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Ethyl acetate fraction of Curcuma longa leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells

  • Authors:
    • Hyun-Jae Jang
    • Yuri Kang
    • Eun-Jae Park
    • Eunmi Park
    • Woonjung Kim
    • Soyoung Lee
    • Seung Woong Lee
  • View Affiliations / Copyright

    Affiliations: Functional Biomaterials Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Jeollabuk 56212, Republic of Korea, Department of Chemistry, Hannam University, Daejeon 34054, Republic of Korea, Department of Food and Nutrition, Hannam University, Daejeon 34054, Republic of Korea, Department of Innovative Pharmaceutical Sciences, Advanced Institute of Science and Technology, Kyungpook National University, Daegu, Gyeongsangbuk 41566, Republic of Korea
    Copyright: © Jang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 35
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    Published online on: January 20, 2026
       https://doi.org/10.3892/br.2026.2108
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Abstract

The interleukin‑6 (IL‑6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays a pivotal role in regulating inflammation and tumorigenesis, making it a significant target for therapeutic intervention. Although the rhizome of Curcuma longa (C. longa) has been extensively investigated, the pharmacological potential of its aerial parts, such as the leaves, remains largely unexplored. The present study investigated the inhibitory effects of the ethyl acetate fraction of C. longa leaf extract (CL‑E) on IL‑6‑induced STAT3 activation in Hep3B hepatoma cells in vitro. CL‑E significantly suppressed IL‑6‑stimulated STAT3 transcriptional activity in a dose‑dependent manner (IC50, 33.9±3.7 µg/ml) without compromising cell viability (up to 60 µg/ml). Mechanistically, CL‑E reduced STAT3 phosphorylation at Tyr705 while enhancing STAT3 phosphorylation at Ser727; this opposing pattern suggests an ERK‑mediated rebalancing of STAT3 signaling that attenuates canonical Tyr705‑dependent activation and augments Ser727‑associated modulation. Additionally, CL‑E inhibited STAT3 nuclear translocation and downregulated the mRNA expression of STAT3 target genes, including C‑reactive protein and suppressor of cytokine signaling 3. Mechanistic investigations revealed that CL‑E promoted extracellular signal‑regulated kinase (ERK)1/2 phosphorylation, leading to increased STAT3 Ser727 phosphorylation. Notably, the inhibitory effect of CL‑E on STAT3 Tyr705 phosphorylation was reversed by MEK1/2 (U0126) or PKC inhibitors (bisindolylmaleimide II), indicating that CL‑E modulates STAT3 signaling through an ERK‑mediated mechanism. Collectively, these in vitro findings identify C. longa leaves as an underutilized botanical resource with the potential to regulate IL‑6/STAT3 signaling, warranting in vivo evaluation to establish its efficacy, safety and pharmacokinetics, and to define potential therapeutic utility in IL‑6/STAT3‑driven conditions.

View Figures

Figure 1

Ethyl acetate fraction of CL-E
inhibits IL-6-induced STAT3 activation in Hep3B cells. (A) Hep3B
cells stably expressing STAT3-responsive luciferase reporter
(pSTAT3-Luc) were treated with IL-6 (10 ng/ml) for 12 h in the
presence or absence of CL-E or an aqueous fraction of C.
longa L. leaves extract at concentrations of 10, 30, and 60
µg/ml. Luciferase activity was measured according to the
manufacturer's instructions. Data are presented as relative
luciferase activity normalized to the untreated control. (B) Hep3B
cells were seeded in 96-well plates and treated with each fraction
for 24 h at the indicated concentrations. Cell viability was
assessed using the MTT assay. Results are expressed as the
percentage of viable cells relative to the untreated control. Data
represent the mean ± SE (n≥3). *P<0.05 and
**P<0.01 vs. the IL-6 alone group;
##P<0.01 indicates a significant difference between
the 10 and 60 µg/ml CL-E doses. CL-E, Curcuma longa L.
extract; IL-6, interleukin 6; STAT3, signal transducer and
activator of transcription 3.

