Anti-inflammatory effect of a pomegranate extract on LPS-stimulated HepG2 cells

Abstract Pomegranate is an important source of bioactive molecules with proven beneficial effects on human health. The aim of this study was to investigate the potential anti-inflammatory effect of a pomegranate extract (PE), obtained from the whole fruit and previously characterized by Reversed Phase-Ultra High-Pressure Liquid Chromatography-High Resolution Mass Spectrometry (RP-UHPLC–HRMS), on HepG2 human hepatocellular carcinoma cells challenged with the lipopolysaccharide (LPS). In LPS-treated cells (1 µg/ml, 24h), the PE treatment (administered at the non-cytotoxic dose of 1 µg/ml, 24h) induced a significant reduction of three key pro-inflammatory cytokines, i.e. interleukin-8 (IL-8), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), at both gene expression (as assayed by real-time PCR) and secretion levels (by Enzyme-linked Immunosorbent Assay, ELISA). Although further in vivo studies are needed to prove its efficacy, this preliminary in vitro study suggests that the PE might be useful for ameliorating liver inflammation. Graphical Abstract


Introduction
The pomegranate (Punica granatum L.) is a fruit tree adaptable to a wide range of agro-climatic conditions; it originates from Iran, but it is currently cultivated in many different regions of the world, including Italy (Shaygannia et al. 2016).
The production and consumption of the pomegranate fruit have significantly intensified over times due to the increasing awareness of people about its promising health benefit-related properties, that suggested its definition as novel 'superfood' (Johanningsmeier and Harris 2011;Caruso et al. 2020).These functions are not only limited to the fruit edible part (the arils), but also recently extended to the inedible parts (mainly peel) which have been proven to contain different biologically active compounds (Kandylis and Kokkinomagoulos 2020).
The major class of phytochemicals contained in the fruit of pomegranate is represented by the polyphenols, including flavonoids, condensed tannins, flavonols, anthocyanins, phenolic and organic acids, and hydrolysable tannins (Melgarejo-Sánchez et al. 2021).Pomegranate also contains fatty acids, mainly the polyunsaturated component (like the punicic acid), that has been gaining growing interest for its wide array of biological properties including antidiabetic, antiobesity, antiproliferative, and anticarcinogenic activity against various forms of cancer (Aruna et al. 2016;Bedel et al. 2017;Shabbir et al. 2017).Increasing experimental in vitro and in vivo data support the promising application of pomegranate extracts, and/or its polyphenolic and fatty acid bioactive compounds, as source of anti-inflammatory, antioxidant, anti-microbial and anticancer bioactives, although they are mostly based on seeds and fresh fruit juice.Data investigating the effects of whole fruit extract are still poor.
In the context of possible applications of pomegranate extracts and/or its bioactive compounds, the impact on liver toxicology has been only preliminarily investigated (Choudhury et al. 2016;Makled et al. 2016;Noori et al. 2017).Liver is one of the targets of pathogenic microorganisms and bacterial byproducts coming from the intestine through the portal vein.A typical bacterial product is the LPS (Guerville and Boudry 2016), an endotoxin composing the wall of Gram-negative bacteria.The human intestinal epithelium is the first line of defense against bacterial infection.Impaired intestinal integrity can contribute to the rise of circulating blood endotoxin and lead to a low-grade endotoxemia.Low-grade endotoxemia and inflammation are associated with an increased risk of chronic degenerative diseases (Ghosh et al. 2020).Through the portal vein, the LPS reaches the liver and, after binding to the Toll-like receptor 4 (TLR4), activates the transcription of key inflammatory genes, such as IL-1, interleukin-6 (IL-6), IL-8, TNF-α, leading to hepatic inflammation (Kanmani and Kim 2018).
As some experimental evidence reports the anti-inflammatory effect of pomegranate in human chondrocytes and intestine cells (Lansky and Newman 2007;Shukla et al. 2008;Larrosa et al. 2010;Mastrogiovanni et al. 2019), we here aimed at investigating whether a pomegranate extract, obtained from the whole fruit, might efficiently inhibit the expression and release of IL-1β, IL-8 and TNF-α on LPS-challenged HepG2 cells.

