Modulation of Endoplasmic Reticulum Stress by Selected Polyphenols from Sambucus ebulus Fruit

1. Introduction

The endoplasmic reticulum is an organelle characteristic only of eukaryotic cells, that performs various functions, including protein synthesis, post-translational protein modification, participation in lipid metabolism, calcium ion storage, and glycogen production. [1] The lumen of this organelle contains chaperones and enzymes necessary for the proper folding of newly synthesized secretory and membrane proteins, such as PDI (protein disulfide isomerase), GRP78/BiP (glucose-regulated protein 78/binding immunoglobulin protein), GRP94 (glucose-regulated protein 94), etc. [2] Endoplasmic reticulum (ER) stress occurs when misfolded or unfolded proteins accumulate within the ER lumen. It can be caused by a variety of factors, including increased demand for protein synthesis, genetic mutations, oxidative stress, and more. [3] There are special signaling pathways in cells, known as the unfolded protein response (UPR), whose purpose is to rid the cell of ER stress and restore homeostasis. This can be achieved through decreased protein synthesis, increased production of folding-facilitating molecules, or enhanced degradation of misfolded proteins. However, if homeostasis cannot be restored, the UPR may ultimately trigger apoptosis (Figure 1). [4] Chronic inflammation can both induce and be a result of ER stress. Activation of the PERK and IRE1 pathways leads to stimulation of inflammatory signaling. [5,6].

ER stress is involved in the pathogenesis of various diseases, such as cancers, metabolic, and neurodegenerative disorders. The regulation of the adaptive response is an important mechanism guaranteeing the survival and growth of tumor cells, as they grow in an unfavorable environment, such as hypoxia, nutrient deficiencies, and increased demand for protein synthesis. [7] These stressors place a significant burden on the endoplasmic reticulum. For example, in conditions of chronic stress, many tumors upregulate the chaperone GRP78, which promotes cell proliferation, metastasis, and drug resistance. [8,9] The transcription factor XBP1s has been reported to drive the pathogenesis of multiple myeloma. [10] Since the activation of the IRE1-XBP1 pathway is related to the expression of genes involved in endoplasmic reticulum-associated degradation (ERAD), which helps eliminate misfolded proteins, Hepatitis C virus successfully suppresses this pathway, thereby stimulating viral protein synthesis and inducing cellular stress. [11] The accumulation of the large surface protein of Hepatitis B virus also leads to ER stress. [12] Ethyl alcohol has also been shown to induce CHOP expression and trigger hepatocyte apoptosis in the murine model of intragastric ethanol feeding. [13] The role of ER stress in the development of non-alcoholic fatty liver disease (NAFLD) has been demonstrated in an animal model of diet-induced obesity in mice, where overexpression of GRP78 in the liver of mice suppressed insulin signaling and ER stress-dependent activation of the transcription factor SREBP-1c, a key regulator of hepatic lipogenesis. [14] ER stress also contributes to the development of type 2 diabetes and obesity by impairing insulin signaling. This occurs through JNK activation, which phosphorylates IRS1 (insulin receptor substrate 1), thereby preventing proper hormone action. [15] In the context of cardiovascular disease, Tsukano et al. reported that CHOP-mediated apoptosis of foam cells in the vessels of the heart contributes to the rupture of atherosclerotic plaques and the onset of acute coronary syndrome. [16] There is a link between ER stress and some neurological diseases as well. Mutations in the gene encoding presenilin-1 (PS1), associated with familial Alzheimer’s disease, increase cellular sensitivity to ER stress by disrupting the UPR. [17] ER stress increases the expression of the gene encoding parkin, an E3 ubiquitin ligase, involved in the degradation of proteins via ubiquitin-proteasome system. Loss of parkin function removes its neuroprotective effect and makes them susceptible to cellular stress. Mutations in this gene are known to cause juvenile Parkinson’s disease. [18] ER stress has also been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). [19].

