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. 2025 Nov 20;15:41008. doi: 10.1038/s41598-025-24845-4

Combined protection against UVB-induced photoaging by oleuropein, hydroxytyrosol, and verbascoside through modulation of inflammation, oxidative stress, and collagen homeostasis

Jing Wang 1,2, Minjia Yuan 1,2, Qi Li 1,2, Chenye Shen 1,2, Xinyi Zhang 1,2, Cuicui Zhu 1,2,✉, Qingqing Cen 3,✉
PMCID: PMC12635131  PMID: 41266576

Abstract

Ultraviolet B (UVB) exposure induces skin photoaging through triggering oxidative stress, inflammation, apoptosis, and collagen degradation. Oleuropein (OLE), Hydroxytyrosol (HT), and Verbascoside (Verb) are polyphenols derived from olive leaves, exhibiting potent antioxidant and anti-inflammatory properties. In the present study, we investigated the protective effects of OLE, HT, and Verb combination (O/H/V) against UVB-induced photoaging using in vitro models. The results showed that O/H/V treatment reduced the expression of senescence and apoptosis-related markers, while concurrently downregulated inflammatory factors and matrix metalloproteinases via suppression of MAPK and NF-κB pathways in UVB-irradiated HDF cells. In UVB-irradiated HaCaT cells, O/H/V treatment attenuated collagen degradation and pro-inflammatory factors expression via upregulation of Nrf2 levels. Furthermore, in a HaCaT-HDFs co-culture system, treatment of UVB-irradiated HaCaT cells with the O/H/V combination led to a significant reduction in pro-inflammatory factors and collagen degradation-related genes in HDFs. Together, these findings indicate that the O/H/V combination exerts protection against UVB-induced photoaging through multiple mechanisms, including inhibition of MAPK/NF-κB pathway and enhancement of Nrf2 expression.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-24845-4.

Keywords: O/H/V, Photoaging, MAPK, NF-κB, Nrf2

Subject terms: Cell biology, Drug discovery, Risk factors

Introduction

Chronic exposure to ultraviolet (UV) radiation is a major extrinsic factor contributing to skin aging, characterized by epidermal thickening, wrinkling, hyperpigmentation, and dryness1–4. UV radiation is divided into three types based on wavelength: UVA (320–400 nm), UVB (280–320 nm), and UVC (200–280 nm), with UVC mostly filtered by the ozone layer. UVA penetrates deep into the dermis, disrupting structural proteins and promoting skin laxity and wrinkle formation5,6. In contrast, UVB primarily affects the epidermis, where it induces DNA damage, oxidative stress, and inflammatory responses primarily triggered by reactive oxygen species (ROS). Although most UVB is absorbed in the epidermis, a small fraction reaches the upper dermis, where it can directly promote fibroblast senescence. Moreover, UVB-exposed keratinocytes release pro-inflammatory cytokines and chemokines that suppress collagen synthesis and activate MMPs in dermal fibroblasts, leading to extracellular matrix (ECM) degradation and impaired skin homeostasis7–11.

Since the pathogenesis of photoaging involves oxidative stress, chronic inflammation, and cellular senescence, multi-targeted therapeutic strategies are promising for mitigating UV-induced damage. Natural botanical compounds are of increasing interest for skin protection. Polyphenolic compounds are among the most widely distributed secondary metabolites in plants12. To date, numerous studies have elucidated the mechanisms through which polyphenols ameliorate photoaging. Their protective effects are primarily exerted via reducing ROS production to mitigate oxidative stress13, cellular senescence and apoptosis, and inhibiting MAPK phosphorylation and NF-κB activity14,15, thereby blocking the secretion of pro-inflammatory cytokines and the degradation of collagen16.

Olea europaea, a globally renowned woody oil crop, yields valuable ingredients for cosmetic use, Oleuropein (OLE), Hydroxytyrosol (HT) and Verbascoside (Verb), which are all active hydrophilic polyphenols derived from olive leaf, exhibit strong antioxidant and anti-inflammatory properties by scavenging ROS17,18, reducing pro-inflammatory cytokines and MMPs release in UVB-exposed skin cells19,20. This study investigated the combined photoprotective effects of OLE, HT, and Verb in in vitro skin cell systems. We evaluated the inhibitory effects of the OLE, HT, and Verb combination (O/H/V) on UVB-induced oxidative stress, inflammation, apoptosis, and collagen degradation, with the aim of providing new insights into the potential application of olive polyphenols for protecting skin from UVB-induced photoaging.

Materials and methods

Cell culture

HDFs and HaCaT obtained from the National Collection of Authenticated Cell Cultures were maintained in Dulbecco’s modified Eagle medium (Sigma, D6171#) with 10% (v/v) fetal bovine serum (Gibco, 10091148#) and 1% (w/v) penicillin/streptomycin (Gibco, 15140122#) at 37℃ in a humidified atmosphere of 5% CO2 (v/v).

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Adamas Life, C8022#). HDFs or HaCaT cells (1 × 10⁴ cells/well) were seeded in 96-well plates. After 24 h, cells were treated with OLE, HT, Verb, or O/H/V for 48 h, and incubated with CCK-8 reagent for 2 h. Absorbance was measured at 450 nm using the PerkinElmer EnSpire multimode reader.

Cell photoaging model

In this study, OLE, HT, and Verb were purchased from Adamas Life (BC000164001#, 12816 A#, BC000093001#). The concentrations of OLE, HT, and Verb utilized to treat cells were all at 20 µM, and their combination (O/H/V) was composed of 10 µM OLE + 10 µM HT + 10 µM Verb. When the confluent rate of HDFs and HaCaT cells reached 50%−60%, and were treated with OLE, HT, Verb, or O/H/V for 24 h, the complete medium was replaced by a thin layer of PBS and irradiated with directly under a UVB light source at 10 cm distance (irradiance: 3 mW/cm2, duration: 27 s, total fluence: 80 mJ/cm2). The irradiated cells were immediately treated with OLE, HT, Verb, or O/H/V for an additional 2–24 h before subsequent experimentation.