Results and discussion
The pomegranate whole fruit dry extract used in this in vitro experimental study (Punicaplus®, from Esserre Pharma srl, Rome, Italy) was previously analyzed and characterized by RP-UHPLC-HRMS, revealing that the main PE compounds belong to the classes of ellagitannins (mainly punicalin), gallotannins, phenolic acids, and flavonol glycosides (Esposito et al. 2022).
In the present study, HepG2 cells viability was first evaluated in response to a wide dose-range of PE by the Cell Counting Kit-8 (CCK-8), to identify the non-cytotoxic concentrations of the extract suitable for further evaluation of the PE anti-inflammatory property.Cells were treated with PE (from 1 to 1000 µg/ml) for 24, 48 and 72 h; as reported in Figure 1A-C, HepG2 cells undergo a dose and time-dependent decrease in cell viability percentage when treated with PE concentrations higher than 10 µg/ml (still inducing an about 25% cell viability reduction at 72 h of continuous PE treatment).The dose of 1 µg/ml was then selected for subsequent analyses, to ensure cell viability was not affected by the PE extract even over a longer treatment time window, and to prevent it might negatively impact on the anti-inflammatory response.Pomegranate extracts have been indeed reported to inhibit proliferation and decrease cancer cells viability at doses as low as 5 µg/ml in different cancer types including breast, prostate, lung, colon, skin, blood cancers, as well as HepG2 cells in vitro, due to its bioactive compounds punicalagin and ellagic acid (Seeram et al. 2005;Dai et al. 2010;Li et al. 2019), that are also abundantly represented in our PE (Esposito et al. 2022).
An inflammatory response in HepG2 cells was then induced by using LPS stimulation.Three inflammatory human cytokines, i.e.IL-1β, IL-8 and TNF-α, were selected to evaluate the anti-inflammatory properties of the PE on LPS-induced HepG2 cells.
To this aim, HepG2 cells underwent the following schedule of administration: 1 µg/ml LPS (for 24h) followed by treatment with PE (1 µg/ml) for further 24h.As shown in Figure 2, the LPS treatment triggers the stimulation of IL-1β, IL-8 and TNF-α expression at mRNA level in HepG2 cells, and such increase is significantly recovered by PE administration.In accordance, the levels of secreted cytokines, which represent the biologically active forms, are significantly reduced when cells are treated with the PE (Figure 3A).The ability of the pomegranate extract to inhibit   the pro-inflammatory cytokines has been compared to Diclofenac, a well-known non-steroidal anti-inflammatory drug.Diclofenac cytotoxicity was preliminarily assayed in HepG2 cells undergoing exposure to the drug for 24 h at the 0.5-500 µg/ ml concentrations, selected according to literature in vitro data (Wang et al. 2002;Duval et al. 2019).As reported in Figure 3B, the drug induces a dose-dependent decline in cell viability; the non-cytotoxic diclofenac concentration of 1 µg/ml was then selected for verifying the anti-inflammatory activity in LPS-challenged cells, in terms of secreted cytokines levels.Figure 3A demonstrates that diclofenac impairs the LPS-induced secretion of IL-1β, IL-8 and TNF-α by about 50% if compared to single LPS challenge.Interestingly, we report that our PE (used at the same Diclofenac concentration) exerts a comparable anti-inflammatory activity in terms of IL-8 and TNF-α reduction, whereas it appears slightly less effective (p = 0.03) then diclofenac positive control in inhibiting the IL-1β secretion.This finding is consistent with previous data (Mastrogiovanni et al. 2019) reporting a pomegranate extract is able to affect cytokine expression levels at different extent, according to the specific gene analysed.
Several studies demonstrate that LPS promotes hepatic transcription of pro-inflammatory cytokines genes by activating the TLR4/NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway (Zhou et al. 2016;Liu et al. 2017;Zarate et al. 2020;Seo et al. 2022).NF-kB is the transcription factor that, in response to the TLR4 activation cascade, translocates to the nucleus and activates the pro-inflammatory genes.We hypothesized that the pomegranate extract, selected for this study, might suppress/interfere with the TLR4-mediated NF-kB signaling pathway that leads to down-stream cytokines genes transcription; further in vitro experiments are necessary to specifically address this molecular hypothesis.We assumed that the punicalagin, the most abundant compound in our extract, may be the active biomolecule responsible for the anti-inflammatory effect of P. granatum, as suggested by previous experimental studies (Du et al. 2019;Xu et al. 2021).Additionally, gallotannins might be involved in such effect, as they have been proven to play an anti-inflammatory role in A549 adenocarcinomic human alveolar basal epithelial cells, via suppression of most cytokines and chemokines expression through inhibition of NF-κB nuclear translocation (Erdèlyi et al. 2005).Interestingly, in LPS-treated macrophage RAW 264.7, both gallotannins and flavonol glycosides display an anti-inflammatory effect through inhibition of the NF-κB signaling pathway (Hyemee et al. 2022;Yang and He 2022), thus extending the panel of potential PE-derived bioactive molecules to be addressed in future studies for identification of the anti-inflammatory specific regulator(s) in our experimental model.

Conclusions
Our findings demonstrate that the pomegranate extract exerts an anti-inflammatory effect on LPS-challenged human HepG2 hepatocarcinoma cells in vitro, and pave the way for further in vitro and in vivo studies that might validate the possible application of such extract to target hepatic inflammation.

Figure 2 .
Figure2.evaluation of Il-1β, Il-8 and tNF-α gene expression carried out after 24 h of lPs challenge (1 µg/ml) followed by 24 h of treatment with the pomegranate extract (1 µg/ml).data are presented as Mean ± sd in N = 3 independent experiments.*p < 0.05, calculated in lPs-treated cells compared to untreated ones; # p < 0.05, calculated in lPs-treated cells compared to pomegranate + lPs-treated ones.