Polyphenols are found in a wide range of fruits, vegetables, drinks, and cereals. They provide protection from ultraviolet radiation and pathogens, and partly determine flavor profiles. Their potential health benefits have been extensively studied in recent decades. Polyphenols are categorized into four main classes: phenolic acids, flavonoids, stilbenes, and lignans. Flavonoids are further divided into six subclasses based on their specific structural characteristics: flavones, flavanones, flavonols, flavanols, anthocyanins, and isoflavones. [20]

Sambucus ebulus, also known as dwarf elder, is present in the folk medicine of many countries in Europe and the Middle East. Тhe fruits are the most commonly utilized part of the plant, as they contain the highest concentration of polyphenolic compounds [21,22] and have traditionally been used in the form of marmalade for the treatment of tuberculosis and haemorrhoids, as well as in syrup form for appetite-stimulating and tonic action. [23] Our research group recently analyzed the phytochemical composition and biological activity of an aqueous-alcoholic extract of Sambucus ebulus fruits. The results of the chemical analysis revealed a high content of the following compounds: neochlorogenic acid, chlorogenic acid, cyanidin-3-O-galactoside or idaein, epicatechin, resveratrol, cyanidin-3-O-glucoside or chrysanthemin. Using the J774A.1 macrophage cell line, the study demonstrated significant anti-inflammatory properties of the extract, accompanied by a reduction in key endoplasmic reticulum stress markers, including peIF2α, ATF6, and CHOP, under conditions of LPS-induced inflammatory activation. The reported decrease in the expression of these proteins suggests that modulation of ER stress is a likely additional mechanism by which the extract exerts its anti-inflammatory effects. [24] However, the specific phytochemical components responsible for the modulation of ER stress were not identified in the cited study. The present review aims to summarize the effects of the most prevalent polyphenolic compounds identified in the extract on mechanisms related to endoplasmic reticulum stress, based on available data from scientific literature. Thus, the potential causal relationship between the polyphenols contained in the extract and the observed biological effects can be elucidated.

2. Materials and Methods

2.1. Search Strategy

We applied the ’’Advanced search’’ option in two databases, Pubmed and ScienceDirect, where we used the following keywords and Boolean operators: ’’Neochlorogenic acid’’ and ’’ER stress’’, ’’Chlorogenic acid’’ and ’’ER stress’’, ’’Resveratrol’’ and ’’ER stress’’, “Cyanidin-3-o-glucoside” OR “Chrysanthemin” and “ER stress”, “Cyanidin-3-o-galactoside” OR “Idaein” and “ER stress”, ’’Epicatechin’’ and ’’ER stress’’. We refined the results by adding supplementary filters to ensure relevance and quality:

• Text availability – free full texts
• Publication date – from 2000/1/1 to 2025/5/1
• Article type – research articles
• Subject area – Biochemistry, Genetics and Molecular Biology
• Access type – open access and open archive

2.2. Inclusion and Exclusion Criteria

The review included in vivo or in vitro studies that focused on the effects of the individual polyphenolic compounds (resveratrol, cyanidin-3-o-glucoside, cyanidin-3-o-galactoside, epicatechin, neochlorogenic acid, and chlorogenic acid) on ER stress. Full text articles discussing markers and pathways, affected by these phytochemicals, and providing sufficient experimental details (e.g., dosage, model system) for evaluation were also defined as inclusion criteria.

Studies were excluded if they examine plant extracts or analogues of the specific isolated compounds. Articles that didn’t evaluate ER stress key markers or molecular pathways were not considered. Records limited to anti-inflammatory, metabolic, antioxidant or other biological effects of ingredients, without any relation to ER stress, were also excluded.

2.3. Statistical Analyses and Presentation

MS Excel 2016 was used for statistical processing and graphical presentation of the collected data. BioRender was used to build a schematic for the ER stress signaling pathways.

3. Results

3.1. Study Selection

We were able to find 100 records from the PubMed and ScienceDirect databases. The study selection process followed the PRISMA flowchart (Figure 2). First, twenty duplicate records were removed. After screening the remaining 80 articles, 20 titles or abstracts didn’t meet the inclusion and exclusion criteria and were therefore excluded. Sixty full-text articles were assessed for eligibility. Studies discussing biological properties of plant extracts, containing the listed phytochemicals or their analogues, weren’t considered. Ultimately, 42 articles underwent literary analysis.

3.2. Presentation of the Results

Available experimental evidence on the effects of the selected compounds on UPR signaling was found for only 4 of the 6 phytochemicals listed in the inclusion criteria – resveratrol, chlorogenic acid, epicatechin, and cyanidin-3-o-glucoside.