Senescence-associated β-galactosidase (SA-β-gal) assay

Cellular senescence was assessed using a SA-β-gal staining kit (Beyotime, C0602#) according to the manufacturer’s protocol. HDFs were seeded in 24-well culture plates at a density of 3 × 104 cells per well. Cells were treated with OLE, HT, Verb, or O/H/V and exposed to UVB (80 mJ/cm2). After 24 h, cells were washed with PBS and fixed with β-galactosidase fixative solution for 15 min at room temperature. The fixative was removed, and cells were gently rinsed three times with PBS. Subsequently, cells were incubated with freshly prepared SA-β-gal staining solution at 37 °C overnight in the dark. Following incubation, nuclei were counterstained with DAPI (Beyotime, P0131#) for 2 min, and cells were mounted using an anti-fade medium.

Bright-field images of SA-β-gal-positive cells (blue staining) and fluorescence images of DAPI-stained nuclei (blue fluorescence) were captured using an Olympus BX53 microscope, and senescence-associated β-galactosidase activity was subsequently quantified by calculating the percentage of SA-β-gal-positive cells relative to DAPI-stained nuclei in 3 randomly selected non-overlapping fields per well (avoiding the outer 2 mm edge region) using ImageJ software, with the ratio expressed as: senescent cells = Number of SA-β-gal⁺ cells/Total DAPI⁺ nuclei.

Real-time PCR

HDFs or HaCaT cells were seeded in 6-well culture plates at a density of 1 × 105 cells per well. Cells were treated with OLE, HT, Verb, or O/H/V and irradiation with UVB (80 mJ/cm2). After 24 h, cells were washed once with ice-cold PBS and lysed for total RNA extraction using the FastPure Complex Tissue/Cell Total RNA Isolation Kit (Vazyme, RC112#) according to the manufacturer’s protocols. cDNA synthesis was performed with 1 µg RNA template using HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme, R333#). RT-qPCR amplification was conducted on a QuantStudio™ 3 system (Applied Biosystems) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711#). Amplification primers were 5- CCACAGACCTTCCAGGAGAATG-3-(forward)5-GTGCAGTTCAGTGATCGTACAGG-3-(reverse) for the IL-1β gene; 5-GAGAGTGATTGAGAGTGGACCAC-3-(forward)5-CACAACCCTCTGCACCCAGTTT-3-(reverse) for the IL-8 gene; 5-GTCTCCTCTGACTTCAACAGCG-3-(forward)5-ACCACCCTGTTGCTGTAGCCAA-3-(reverse) for the GAPDH gene; 5-CTCTTCTGCCTGCTGCACTTTG-3-(forward)5-ATGGGCTACAGGCTTGTCACTC-3-(reverse) for the TNFα gene; 5-AGACAGCCACTCACCTCTTCAG-3-(forward)5-TTCTGCCAGTGCCTCTTTGCTG-3-(reverse) for the IL-6 gene; 5-TCAGGATGCGTCCACCAAGAAG-3-(forward)5-TGTGTCCACGGCGGCAATCATC-3-(reverse) for the Bax gene; 5-ATCGCCCTGTGGATGACTGAGT-3-(forward)5-GCCAGGAGAAATCAAACAGAGGC-3-(reverse) for the Bcl2 gene; 5-CACATCCAGTCAGAAACCAGTGG-3-(forward)5-GGAATGTCTGCGCCAAAAGCTG-3-(reverse) for the Nrf2 gene; 5-ATGAAGCAGCCCAGATGTGGAG-3-(forward)5-GTGCAGTTCAGTGATCGTACAGG-3-(reverse) for the MMP1 gene; 5-GCCACTACTGTGCCTTTGAGTC-3-(forward)5-CCCTCAGAGAATCGCCAGTACT-3-(reverse) for the MMP9 gene; 5-GATTCCCTGGACCTAAAGGTGC-3-(forward)5-AGCCTCTCCATCTTTGCCAGCA-3-(reverse) for the Col1a gene; and 5- TGGTCTGCAAGGAATGCCTGGA-3-(forward)5-TCTTTCCCTGGGACACCATCAG-3-(reverse) for the Col3a gene. Each sample’s threshold cycle number (CT) was determined in triplicate. The CT values for genes were normalized against GAPDH as described previously.

Enzyme-linked immunosorbent (ELISA) assay

HDFs and HaCaT cells were seeded in 6-well culture plates at a density of 1 × 105 cells per well. Cells were treated with OLE, HT, Verb, or O/H/V and exposed to UVB (80 mJ/cm2). After 24 h, cell culture supernatants were collected, centrifuged at 1,000 × g for 5 min to remove cellular debris, and stored at − 80 °C prior to ELISA analysis. The amount of pro-inflammatory cytokines (IL-6, P9991#), 8-oxo-dG (LV11468M#), matrix metalloproteinase-related biomarkers (MMP1, P9844#), and pro-collagen type I (Col1a, P8031#), which reflect the level of collagen synthesis and photoaging, was measured using commercial ELISA kits following the manufacturer’s instructions. The ELISA assay was performed according to the manufacturer’s instructions (Adamas Life). Finally, use the PerkinElmer EnSpire system to measure the absorbance at 450 nm.