Figure 2

CL-E inhibits IL-6-induced STAT3
phosphorylation and nuclear translocation in Hep3B cells. (A) Hep3B
cells were treated with IL-6 (10 ng/ml) for the indicated time
periods (0-60 min). Total cell lysates were analyzed by western
blotting using antibodies against P-STAT3 (Tyr705), P-STAT3
(Ser727) and total STAT3. (B) Cells were pretreated with the CL-E
at 10, 30 and 60 µg/ml for 1 h and then stimulated with IL-6 (10
ng/ml) for 30 min. Proteins were extracted and analyzed by western
blotting as described above. (C) STAT3 nuclear translocation was
assessed by immunofluorescence microscopy. Hep3B cells were
cultured on Nunc Lab-Tek II 8-well chamber slides and treated with
IL-6 (10 ng/ml) for 4 h in the absence or presence of CL-E (60
µg/ml) or genistein (60 µM, positive control). Cells were fixed,
permeabilized and stained with anti-STAT3 antibody followed by
FITC-conjugated secondary antibody. Subcellular localization of
STAT3 was visualized using fluorescence microscopy (Scale bar: 20
µm). CL-E, Curcuma longa L. extract; IL-6, interleukin 6;
STAT3, signal transducer and activator of transcription 3; P-,
phosphorylated.

Figure 3

ERK signaling pathway contributes to
CL-E-mediated regulation of IL-6-induced STAT3 activation. (A)
Hep3B cells were stimulated with IL-6 (10 ng/ml) for the indicated
times (0, 5, 10, 30 and 60 min). Protein lysates underwent western
blotting analysis using antibodies against P-ERK1/2 and total
ERK1/2 to evaluate time-dependent ERK1/2 activation. (B) Cells were
pretreated with CL-E at 10, 30 and 60 µg/ml for 1 h, followed by
IL-6 stimulation (10 ng/ml) for 30 min. ERK1/2 phosphorylation was
analyzed by western blotting analysis. (C) To investigate the role
of ERK in STAT3 Ser727 phosphorylation, cells were treated with
CL-E (60 µg/ml), PKC inhibitor bisindolylmaleimide II (20 µM),
and/or the MEK1/2 inhibitor U0126 (20 µM) for 1 h in the absence of
IL-6. Phosphorylation of STAT3 (Ser727) and total STAT3 were
examined by western blotting analysis. (D) To determine whether ERK
activation contributes to regulation of STAT3 Tyr705
phosphorylation, cells were pretreated with CL-E, U0126 and/or
bisindolylmaleimide II (20 µM) for 1 h before IL-6 stimulation (10
ng/ml, 30 min). STAT3 Tyr705 phosphorylation was analyzed by
western blotting. CL-E, Curcuma longa L. extract; IL-6,
interleukin 6; STAT3, signal transducer and activator of
transcription 3; P-, phosphorylated.

Figure 4

CL-E inhibits IL-6-induced expression
of CRP and SOCS3 mRNA in Hep3B cells. Hep3B cells were pretreated
with CL-E at concentrations of 10, 30, and 60 µg/ml for 1 h,
followed by IL-6 stimulation (10 ng/ml) for 6 h. Total RNA was
extracted, and the mRNA levels of (A) CRP and (B) SOCS3 were
measured using quantitative real-time PCR. Gene expression levels
were normalized to 18S rRNA and are presented as fold change
relative to the untreated control. Data represent the mean ± SE
(n≥3). *P<0.05, **P<0.01 and
***P<0.001 vs. the IL-6-treated group;
###P<0.001. CL-E, Curcuma longa L. extract;
IL-6, interleukin 6; CRP, C reactive protein; P-, phosphorylated;
SOCS3, suppressor of cytokine signaling 3.
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Spandidos Publications style
Jang H, Kang Y, Park E, Park E, Kim W, Lee S and Lee SW: <p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>. Biomed Rep 24: 35, 2026.
APA
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., & Lee, S.W. (2026). <p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>. Biomedical Reports, 24, 35. https://doi.org/10.3892/br.2026.2108
MLA
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., Lee, S. W."<p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>". Biomedical Reports 24.3 (2026): 35.
Chicago
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., Lee, S. W."<p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>". Biomedical Reports 24, no. 3 (2026): 35. https://doi.org/10.3892/br.2026.2108
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Spandidos Publications style
Jang H, Kang Y, Park E, Park E, Kim W, Lee S and Lee SW: <p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>. Biomed Rep 24: 35, 2026.
APA
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., & Lee, S.W. (2026). <p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>. Biomedical Reports, 24, 35. https://doi.org/10.3892/br.2026.2108
MLA
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., Lee, S. W."<p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>". Biomedical Reports 24.3 (2026): 35.
Chicago
Jang, H., Kang, Y., Park, E., Park, E., Kim, W., Lee, S., Lee, S. W."<p>Ethyl acetate fraction of <em>Curcuma longa</em> leaves suppresses IL‑6‑induced STAT3 activation via ERK signaling in Hep3B cells</p>". Biomedical Reports 24, no. 3 (2026): 35. https://doi.org/10.3892/br.2026.2108
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