33]. Like VGCCs, NCX is also capable of regulating intracellular calcium concentration. While expressed mainly in the heart and brain, little is known regarding its role in the liver. Mechanistically, NCX has two working modes: (1) forward-mode, which pumps Ca2+ out of the cell in exchange for Na+, and (2) reverse-mode, which brings Ca2+ into the cell and pumps Na+ out in a concentration-dependent fashion. In reverse-mode, it has been proposed (through a stepwise process) that NCX plays a critical role in downstream ERK1/2 phosphorylation in endothelial cells [34]. It’s also worth noting that the inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), which is expressed in the endoplasmic reticulum and serves to release calcium to the mitochondria as part of senescence was identified as part of the downregulated core gene battery as well (Table 2) [35]. This further implicates the likelihood that quercetin exposure alters intracellular calcium signaling.
Two additional targets identified by GEO and DAVID as differentially expressed following quercetin treatment were TGFB and integrins, specifically, TGFB1, TGFB2, ITGA3, ITGA4, ITGA7, ITGA9, and ITGB5. While these targets have no direct effect on calcium flux in or out of the cell, they are both capable of initiating signals leading to ERK activation. For example, it is well-established that calcium signals mediate tyrosine kinase receptor signaling, which in turn activates ERK-mediated signaling; thus, providing a possible connection between TGFB and ERK [36, 37, 38]. And lastly, integrins, which are heterometric transmembrane cell adhesion proteins, are well-characterized regulators of cell growth, proliferation, migration, signaling, and cytokine activity. Integrins are formed by the pairing integrin alpha and beta subunits. There are 18 different α subunits which are capable of combining with eight different β subunits to form several unique integrin heterodimers [39]. Despite the complexities associated with integrin dimers, strong evidence exists supporting the fact that integrin-mediated signaling regulates ERK phosphorylation and subsequent ERK activity [40].
By altering the gene expression of membrane proteins, and potentially their composition and localization, quercetin has the potential to alter calcium signals and ERK-mediated signaling in the liver. Perhaps it was foreseeable that the three membrane proteins and the one cytokine that were highlighted herein from the comparative microarray study were all capable of modulating ERK activity since it has well established that growth factors and adhesion signals activate ERK. These effects align with quercetin’s purported common pharmacodynamic effects that have been reported in the literature related to cell proliferation, regeneration, and adhesion. A confounding factor that was challenging to address was the discovery of simultaneous up and downregulation of specific subunits of VGCCs and integrins. Making sense of how the inconsistent, yet significant, regulation of these subunits relates to a predictive cellular response is challenging because each specific combination of these variable subunits gives rise to a unique mode of activity.
In summary, the GEO- and DAVID-based results identified cell signaling pathways that may contribute to how quercetin affects hepatocellular health. These results highlight how bioinformatic approaches can be leveraged to generate meaningful and testable hypotheses in the laboratory. Next steps should involve validation through protein expression and activity studies related to calcium signaling. While the results from this study implicate membrane proteins related to calcium signaling and potentially ERK, additional efforts should be directed at investigating effects stemming from quercetin’s structure, such as its chemical ability to redox cycle and its reported ability to serve as both a direct and indirect antioxidant. This would include future cytoprotection studies to investigate quercetin’s ability to protect against known toxins capable of causing oxidative stress. While these cell-based assays are necessary for validating quercetin as a useful therapeutic agent for hepatoprotection, the preliminary bioinformatic results from this pilot study have provided intriguing targets and potential mechanisms related to quercetin’s future as an agent capable of hepatoprotection. Dissemination of hypothesis-generating results and the identities of these gene targets and mechanistic pathways, provides a record and a springboard on which the scientific community can build.
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Written By
Nhung Au and Brendan D. Stamper
Submitted: 07 September 2023Reviewed: 11 September 2023Published: 06 October 2023
Open access peer-reviewed chapter