Western blot

HaCaT and HDFs were seeded in 6-well culture plates at a density of 1 × 105 cells per well. Cells were treated with OLE, HT, Verb, or O/H/V and exposed to UVB (80 mJ/cm2). After 2 h, cells were lysed on ice for 10 min using RIPA buffer (Beyotime Biotechnology, P0013B#) containing 100× protease inhibitor cocktail (Cell Signaling Technology, 5871#) and 100× phosphatase inhibitor cocktail (Cell Signaling Technology, 5870#). Lysates were centrifuged at 12,000 ×g for 15 min at 4 °C, and protein concentrations in the supernatants were quantified via BCA assay (TaKaRa BCA Protein Assay Kit). Protein samples (20 µg per lane, n = 3) were denatured in 4× Laemmli Sample Buffer by boiling at 95 °C for 5 min, separated on 10% SDS-polyacrylamide gels, and transferred to PVDF-FL membranes (Millipore) using a semi-dry transfer system. Membranes were blocked with 5% non-fat milk in TBS and probed overnight at 4 °C with the following primary antibodies: Rabbit antibodies: p38 (Cell Signaling Technology, 8690#, 1:1000), ERK2 (Cell Signaling Technology, 4696#, 1:1000), p-p65 (Cell Signaling Technology, 8690#, 1:1000), COX-2 (Cell Signaling Technology, 12282#, 1:1000), p53 (Cell Signaling Technology, 9282#, 1:1000), p21 (Cell Signaling Technology, 2974#, 1:1000), p16 (Cell Signaling Technology, 80772#, 1:1000), Nrf2 (Cell Signaling Technology, 1272#, 1:1000), cleaved caspase-3 (CC3, Cell Signaling Technology, 9662#, 1:1000), β-actin (Cell Signaling Technology, 8457#, 1:5000); Mouse antibodies: p-p38 (Cell Signaling Technology, 9216#, 1:1000), p-ERK2 (Cell Signaling Technology, 4377#, 1:1000), p65 (Cell Signaling Technology, 6956#, 1:1000). After washing, membranes were incubated with species-matched IRDye fluorescent secondary antibodies (Cell Signaling Technology, 5257#, Cell Signaling Technology, 5470#, 1:10000) for 1 h at room temperature. Blots were scanned using an Odyssey CLx Infrared Imaging System (LI-COR Biosciences) and quantified with ImageJ software. Protein expression levels were normalized to β-actin.

TUNEL assay

The TUNEL assay was performed according to the manufacturer’s instructions (Beyotime Biotechnology kit, C1088#). HDFs were seeded in 24-well culture plates at a density of 3 × 104 cells per well. Cells were treated with OLE, HT, Verb, or O/H/V and exposed to UVB (80 mJ/cm2). After 24 h, cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.3% Triton X-100 in PBS for 5 min. The TUNEL detection solution was prepared as per the kit protocol, and cells were incubated with the reaction mixture at 37 °C for 60 min in the dark. After PBS washing to remove unbound reagents, nuclei were counterstained with DAPI(Beyotime, P0131#)for 10 min. Cells were mounted using an anti-fluorescence quenching medium and imaged under a fluorescence microscope (Olympus BX53) with appropriate filters (DAPI: 358 nm; TUNEL probe: 488 nm). TUNEL-positive cells were quantified using ImageJ software, with apoptosis rates calculated as the ratio of TUNEL-positive to DAPI-stained nuclei across multiple fields.

Keratinocyte-human fibroblast transwell co-culture

HaCaT (3 × 105 cells/well) were seeded into 6-well culture inserts, and HDFs (1 × 105 cells/well) were seeded into 6-well culture plates (Corning, 3450#)21. After 24 h, HaCaT in the upper chamber were treated with OLE, HT, Verb, and O/H/V for 24 h. When the confluent rate of HaCaT cells reached 80%, the upper chamber with HaCaT into a new cell-free 6-well culture plate, and then cells culture media were replaced with PBS and exposed to UVB light (80 mJ/cm²). following UVB irritation, the PBS was replaced by new media with OLE, HT, Verb, and O/H/V, and HaCaT were then co-cultured with HDFs for 24 h at 37℃.

ROS measurement by DCFH-DA fluorescence

The intracellular ROS levels were quantified using a 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe assay (Beyotime Biotechnology, China) following the manufacturer’s protocol with UVB irradiation adjustments. Briefly, HaCaT cells were seeded in 96-well black-walled plates (1 × 10⁴ cells/well). Cells were treated with OLE, HT, Verb, or O/H/V and exposed to UVB (80 mJ/cm2). After 2 h, cells were gently washed three times with PBS to remove residual culture medium. The DCFH-DA working solution (10 µM) was prepared by diluting the stock solution (10 mM) in pre-warmed PBS. Cells were incubated with 100 µL of DCFH-DA working solution at 37 °C in a humidified 5% CO₂ incubator for 30 min. Unincorporated probes were removed by three additional PBS washes. Fluorescence intensity was measured immediately using a multimode microplate reader (PerkinElmer EnSpire) with excitation/emission wavelengths set at 488 nm and 525 nm, respectively.

Statistical analysis

All data values are expressed as the means ± standard error of mean (means ± SEM), and all experiments were performed in triplicate. The data were analyzed using one-way ANOVA, and each data group was compared with the UVB irradiation treatment alone. P < 0.05 was considered statistically significant.

Results

O/H/V attenuates UVB-induced cytotoxicity and apoptosis in HDFs

To evaluate the protective effects of OLE, HT, and Verb against UVB-induced cytotoxicity, HDFs were first treated with increasing concentrations of each compound individually or in combination, for 48 h. No significant cytotoxicity was observed at concentrations up to 20 µM for the individual compounds or up to 10 µM each for the combined treatment (Fig. 1a-b). The combination was applied in a 1:1:1 molar ratio, selected due to the similar cytotoxicity profiles of the individual compounds, allowing equal contribution from each component and enabling comparison at biologically relevant total concentrations. Based on these findings, 20 µM of each compound was selected for individual treatments, and 10 µM of each compound (i.e., 30 µM total) was used for the combination treatment (O/H/V) in all subsequent experiments. As shown in Fig. 1c, UVB exposure markedly reduced cell viability, which was substantially restored by all treatments, with the O/H/V combination exhibiting the strongest protective effect. To determine whether this improvement in viability was associated with anti-apoptotic effects, the CC3 protein level was assessed by western blot. UVB irradiation led to a robust increase in CC3 levels, which was attenuated by all treatments. Among them, the O/H/V combination showed the greatest reduction in UVB-induced CC3 level (Fig. 1d). To further validate these findings, TUNEL staining was used to detect UVB-induced DNA fragmentation. UVB exposure resulted in a substantial increase in TUNEL-positive nuclei. Treatment with OLE or Verb reduced the UVB-induced proportion of apoptotic cells, whereas HT alone had no substantial effect. In contrast, the O/H/V combination produced a more pronounced decrease in UVB-induced TUNEL-positive nuclei (Fig. 1e).

