## 4 Discussion
We observed that the treatment with DL extract produced the most intensive staining, and this extract contained the highest amount of quercetin out of all four extracts, so it is possible that this flavonoid contributed to mucin upregulation. Our results are in line with the report that the Correlian cherry iridoid-polyphenol extract prevented the loss of the mucus layer during chemically induced colitis in rats [36]. Sea buckthorn water extracts administered in the concentration of 500 \(\mu\)g/mL improved the Caco-2 barrier functions by inhibiting the translocation of LPS added to the apical chamber of the insert to the basal chamber [37]. Similarly, various polyphenols, including resveratrol, rutin and quercetin, were shown to protect the epithelial layer's integrity in the Caco-2 or IPEC-J2 intestinal mucosa models challenged with mycotoxins or indomethacin [38; 39]. In these reports, the polyphenols restored the TEER values and barrier functions of the cell monolayer. We did not detect any significant changes in TEER values in the Caco-2/HT-29-MTX co-cultures, which reflected a good barrier integrity. However, in our experiments, no external challenge, such as a drug, chelator, toxin or LPS, was used to disrupt the epithelial barrier, so we could not evaluate any eventual protective activity of the tested extracts. Numerous polyphenols have been tested so far in the context of their protective role against NAFLD development or alleviation of NAFLD or NASH symptoms. [PERSON] and co-authors observed that kaempfer and kaempfer isolated from sea buckthorn alleviated oleic acid-induced lipid accumulation in HepG2 cells [40]. The fatty acid induced lipid droplet accumulation in primary hepatocytes or HepG2 cells was also decreased after the treatment with pure quercetin, resveratrol, myciretin, rutin, ferulic acid, caffeic acid, cinnamic acid and \(p\)-coumaric acid [21; 41; 42; 43; 44; 45; 46; 47; 48; 49]. Our data showing that the RL (rich in rutin), RO (rich in myricetin) and DO (rich in rutin and \(p\)-coumaric acid) extracts decreased lipid droplet size in HepG2 cells challenged with hyperglycaemic glucose concentration and palmitate confirms these results. Additionally, we analysed other conditions that were meant to model diabetes (hyperglycaemia), insulin resistance (hyperglycaemia with insulin) and high fat diets with insulin resistance (hyperglycaemia, insulin and palmitate)in our HepG2 experiments, as it is known that the aforementioned metabolic conditions contribute to NAFLD development. All the tested extracts decreased the lipid droplet size in at least two of these conditions (Table 2). All the extracts restricted overall intracellular lipid storage in diabetes and insulin resistance--mimicking those conditions (Figure 3b). In some cases, the extracts (e.g., RO extract in HG + Ins + Palm condition, DO extract in HG + Palm, Figure 3c) decreased the droplet size but increased their number. This may indicate stimulation of lipid droplet dynamics and their emulsification, which often leads to their easier breakdown, as the interface of lipid to cytosol is increased [50]. In terms of the protein level of the enzymes involved in lipid metabolism, it is hard to get a clear picture of generally increased or decreased levels of enzymes taking part in fatty acid synthesis or \(\beta\)-oxidation. This might be caused by the application of extracts that have a complex composition and the timeline of the changes induced by individual phenolic compounds interfering with each other, therefore we observe a dynamic resulting effect. It would be better to collect lysates from several time points and compare the results.