3.2.1. Modulation of ER Stress by Resveratrol

The influence of resveratrol on endoplasmic reticulum stress has been widely investigated by multiple groups. Analysis of the studies revealed that resveratrol modulates the ER stress in different ways depending on the dose and the type of experimental model used, as presented in Table 1. Under conditions of induced ER stress, and when administered at low doses, resveratrol suppresses UPR signaling and reduces the expression of key markers such as GRP78, GRP94, CHOP, p-PERK, IRE1, p-eIF2α and ATF6, thus exhibiting a protective effect. [25,26,27,28,29,30,31,32,33,34,35] Conversely, at higher doses – particularly in tumor cell lines – resveratrol enhances ER stress and apoptosis, which is an indication of its cytotoxic action. [31,36,37,38,39,40] The data show that this polyphenolic compound primarily affects the PERK and IRE1 signaling cascades and modulates the expression of proteins and chaperones involved in the proper folding of newly synthesized polypeptide chains. [27,35,36,37,41,42,43,44,45]

3.2.2. Modulation of ERS by Cyanidin-3-o-Glucoside, Chlorogenic Acid and Epicatechin

According to literature, in experimental models of induced ER stress cyanidin-3-o-glycoside decreases the expression of GRP78, p-PERK, p-eIF2α, ATF4, CHOP, IRE1, XBP1, and ATF6, which indicates its potential to suppress UPR signaling. A similar effect is observed for chlorogenic acid, but its impact on the IRE1 pathway is less characterized. Although data on epicatechin are scarce, it also has the ability to regulate protein homeostasis by acting on IRE1 and PERK signaling pathways. These polyphenols have a protective effect at low doses, similar to the effects of resveratrol.

4. Discussion

Our study investigates and summarizes the known effects of specific polyphenols on endoplasmic reticulum (ER) stress, a process implicated in the development of a wide range of diseases, as mentioned above. This research builds upon our previous work, in which we observed the ER stress-modulating activity of an aqueous extract from the Sambucus ebulus fruit. However, information on the main bioactive components responsible for this effect is still lacking. [24] Future experimental work would enrich our understanding of the mechanisms underlying the biological activity of dwarf elderberry fruits.

In general, the four examined polyphenols act on one or more branches of the UPR signaling. While some studies do not specify the exact branch affected, altered expression levels of certain molecules suggest the pathway involved. Considering Table 2 and Table 3, the polyphenols most frequently influence the PERK signaling cascade, along with a reduction of CHOP, a critical mediator of ER stress-induced apoptosis. Since all four polyphenols affect PERK and IRE1, this may explain the observed anti-inflammatory properties of the dwarf elderberry extract, as these pathways can directly activate inflammatory cascades. [6] Data from two independent studies suggest that epicatechin does not significantly modulate the ATF6 signaling pathway. However, there are some key methodological differences – one study used antibodies against cleaved ATF6 as part of the protocol, while the other did not include such antibodies. [52,53] Thus, the influence of this polyphenol on the ATF6 branch of the UPR is still unexplored, highlighting the need for furhter investigation. Experimental studies are also needed to address the current lack of data on the effects of neochlorogenic acid and idaein on ER stress.

The precise molecular mechanisms by which these phenolic compounds exert their effects on ER stress are not fully understood. It is known, for instance, that the activation of sirtuin 1 (SIRT1) is key to resveratrol’s activity. SIRT1 is an NAD+-dependent protein deacetylase that modulates the activity of molecules involved in the UPR pathway. [25,30,31,47] A study by Luo et al. (2023) clearly highlighted the role of SIRT1 as a mediator of resveratrol’s effects, as inhibition of this enzyme abolished the compound’s protective action against bupivacaine-induced neurotoxicity in PC12 cells. [35] SIRT1 stimulation has also been reported for cyanidin-3-O-glucoside. [63] Resveratrol’s protective effect under induced endoplasmic reticulum (ER) stress may also be mediated by clusterin, a molecular chaperone induced by stress, аlthough its connection to SIRT1 remains unclear. [30] Moreover, a mechanism independent of SIRT1 has been reported by Graham et al., who suggest that resveratrol stimulates the enzyme protein phosphatase 1α (PP1α), which in turn inhibits protein kinase B (Akt), a modulator of protein synthesis and endoplasmic reticulum stress. [45] Furthermore, in prostate cancer cell lines, resveratrol has also been shown to deplete calcium stores in the ER and suppress the store-operated Ca²⁺ entry (SOCE) mechanism, ultimately leading to ER stress and cell death. [39] Further research should focus on elucidating the mechanisms by which phytochemicals modulate the expression of key markers of UPR signaling, especially in the case of epicatechin and chlorogenic acid.