Fig. 1.

Fig. 1

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OLE, HT, and Verb, individually or in combination, protect HDFs from UVB-induced cytotoxicity by suppressing apoptosis. (a) The chemical structures of OLE, HT, and Verb. (b) Cell viability of HDFs treated with increasing concentrations of OLE, HT, Verb, or O/H/V for 48 h. Cell viability was assessed by the CCK-8 assay and normalized to untreated controls. (c-e) Following treatment with OLE, HT, Verb, or the O/H/V combination and subsequent UVB irradiation (80 mJ/cm²), the viability of HDFs was assessed using the CCK-8 assay(c). (d) Western blot analysis of CC3 level. Quantification was performed relative to β-actin. (e) Representative TUNEL staining images of HDFs. Nuclei were counterstained with DAPI (blue), and apoptotic cells were visualized as TUNEL-positive (green), the write arrow meaning TUNLE-positive nuclei (DAPI/TUNEL merge). Apoptosis was quantified as the percentage of TUNEL-positive nuclei among total DAPI-stained nuclei. Scale bar = 100 μm. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. UVB-treated group; ##p < 0.01, ###p < 0.001 vs. untreated control.

O/H/V combination alleviates UVB-induced senescence in HDFs

Following the observed anti-apoptotic effects, we next investigated whether OLE, HT, and Verb could mitigate cellular senescence in HDFs, another hallmark of photoaging. As shown in Fig. 2a, UVB exposure significantly increased the proportion of SA-β-gal-positive HDFs. Treatment with OLE, HT, Verb, or the O/H/V combination substantially reduced the number of senescent cells, with the O/H/V yielding the greatest effect. In order to elucidate the mechanism underlying this effect, the expression of three key senescence-associated proteins, including p53, p21 and p16, was analyzed by western blot (Fig. 2b). UVB irradiation strongly upregulated all three markers. Treatment with OLE or HT individually suppressed UVB-induced p53 and p16 expression but had a limited effect on p21. In contrast, the O/H/V combination significantly inhibited the expression of all three proteins, demonstrating broader suppression of senescence-associated signaling. These results indicate that O/H/V provides a more comprehensive and potent inhibitory effect on UVB-induced senescence than the individual compounds.

Fig. 2.

Fig. 2

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OLE, HT, and Verb, individually or in combination, attenuate UVB-induced senescence in HDFs. (a) Representative images of SA-β-gal staining in HDFs. Senescent cells stained positively in blue. Quantification of SA-β-gal-positive cells is presented as the normalized ratio of positive cells. Scale bar = 100 μm. (b) Western blot analysis of the senescence-associated markers p53, p21, and p16. Quantification of protein expression was normalized to β-actin. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs. UVB-treated group; #p < 0.05, ##p < 0.01 vs. untreated control.

O/H/V combination suppresses UVB-induced inflammation and preserves collagen homeostasis in HDFs

To examine the transcriptional regulation of pro-inflammatory and extracellular matrix (ECM)-related genes by OLE, HT, Verb, or the O/H/V combination, mRNA levels of TNF-α, IL-1β, IL-6, IL-8, MMP1, MMP9, Col1a, and Col3a were quantified by RT-qPCR. Figure 3a showed that UVB upregulated TNF-α, IL-1β, IL-6, IL-8, MMP1 and MMP9 expression, while suppressed Col1a and Col3a. Treatment with the O/H/V combination robustly suppressed UVB-induced cytokines and MMPs expression and significantly restored Col1a and Col3a. Among individual treatments, HT significantly suppressed IL-1β expression, and all three compounds significantly reduced IL-6, IL-8, and MMP9 expression, but exhibited limited or no effects on TNF-α, MMP1, or collagen genes. Sebquently ELISA assay was performed to measure secreted MMP1, IL-6, and Col1a protein levels (Fig. 3b). O/H/V treatment significantly reduced MMP1 and IL-6 and restored Col1a protein levels comparing to the UVB-treated group. All three individual compounds significantly reduced UVB-induced IL-6 secretion, but had no appreciable effect on MMP1 or Col1a protein levels, suggesting that the combination might be necessary to achieve a coordinated modulation of matrix degradation and repair at the protein level. To further elucidate the upstream signaling mechanisms, phosphorylation of MAPK components (p38, ERK2) and NF-κB p65, as well as COX-2 expression, was assessed by western blot (Fig. 3c). UVB irradiation strongly induced phosphorylation of p38, ERK2 and p65, and upregulated COX-2 expression. O/H/V treatment significantly inhibited these UVB-induced changes, indicating coordinated suppression of MAPK and NF-κB signaling. Among individual compounds, OLE significantly reduced phosphorylation of p38, and HT significantly suppressed phosphorylation of p65, while other effects were partial or inconsistent.

Fig. 3.

Fig. 3

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Effects of OLE, HT, and Verb, individually or in combination, on UVB-induced inflammation and collagen homeostasis in HDFs. (a) The mRNA levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8), matrix metalloproteinases (MMP1, MMP9), and collagen-related genes (Col1a, Col3a) were assessed by RT-qPCR, all gene expression levels were normalized to GAPDH. (b) Protein levels of MMP1, IL-6, and Col1a were quantified by ELISA. (c) Western blot analysis of MAPK and NF-κB signaling pathways, including phosphorylated and total forms of p38, ERK2, and p65, and downstream effector COX-2. Densitometric quantification was normalized to β-actin. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs. UVB-treated control; #p < 0.05, ##p < 0.01 vs. untreated control.

