Anti-inflammatory and antioxidant properties of Camellia sinensis L. extract as a potential therapeutic for atopic dermatitis through NF-κB pathway inhibition

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by immune dysregulation and excessive cytokine production. This study aimed to explore the potential of Camellia sinensis L. water extract (CSE) as a treatment for AD by the impact of CSE on inflammatory responses in keratinocytes, particularly concerning the production of inflammatory cytokines and the modulation of signaling pathways relevant to AD pathogenesis. CSE was obtained via hot water extraction from Camellia sinensis L. Ultra-high-performance liquid chromatography (UPLC) analyzed catechin and caffeine content. Cell viability was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), polyphenol and flavonoid content were determined. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay measured antioxidant activity. Enzyme-Linked Immunosorbent Assay (ELISA), western blotting, and Immunofluorescence (IF) assays examined cytokines, pathways, and protein localization, respectively. Molecular docking assessed compound binding with inflammation-related proteins. UPLC identified six CSE components including epigallocatechin (EGC) epicatechin (EC), caffeine (CF), catechin (C), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). CSE demonstrated a significant reduction in the production of inflammatory cytokines interleukin (IL)-2 and IL-6 in TNF-α/IFN-γ activated keratinocytes. Treatment with CSE inhibited the mitogen-activated protein kinase (MAPK) pathway, which resulted in decreased phosphorylation of p38, Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Exposure of TNF-α/IFN-γ- stimulated human keratinocytes (HaCaT) cells to CSE resulted in a 200 µg/mL dependent inhibition of p65 and signal transducer and activator of transcription 1 (STAT-1) translocation from the cytosol to the nucleus, as confirmed through immunofluorescence (IF) staining. Molecular docking simulations provided insights into the underlying mechanisms of CSE action, which supported its potential as a therapeutic agent for AD. CSE might be a potential candidate for its therapeutic efficacy for inflammatory skin conditions like AD. Thus, based on this evidence, the authors suggest that CSE should be studied further for its anti-inflammatory activities and topical application in the treatment of AD.

Keywords: Camellia sinensis L.; Anti-inflammation; Antioxidant; Atopic dermatitis; Ultra performance liquid chromatography.

Fig. 1

Effect of CSE on HaCaT keratinocyte viability. (A) MTT assay and (B) WST-8 assay results showing the effect of CSE on HaCaT cell viability. HaCaT cells were treated with varying concentrations of CSE (0–300 µg/mL) for 24 h. CSE did not substantially reduce HaCaT keratinocytes by 200 µg/mL. Dexa:10 µg/mL. The results obtained from three independent experiments were expressed as means ± standard deviation (SD), compared with the control group. *p < 0.05 vs. control group.

Fig. 2

Dose-dependent suppression of TNF-α/IFN-γ-stimulated proinflammatory release by CSE. HaCaT cells were pretreated with TNF-α/IFN-γ (each 10 ng/mL) for 1 h and then stimulated with CSE for 24 h. Release concentration of (A) IL-6 and (B) IL-2 were measured by ELISA. Results are presented as mean ± standard error of the mean (SEM) of three independent experiments. ###p < 0.001 vs. TNF-α/IFN-γ-stimulated group; ***p < 0.001 vs. control group. Dexa: dexamethasone (positive control).

Fig. 3

Inhibition of TNF-α/IFN-γ-stimulated iNOS and COX-2 expression in HaCaT cells by CSE. HaCaT cells were incubated with after a 1h pretreatment with TNF-α/IFN-γ (10 ng/mL each) and then stimulated with CSE for 24 h. (A) A typical Western blot showing COX-2 and iNOS expression under the indicated treatment conditions. The expression β-actin was measured as the gel loading control. (B) Bar graphs show the densities of the COX-2 band and iNOS band normalized to β-actin band density. Results are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. TNF-α/IFN-γ-stimulated group; ##p < 0.01 vs. control group. Dexa: dexamethasone (positive control).

Fig. 4

Inhibition of TNF-α/IFN-γ-stimulated NF-κB and STAT-1 signaling in HaCaT cells by CSE. (A) Typical Western blots showing the time courses of NF-κB and STAT-1 phosphorylation induced by TNF-α/IFN-γ. (B) Treatment with TNF-α/IFN-γ induced a transient rise in p-p65, and p-STAT-1 expression levels peaked at around 30 min. These responses were suppressed dose-dependently by 1 h of CSE pretreatment. (C) Bar graphs of p-p65 and p-STAT-1 band densities relative to β-actin band density (the gel loading control). Treatment with TNF-α/IFN-γ for 30 min induced transient increases in p-p65 and p-STAT-1 expression levels that peaked. These increases in phosphorylation were dose-dependently reversed by CSE pretreatment. All values are presented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. TNF-α/IFN-γ-stimulated group; ###p < 0.001 vs. control group. Dexa: dexamethasone.

Fig. 5

Inhibition of MAPKs phosphorylation/activation by CSE in TNF-α/IFN-γ-stimulated HaCaT cells. (A) Typical Western blots showing the time course of MAPKs phosphorylation (p-JNK, p-ERK, and p-p38) in HaCaT cells during stimulation with TNF-α/IFN-γ (10 ng/mL each) alone and following pretreatment with CSE. (B) Treatment with TNF-α/IFN-γ induced a transient rise in p-JNK, p-ERK, and p-p38 expression levels that peaked at around 30 min. These responses were suppressed dose-dependently by 1 h of CSE pretreatment. (C) Bar graphs of p-JNK, p-ERK and p-p38 band densities relative to total form band density (the gel loading control). All values are the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. TNF-α/IFN-γ-stimulated group; #p < 0.05 vs. control group. Dexa: dexamethasone (positive control).

Fig. 6

Inhibition of TNF-α/IFN-γ-stimulated p-p65 and p-STAT-1 nuclear translocation by CSE. (A) Immunofluorescence images of HaCaT keratinocyte cells stained with an antibody against p-p65 (red) and counterstained with DAPI (blue). (B) Immunofluorescence images of HaCaT cells stained with an antibody against p-STAT-1 (green) and counterstained with DAPI (blue). Treatment with TNF-α/IFN-γ for 30 min enhanced nuclear translocation as evidenced by increased dual staining with DAPI in merged images. Translocation of both factors was blocked by 1 h preincubation with 200 µg/mL CSE. After the indicated treatments, cells were fixed in 4% formaldehyde, stained with the indicated primary antibody, and counterstained with DAPI. Merged images indicate colocalization. scale bar = 10 μm. Dexa: dexamethasone (positive control).

Fig. 7

Concentration-dependent DPPH and ABTS free radical remaining by CSE. Results are the mean ± standard deviation (SD) of three independent experiments. *** p < 0.001 vs. the CSE control group. Ascorbic acid (AA): 100 µg/mL ascorbic acid (positive control).

Fig. 8

Chromatograms identifying five catechins and caffeine in CSE powder based on retention time matching with known standards. Eluents were monitored at 240 nm. Shown are typical UPLC chromatograms of (A) standard compounds and (B) a CSE sample. (C) Molecular structures of the identified compounds.

Fig. 9

Molecular docking analysis of NF-кB and polyphenolic compounds in CSE. The 3D structure of NF-кB bound efficiently to (A) EGC, (B) C, (C) CF, (D) EC, (E) EGCG, and (F) ECG, as well as to the known inhibitor (G) CPUY192018 (positive control).