The Protective Role of Feruloylserotonin in LPS-Induced HaCaT Cells

Abstract

Epidermal inflammation is caused by various bacterial infectious diseases that impair the skin health. Feruloylserotonin (FS) belongs to the hydroxycinnamic acid amides of serotonin, which mainly exists in safflower seeds and has been proven to have anti-inflammatory and antioxidant activities. Human epidermis mainly comprises keratinocytes whose inflammation causes skin problems. This study investigated the protective effects of FS on the keratinocyte with lipopolysaccharides (LPS)-induced human HaCaT cells and elucidated its underlying mechanisms of action. The mechanism was investigated by analyzing cell viability, PGE₂ levels, cell apoptosis, nuclear factor erythroid 2-related factor 2 (Nrf2) translocation, and TLR4/NF-κB pathway. The anti-inflammatory effects of FS were assessed by inhibiting the inflammation via down-regulating the TLR4/NF-κB pathway. Additionally, FS promoted Nrf2 translocation to the nucleus, indicating that FS showed anti-oxidative activities. Furthermore, the antioxidative and anti-inflammatory effects of FS were found to benefit each other, but were independent. Thus, FS can be used as a component to manage epidermal inflammation due to its anti-inflammatory and anti-oxidative properties.

Keywords: HaCaT cell; anti-inflammation; anti-oxidation; epidermal inflammation; feruloylserotonin.

Figure 1

The effects of lipopolysaccharides (LPS) and feruloylserotonin (FS) on HaCaT cells. Cell viability was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). (a) HaCaT cells were treated with 0–2 µg/mL LPS for 12, 24, and 48 h. (b) COX-2 levels in 0–1 µg/mL LPS-induced HaCaT cells were detected with Western blotting. (c) The HaCaT cells were treated with 0–100 µM FS for 24 h. (d) The cell viabilities in only LPS group and FS-LPS group were compared under the treatment of 0 to 1 µg/mL LPS. (e) The released prostaglandin E2 (PGE₂) levels in only LPS group and FS-LPS group were measured with the prostaglandin E₂ assay kit. * p-value < 0.05.

Figure 2

Comparison between FS and COX-1/COX-2 inhibitors in HaCaT cells. (a) In order to compare the activity of FS on COX-1 and COX-2, 10 µM piroxicam, 10 µM celecoxib, and 10 µM FS were used to inhibit COX-1 and COX-2 respectively. (b) The effects of piroxicam, celecoxib, and FS on PGE₂ release were detected. (c) To compare the anti-oxidative activities of piroxicam, celecoxib, and FS, DCF-DA staining was used to detect the reactive oxygen species (ROS). A total of 5 µM NAC (N-acetyl-L-cysteine) was used to scavenge ROS as a positive control. * p-value < 0.05.

Figure 3

Inhibition of FS on the TLR4/NF-kB pathway. The expressions of proteins on TLR4/NF-κB pathway were detected by Western blotting. The main protein levels in the results of Western blotting were measured by Image J software and shown in the diagram. * p-value < 0.05.

Figure 4

The inhibition of FS on ROS and apoptosis. (a) The intracellular ROS was detected with DCF-DA staining. (b) To investigate the effect of FS on cell apoptosis, HaCaT cells stained with annexin V/propidium iodide were analyzed by flow cytometry. * p-value < 0.05.

Figure 5

The effects of FS on Nrf2 translocation and stability. (a) In order to confirm the translocation of Nrf2, the immunofluorescence staining was used to observe Nrf2 location via green fluorescence. 4‘,6-diamidino-2-2phenylindole (DAPI) was used to show the location of nucleus via blue fluorescence. (b) Nuclear Nrf2 (nuclear factor erythroid 2-related factor 2) levels in HaCaT cells were measured with Western blotting. (c) 50 µM cycloheximide (CHX) was used to block the synthesis of Nrf2 in HaCaT cells to evaluate the effect of FS on Nrf2 stability. * p-value < 0.05.

Figure 6

The action of FS under the condition of inflammation or oxidation. (a) Scavenging ROS with 5 μM NAC to avoid the effect of oxidation on TLR4/NF-κB pathway. The proteins expressions were measured with Western blotting. (b) TLR4-IN-C34 as the inhibitor of TLR4 was used to discard the influence of TLR4 pathway on Nrf2 translocation. The action of FS on Nrf2 translocation was investigated by Western blotting. * p-value < 0.05.