O/H/V combination attenuates oxidative stress and inflammatory responses in UVB-irradiated HaCaT cells

Following the demonstrated protective effects of OLE, HT, and Verb in dermal fibroblasts, their efficacy was next examined in keratinocytes. Cell viability assays confirmed that treatment with OLE, HT, Verb (1–50 µM), or their combination (O/H/V, 1:1:1 molar ratio) for 48 h did not affect HaCaT cell viability. Therefore, 20 µM dose was selected for individual compounds and 10 µM each for the combination in all subsequent experiments (Fig. 4a), in order to ensure the consistency with fibroblast assays. Treatment with the O/H/V combination remarkably suppressed ROS accumulation and reduced 8-oxo-dG levels relative to the UVB group. In contrast, none of the individual compounds showed significant inhibitory effects on either of the two markers (Fig. 4b–c). The expression of Nrf2, a key regulator of the antioxidant response, was then evaluated. UVB irradiation significantly downregulated Nrf2 expression at both the mRNA and protein levels. Although individual treatments restored the mRNA level of Nrf2, they did not significantly affect its protein expression. Notably, the O/H/V combination significantly upregulated Nrf2 at both the mRNA and protein levels following UVB exposure, indicating effective reactivation of the Nrf2 pathway under oxidative stress conditions (Fig. 4d).

Fig. 4.

Fig. 4

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OLE, HT, and Verb, individually or in combination, alleviate UVB-induced oxidative stress, inflammation, and collagen degradation in HaCaT cells via Nrf2 activation. (a) Cell viability of HaCaT cells treated with various concentrations (1–50 µM) of OLE, HT, Verb, or their combination (O/H/V, 1:1:1) for 48 h, assessed using the CCK-8 assay. (b) Intracellular ROS levels were measured using the DCFH-DA fluorescence assay. (c) Oxidative DNA damage was evaluated by ELISA detection of 8-oxo-dG. (d) Nrf2 protein levels were analyzed by western blot, the Nrf2 protein level was normalized to β-actin; Nrf2 mRNA levels were determined by RT-qPCR, the Nrf2 expression level was normalized to GAPDH. (e) The mRNA levels of c-Fos, c-Jun, MMP1, MMP9, Col1a, TNF-α, IL-1β, and IL-6 were measured by RT-qPCR; all gene expression levels were normalized to GAPDH. (f) Protein levels of MMP1, Col1a, IL-1β, and IL-6 were quantified by ELISA. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs. UVB-treated control (UVB); #p < 0.05, ##p < 0.01 vs. untreated control (CTR).

Given the link between oxidative stress and inflammation, the effects of O/H/V on UVB-induced inflammatory and matrix-degrading responses were examined. UVB irradiation activated AP-1 transcription factors (c-Fos, c-Jun), upregulated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and matrix metalloproteinases (MMP1, MMP9), while suppressing Col1a expression. Treatment with the O/H/V combination significantly attenuated these UVB-induced gene expression changes and restored Col1a mRNA levels (Fig. 4e). Among the individual treatments, OLE, HT, and Verb each significantly downregulated UVB-induced expression of c-Fos, c-Jun, TNF-α, and IL-1β, and restored Col1a mRNA levels to varying degrees. However, none suppressed MMP9 expression. Both OLE and Verb, but not HT, significantly reduced MMP1 and IL-6 gene expression. These findings indicate that while individual compounds exhibit partial anti-inflammatory and collagen-preserving effects, their combined use yields more comprehensive protection. ELISA assay results showed that the O/H/V combination significantly reduced secretion of MMP1, IL-1β, and IL-6, and restored Col1a protein levels (Fig. 4f). In summary, these findings demonstrate that the O/H/V combination effectively attenuates UVB-induced oxidative stress, inflammation, and matrix degradation in keratinocytes.

O/H/V combination prevents UVB-induced paracrine photoaging signals from keratinocytes to fibroblasts in a co-culture system

To determine whether the protective effects of OLE, HT, and Verb against UVB-induced stress involve intercellular signaling, we used a transwell co-culture system. HaCaT cells were cultured in the upper chamber and HDFs in the lower chamber. Both cell types were treated with OLE, HT, or Verb (20 µM each), or their combination (O/H/V, 10 µM each) for 24 h. Subsequently, only the HaCaT cells were exposed to UVB irradiation (80 mJ/cm²), after which the co-culture was maintained in the continued presence of the same treatments for an additional 24 h (Fig. 5a).

Fig. 5.

Fig. 5

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OLE, HT, and Verb, individually or in combination, modulate paracrine signaling from UVB-irradiated keratinocytes to underlying fibroblasts in a co-culture model. (a) Schematic representation of the transwell co-culture system and experimental workflow corresponding to panels. (b) HDFs viability was assessed by CCK-8 assay and normalized to the untreated control. (c) RT-qPCR analysis of TNF-α, IL-1β, Bcl2, Bax, MMP1, and Col1a mRNA levels in HDFs, reflecting paracrine-induced inflammatory, apoptotic, and ECM-related responses. All gene expression levels were normalized to GAPDH. (d) Western blot analysis of CC3, phosphorylated and total JNK, and phosphorylated and total NF-κB p65 in HDFs. Protein expression was normalized to β-actin. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 vs. UVB-treated group (UVB); #p < 0.05, ##p < 0.01 vs. untreated control (CTR).

UVB irradiation of HaCaT cells led to a marked decrease in HDFs viability, despite the HDFs cells not being directly irradiated. It is plausible that soluble paracrine mediators released by UVB-stressed keratinocytes exerted cytotoxic effects on neighboring fibroblasts. Among all treatment groups, only the O/H/V combination significantly preserved HDFs viability (Fig. 5b). These findings indicate that the protective effect of the O/H/V combination maybe linked to suppression of paracrine factors production by UVB-irradiated HaCaT cells, therefore, mRNA expression of inflammation-, apoptosis-, and ECM-related genes was assessed in HDFs. UVB exposure of HaCaT cells significantly increased TNF-α, IL-1β, Bax, and MMP1 expression in HDFs. No significant changes in Bcl2 or Col1a expression were observed. O/H/V treatment significantly suppressing the upregulation of TNF-α, IL-1β, Bax, and MMP1, while concurrently enhancing the expression of Bcl2 and Col1a (Fig. 5c). In contrast, individual compounds yielded only partial improvements: all three reduced IL-1β and Bax expression and increased Bcl2 to varying degrees, but none significantly affected TNF-α, MMP1, or Col1a.