The effects of polyphenols on endoplasmic reticulum stress in various in vivo and in vitro models suggest that these substances could be used as therapeutic agents. Consequently, plant sources containing these compounds may also be of interest as functional foods. For example, resveratrol, a stilbene, is found in grapes, red wine, berries, and peanuts, while chrysanthemin, an antocyanin, is present in berries, black beans, and black rice. [64,65,66,67] Epicatechin is abundant in green tea. [68] Coffee, apples, berries, and potatoes are sources of chlorogenic acid, a type of phenolic acid. [69] Models of induced ER stress clearly show that, when administered at low doses, chlorogenic acid, chrysanthemin, and resveratrol exert a protective effect by dose-dependently downregulating the expression of molecules associated with UPR signaling or apoptosis. In a pathological context, this protective function may slow disease progression and alleviate symptoms. As shown in Table 1, high doses of resveratrol, on the other hand, induce endoplasmic reticulum stress and activate apoptotic mechanisms, especially in tumor cell lines, which may be therapeutically advantageous. Thus, resveratrol exerts a complex dual effect on ER stress. Some studies further show that combining resveratrol with other chemicals stimulates ER stress in tumor cells, even at lower concentrations. [41,45,49] A study by Wang et al. (2011) reported a paradoxical finding: in multiple myeloma cell lines resveratrol activated the unfolded protein response (UPR) via the IRE1 signaling cascade, promoting XBP1 splicing, while simultaneously suppressing the activity of XBP1s as a transcriptional factor through SIRT1. This suggests that resveratrol may stimulate stress while hindering the cell’s ability to adapt. [37] In another study, low doses of resveratrol were shown to reduce the levels of p-PERK and ATF4, but activated the ATF6 pathway. This suggests that apoptosis-promoting pathways are supressed, while activation of ATF6 may play a protective role, as it is responsible for the production of chaperones and ERAD factors. At high doses, the effect was reversed. [31]

The interpretation of available data has several limitations:

• insufficient information on the effects of some polyphenols across all UPR branches.
• heterogeneity in experimental models limits direct comparisons and generalization.
• dose variation and dual effect of resveratrol on ER stress – determining the precise therapeutic dose and anticipating potential side effects is challenging.
• lack of clinical trials and data on long-term effects of polyphenol intake – it is difficult to determine the therapeutic potential of polyphenols, as well as to assess the long-term safety and efficacy of these compounds.
• although the results highlight the significant contribution of the polyphenols found at the highest concentrations in the aqueous dwarf elder extract, the possible biological activity of other present phytochemicals in lower amounts cannot be excluded.

Other future research should focus on pro-apoptotic effects of other polyphenols, given that high dose resveratrol has been found to have the same effect in cancer cell lines due to the malfunction of ER. More studies investigating tissue-specific and dose-dependent effects of individual polyphenols or combinations in different disease models are essential. Clinical trials and long-term safety studies are also needed to determine their potential as therapeutic agents.

5. Conclusions

The review identifies a possible link between the most represented polyphenols in the dwarf elder fruit aqueous extract and the modulation of ER stress. Among these, only four of the most abundant polyphenols – resveratrol, chrysanthemin, chlorogenic acid, and epicatechin – have been found to influence UPR pathways, with a predominant effect on the PERK/eIF2α/ATF4/CHOP pathway. The data indicate that these phytochemicals reduce ER stress markers in a dose-dependent manner across various in vivo and in vitro experimental models. Notably, resveratrol is the only compound among them that can amplify ER stress and provoke programmed cell death at high doses. This dual role underscores the potential of dwarf elderberry extract as a remedy for conditions associated with chronic cellular stress. Nevertheless, further research is essential to elucidate the molecular mechanisms by which phytochemicals act, determine their optimal dosages, identify potential side effects, and explore their synergistic interactions with other phytochemicals and drugs.