To further characterize activation of downstream stress signaling in HDFs, levels of CC3, p-JNK, and p-p65 were analyzed by western blot. All three markers were significantly increased in HDFs, indicating the activation of apoptotic and inflammatory cascades. All individual treatments reduced p-JNK level; specifically HT alone significantly attenuated p65 phosphorylation, while OLE decreased CC3 level. In contrast, O/H/V combination robustly suppressed these responses, significantly reducing CC3 level as well as phosphorylation of JNK and p65 (Fig. 5d), thereby reinforcing the superior multi-targeted effect of the O/H/V combination.

Discussion

Prior studies have demonstrated the protective efficacy of each individual compound in the context of cellular damage. OLE exerts anti-inflammatory effects by inhibiting the NF-κB and MAPK signaling pathways, thereby suppressing the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-622. As another major polyphenol derived from olive leaves, HT has been shown to mitigate inflammation in UVA-induced fibroblasts via the NF-κB signaling pathway and exerts anti-aging effects by reducing the expression of MMP1 and MMP323. Verb, a key phenylethanoid glycoside, exhibits substantial antioxidant and anti-inflammatory activities by inhibiting IL-8, IL-1β, NO and TNF-α production in in vitro cell models24,25.Consistent with these findings, our study confirmed that individual treatment with each of the three compounds (OLE, HT, Verb) inhibits the expression of TNF-α, IL-1β by targeting the NF-κB signaling pathway, with O/H/V treatment exerting a more pronounced anti-inflammatory effect. Additionally, individual OLE, HT, and Verb also exerts antioxidant effects by regulating the Keap1-Nrf2 signaling pathway to reduce ROS levels24,26,27. In the keratinocytes, our results further showed that treatment with the three compounds reduces UVB-induced ROS accumulation and upregulates Nrf2 protein expression, with the combined treatment achieving a more potent effect than individual treatments.

Exposure to UVB radiation induces DNA damage, thereby triggering the DNA damage response (DDR) in skin cells, thus trigers apoptosis8,28, In the current study, O/H/V treatment substantially restored cell viability and reduced CC3, Bax level and TUNEL-positive nuclei, while upregulated Bcl2, further supporting its cytoprotective effect. The DDR pathway activation triggers senescence-associated signaling cascades. We found that UVB induced senescence in HDFs, marked by increased SA-β-gal activity and upregulation of p53, p21, and p16. These changes were substantially reversed by O/H/V, whereas individual treatments were less effective. Once fibroblasts become senescent, MMPs production is enhanced, which sebquently supress their capacity for collagen synthesis. The results showed that the expression of TNF-α, IL-1β, IL-6, IL-8, MMP1, and MMP9 increased, while that of Col1a and Col3a reduced in UVB damaged HDFs. The MAPK/NF-κB pathway plays a central role in regulating collagen metabolism and inflammatory cytokine balance29. UVB irradiaton-activated ERK1/2 and p38 translocate to the nucleus, forming AP-1 transcription complexes with c-Fos/c-Jun to upregulate MMP1/MMP3 expression30–32. Furthermore, NF-κB, another activated factor, enables nuclear translocation of p65/p50 dimers to drive MMP9 and pro-inflammatory cytokines production33–36. Mechanistically, O/H/V inhibited the phosphorylation of p38, ERK2, and p65, and reduced COX-2 expression, indicating suppression of both MAPK and NF-κB signaling. In contrast, individual compounds had less effects. Alternatively, collagen dysregulation was mechanistically linked to oxidative stress. In our study, O/H/V treatment reduced UVB-induced ROS accumulation, oxidative DNA damage and restored Nrf2 expression at both mRNA and protein levels. To determine whether the modulation of MAPK, NF-κB, and Nrf2 is a direct effect of the compounds or a secondary consequence of their antioxidant activity, we plan to inhibit ROS generation in subsequent experiments and then examine the expression levels of MAPK/NF-κB and Nrf2.

In the transwell co-culture system, UVB-irradiated keratinocytes triggered paracrine-mediated damage in fibroblasts, as indicated by reduced viability and increased TNF-α, IL-1β, Bax, MMP1, cleaved caspase-3, and phosphorylation of JNK and p65. These findings reflect the action of keratinocyte-derived cytokines and proteases on fibroblasts. These observations reflect the regulatory effects of cytokines and proteases secreted by keratinocytes on fibroblasts. Consistent with the aforementioned fibroblast damage phenotypes, we observed a significant increase in the phosphorylation of JNK and p65 in fibroblasts. To elucidate the underlying mechanism of fibroblast response to keratinocyte-derived signals, JNK and NF-κB signaling were specifically examined. Inflammatoy mediators, such as TNF-α37–40, IL-1β and IL-641,42,43,44, are well-established activators of the NF-κB and JNK pathways. The observed phosphorylation of p65 and JNK in fibroblasts supports the conclusion that these pathways mediate dermal inflammation. O/H/V treatment substantially attenuated this activation, reinforcing its protective role. Notably, Col1a and Bcl2 expression in fibroblasts was not substantially decreased at the 24 h time point. This may be due to insufficient time for keratinocyte-derived mediators to accumulate and exert transcriptional effects on these targets. A longer exposure period may be necessary to capture delayed alterations in ECM synthesis and survival signaling. Furthermore, the transwell co-culture model only simulates the simple cell-cell interaction and cannot fully replicate the human skin tissue. Therefore, subsequent studies will utilize 3D skin equivalents that more closely mimic in vivo skin physiology to further validate the protective effects and mechanisms of O/H/V.

Conclusion

In conclusion, our study reveals that the O/H/V combination exerts combined protective effects against UVB-induced photoaging in HDFs and HaCaT-HDFs co-culture system. The O/H/V combination inhibits NF-κB/MAPK signaling, and promotes Nrf2 expression, thereby suppresses the production of ROS, pro-inflammatory factors, and apoptosis markers. These findings provide insights for the development of O/H/V-based formulations in cosmetic applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (312.9KB, pdf)

Author contributions

JW conceived and designed the study under supervision of CCZ, QQC. JW, CYS and XYZ performed the experiments, and collected and analyzed the data, and conducted statistical analysis. JW, MJY, and QL contributed to the interpretation of the results. JW and CCZ wrote the initial draft of the manuscript. All authors read and approved the final version of the manuscript.

Funding

There is no external funding resources for this research.

Data availability

Regarding data availability, all data generated or analysed during this study are included in this published article. The raw western blot data referenced in the manuscript are provided in the supplementary files. The remaining data are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Cuicui Zhu, Email: cuicui.zhu@outlook.com.

Qingqing Cen, Email: cenqingqing1995@163.com.

References

  • 1.Gu, Y., Han, J., Jiang, C. & Zhang, Y. Biomarkers, oxidative stress and autophagy in skin aging. Ageing Res. Rev.59, 101036 (2020). [DOI] [PubMed] [Google Scholar]
  • 2.Rittié, L. & Fisher, G. J. Natural and sun-induced aging of human skin. Cold Spring Harb Perspect. Med.5, a015370 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Rachmin, I., Ostrowski, S. M., Weng, Q. Y. & Fisher, D. E. Topical treatment strategies to manipulate human skin pigmentation. Adv. Drug Del. Rev.153, 65–71 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Aguirre-Cruz, G., León-López, A., Cruz-Gómez, V. & Jiménez-Alvarado, R. Aguirre-Álvarez, G. Collagen hydrolysates for skin protection: oral administration and topical formulation. Antioxidants9, 181 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Liao, A. H. et al. Application of ultrasound-mediated adapalene-coated lysozyme-shelled microbubbles in UVA-induced skin Photoaging. PLoS One. 15, e0232617 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Liu, S. et al. Oral supplementation of sea cucumber and its hydrolysate mitigates ultraviolet A-induced Photoaging in hairless mice. J. Sci. Food Agric.102, 1987–1994 (2022). [DOI] [PubMed] [Google Scholar]
  • 7.Lee, J. Jr. et al. Galangin reverses H2O2-Induced dermal fibroblast senescence via SIRT1-PGC-1α/Nrf2 signaling. Int. J. Mol. Sci.23, 1387 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kim, D. J., Iwasaki, A., Chien, A. L. & Kang, S. UVB-mediated DNA damage induces matrix metalloproteinases to promote p Hotoaging in an AhR- and SP1-dependent manner. JCI Insight. 7, e156344 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Bang, E., Kim, D. H. & Chung, H. Y. Protease-activated receptor 2 induces ROS-mediated inflammation through Akt-mediated NF-κB and FoxO6 modulation during skin Photoaging. Redox Biol.44, 102022 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Chen, J. et al. Panax ginseng extract prevents UVB-induced skin photodamage by modulating VMP1-mediated ER stress. Phytomedicine134, 156010 (2024). [DOI] [PubMed] [Google Scholar]
  • 11.Rodríguez-Luna, A. et al. Fucoxanthin-Containing cream prevents epidermal hyperplasia and UVB-Induced skin erythema in mice. Mar. Drugs. 16, 378 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Singh, S., Kaur, I. & Kariyat, R. The multifunctional roles of polyphenols in Plant-Herbivore interactions. Int J. Mol. Sci22 (2021). [DOI] [PMC free article] [PubMed]
  • 13.Klarić, D. A. et al. Polyphenol content and antioxidant activity of phytoestrogen containing food and dietary supplements: DPPH free radical scavenging activity by HPLC. Acta Pharm.72, 375–388 (2022). [DOI] [PubMed] [Google Scholar]
  • 14.Ratz-Łyko, A. & Arct, J. Resveratrol as an active ingredient for cosmetic and dermatological applications: a review. J. Cosmet. Laser Ther.21, 84–90 (2019). [DOI] [PubMed] [Google Scholar]
  • 15.Yu, C., Wang, D., Yang, Z. & Wang, T. Pharmacological effects of polyphenol phytochemicals on the intestinal inflammation via targeting TLR4/NF-κB signaling pathway. Int J. Mol. Sci23 (2022). [DOI] [PMC free article] [PubMed]
  • 16.Li, Y., Tao, L., Xu, Y. & Guo, R. Taxifolin ameliorates abdominal aortic aneurysm by preventing inflammation and apoptosis and extracellular matrix degradation via inactivating TLR4/NF-κB axis. Int. Immunopharmacol.119, 110197 (2023). [DOI] [PubMed] [Google Scholar]
  • 17.Georgiev, M. et al. Verbascum xanthophoeniceum-derived phenylethanoid glycosides are potent inhibitors of inflammatory chemokines in dormant and interferon-gamma-stimulated human keratinocytes. J. Ethnopharmacol.144, 754–760 (2012). [DOI] [PubMed] [Google Scholar]
  • 18.Li, Y., Yu, H., Jin, Y., Li, M. & Qu, C. Verbascoside alleviates atopic Dermatitis-Like symptoms in mice via its potent Anti-Inflammatory effect. Int. Arch. Allergy Immunol.175, 220–230 (2018). [DOI] [PubMed] [Google Scholar]
  • 19.Katsiki, M., Chondrogianni, N., Chinou, I., Rivett, A. J. & Gonos, E. S. The Olive constituent Oleuropein exhibits proteasome stimulatory properties in vitro and confers life span extension of human embryonic fibroblasts. Rejuvenation Res.10, 157–172 (2007). [DOI] [PubMed] [Google Scholar]
  • 20.Li, H. et al. Dietary polyphenol Oleuropein and its metabolite Hydroxytyrosol are moderate skin permeable elastase and collagenase inhibitors with synergistic cellular antioxidant effects in human skin fibroblasts. Int. J. Food Sci. Nutr.73, 460–470 (2021). [DOI] [PubMed] [Google Scholar]
  • 21.Ko, H. J. et al. Ergothioneine alleviates senescence of fibroblasts induced by UVB damage of keratinocytes via activation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes. Exp. Cell. Res.400, 112516 (2021). [DOI] [PubMed] [Google Scholar]
  • 22.Cui, Y. et al. Oleuropein attenuates Lipopolysaccharide-Induced acute kidney injury in vitro and in vivo by regulating Toll-Like receptor 4 dimerization. Front. Pharmacol.12, 617314 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Jeon, S. & Choi, M. Anti-inflammatory and anti-aging effects of Hydroxytyrosol on human dermal fibroblasts (HDFs). Biomed. Dermatol.2, 21 (2018). [Google Scholar]
  • 24.Khorashadizadeh, N., Neamati, A., Moshiri, M. & Etemad, L. Verbascoside inhibits paraquate-induced pulmonary toxicity via modulating oxidative stress, inflammation, apoptosis and DNA damage in A549 cell. Drug Chem. Toxicol.45, 2212–2220 (2022). [DOI] [PubMed] [Google Scholar]
  • 25.Wisuitiprot, V. et al. Effects of acanthus ebracteatus Vahl. Extract and Verbascoside on human dermal papilla and murine macrophage. Sci. Rep.12, 1491 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Pirković, A. et al. Oleuropein attenuates oxidative stress in human trophoblast cells. Antioxidants12 (2023). [DOI] [PMC free article] [PubMed]
  • 27.Wen, X. et al. The PI3K/Akt-Nrf2 signaling pathway and mitophagy synergistically mediate Hydroxytyrosol to alleviate intestinal oxidative damage. Int. J. Biol. Sci.20, 4258–4276 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Drigeard Desgarnier, M. C., Fournier, F., Droit, A. & Rochette, P. J. Influence of a pre-stimulation with chronic low-dose UVB on stress response mechanisms in human skin fibroblasts. PLoS One. 12, e0173740 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ding, Y., Jiratchayamaethasakul, C. & Lee, S. H. Protocatechuic aldehyde attenuates UVA-Induced Photoaging in human dermal fibroblast cells by suppressing MAPKs/AP-1 and NF-κB signaling pathways. Int. J. Mol. Sci.21, 4619 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Oh, J. H., Karadeniz, F., Kong, C. S. & Seo, Y. Antiphotoaging effect of 3,5-Dicaffeoyl-epi-quinic acid against UVA-Induced skin damage by protecting human dermal fibroblasts in vitro. Int. J. Mol. Sci.21, 7756 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kusumaningrum, N. et al. Gasdermin C is induced by ultraviolet light and contributes to MMP-1 expression via activation of ERK and JNK pathways. J. Dermatol. Sci.90, 180–189 (2018). [DOI] [PubMed] [Google Scholar]
  • 32.Zeng, L., Rong, X. F., Li, R. H. & Wu, X. Y. Icariin inhibits MMP–1, MMP–3 and MMP–13 expression through MAPK pathways in IL–1β–stimulated SW1353 chondrosarcoma cells. Mol. Med. Rep.15, 2853–2858 (2017). [DOI] [PubMed] [Google Scholar]
  • 33.Kim, H. K. Protective effect of Garlic on cellular senescence in UVB-Exposed HaCaT human keratinocytes. Nutrients8, 464 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wang, M. L. et al. Andrographolide sodium bisulfate attenuates UV–induced photo–damage by activating the keap1/Nrf2 pathway and downregulating the NF–κB pathway in HaCaT keratinocytes. Int. J. Mol. Med.45, 343–352 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang, K. et al. Phytocosmetic potential of Blumea balsamifera oil in mitigating UV-induced photoaging: evidence from cellular and mouse models. J. Ethnopharmacol.334, 118535 (2024). [DOI] [PubMed] [Google Scholar]
  • 36.Wang, J., Ke, J., Wu, X. & Yan, Y. Astragaloside prevents UV-induced keratinocyte injury by regulating TLR4/NF-κB pathway. J. Cosmet. Dermatol.21, 1163–1170 (2022). [DOI] [PubMed] [Google Scholar]
  • 37.Schütze, S., Wiegmann, K., Machleidt, T. & Krönke, M. TNF-Induced activation of NF-κB. Immunobiology193, 193–203 (1995). [DOI] [PubMed] [Google Scholar]
  • 38.Wu, Y. & Zhou, B. P. TNF-α/NF-κB/Snail pathway in cancer cell migration and invasion. Br. J. Cancer. 102, 639–644 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Li, H. & Lin, X. Positive and negative signaling components involved in TNFα-induced NF-κB activation. Cytokine41, 1–8 (2008). [DOI] [PubMed] [Google Scholar]
  • 40.Sabio, G. & Davis, R. J. TNF and MAP kinase signalling pathways. Semin Immunol.26, 237–245 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Li, Y. et al. IL-1β/NF-κB signaling inhibits IGF-1 production via let-7f-5p in dendritic epidermal T cells. J. Leukoc. Biol.112, 1677–1690 (2022). [DOI] [PubMed] [Google Scholar]
  • 42.Kitanaka, T. et al. JNK activation is essential for activation of MEK/ERK signaling in IL-1β-induced COX-2 expression in synovial fibroblasts. Sci. Rep.7, 39914 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wang, L. et al. IL-6 induces NF-kappa B activation in the intestinal epithelia. J. Immunol.171, 3194–3201 (2003). [DOI] [PubMed] [Google Scholar]
  • 44.Feng, W. et al. Combination of IL-6 and sIL-6R differentially regulate varying levels of RANKL-induced osteoclastogenesis through NF-κB, ERK and JNK signaling pathways. Sci. Rep.7, 41411 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (312.9KB, pdf)

Data Availability Statement

Regarding data availability, all data generated or analysed during this study are included in this published article. The raw western blot data referenced in the manuscript are provided in the supplementary files. The remaining data are available from the corresponding author upon reasonable request.

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