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Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells

  • Authors:
    • Yeyang Chen
    • Hua Wu
    • Xiangjian Wu
    • Likun Che
    • Xiaoyan Fu
    • Ruihan Qi
    • Dan Jia
    • Minfang Li
    • Wei Xie
    • Wenxiang Zhu
  • View Affiliations / Copyright

    Affiliations: School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, SAR 999077, P.R. China, School of Life Sciences, Beijing University of Chinese Medicine, Beijing 100029; P.R. China, The Fourth Clinical Medical College, Guangzhou University of Chinese Medicine/Shenzhen Hospital of Traditional Chinese Medicine, Shenzhen, Guangdong 518033, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 158
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    Published online on: April 15, 2026
       https://doi.org/10.3892/ijmm.2026.5829
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Abstract

Influenza A (H1N1) virus‑induced pneumonia poses a significant clinical challenge because of excessive immune activation and limited targeted therapies. While current antivirals suppress viral replication, they fail to control absent in melanoma 2 (AIM2) inflammasome‑mediated hyperinflammation, a key driver of immunopathology and mortality. The present study investigated whether triptolide (TP) ameliorates H1N1‑induced pneumonia by targeting this unmet need. The results demonstrated that H1N1 infection reduced the levels of inflammatory cytokines in both human bronchial epithelial cells (HBEpiCs) and THP‑1 cells and decreased the adhesion between the two cell types. Furthermore, high‑dose H1N1 activation triggered the AIM2 signaling pathway in THP‑1 cells but not in HBEpiCs and decreased the viability of THP‑1 cells. In vitro and in vivo experiments both confirmed that TP effectively inhibited inflammation in alveolar epithelial cells and immune cells, as well as the adhesion between these cell types. Additionally, TP reduced the viral load of H1N1 in a murine pneumonia model. Further studies revealed that TP inactivated the AIM2 signaling pathway in THP‑1 cells but not in HBEpiCs. However, overexpression of AIM2 in THP‑1 cells markedly reversed the anti‑inflammatory effects of TP. Based on these findings, it was hypothesized that TP modulates the immune response by inactivating the AIM2 signaling pathway in immune cells, which reduces excessive immune cell activation and decreases the harmful interactions between immune cells and alveolar epithelial cells, ultimately alleviating the lung inflammation induced by H1N1 virus infection. The present study proposed a novel mechanism by which TP alleviates lung damage by suppressing AIM2‑driven immune hyperactivation in immune cells, thereby reducing harmful crosstalk with epithelial cells. This is the first evidence of AIM2‑targeted therapy positioning TP as a promising candidate for treating viral pneumonia.
View Figures

Figure 1

H1N1 infection affects cell
viability, inflammatory cytokine secretion and interactions between
HBEpiCs and THP-1 cells. (A) CCK-8 assay revealed that HBEpiC
viability decreased in a concentration-dependent manner following
H1N1 infection. (B) ELISA revealed that the levels of IL-1β, IL-6,
TNF-α, and IL-8 in HBEpiCs decreased with increasing H1N1
infection. (C) CCK-8 assay indicated that supernatants from
H1N1-infected HBEpiC cultures reduced the viability of THP-1 cells
in a dose-dependent manner. (D) ELISA results suggested that the
levels of inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8) in
THP-1 cells were decreased following exposure to supernatants from
H1N1-infected HBEpiC cultures. (E) Cell adhesion assay revealed
that the number of THP-1 cells adhering to HBEpiCs increased with
increasing H1N1 concentration (scale bar, 10 μm). Arrow
indicates THP-1 cells that remain attached to the surface of
HBEpiCs, highlighting the adhesion interaction between the two cell
types. (F) Transwell assay suggested that H1N1 infection enhanced
the migration capacity of THP-1 cells, with increased migration
observed at higher virus concentrations (scale bar, 50 μm).
The data are presented as the mean ± standard deviation;
**P<0.01, ***P<0.001 vs. control. H1N1,
influenza A; HBEpiCs, human bronchial epithelial cells; ELISA,
enzyme-linked immunosorbent assay; IL, interleukin; TNF-α, tumor
necrosis factor-α; Con, control; MOI, multiplicity of
infection.

Figure 2

Differential activation of AIM2
signaling in THP-1 cells and HBEpiCs following H1N1 infection. (A)
RT-qPCR analysis revealed that H1N1 infection did not alter AIM2
mRNA levels in HBEpiCs but markedly increased AIM2 mRNA levels in
THP-1 cells. (B) LDH release assay indicated that H1N1 infection
did not affect LDH release in HBEpiCs but markedly increased LDH-1
release in THP-1 cells. (C) Dual-immunofluorescence staining
demonstrated the formation of ASC-AIM2 complexes in THP-1 cells
post-H1N1 infection (scale bar, 10 μm). (D) Western blot
analysis revealed no significant changes in AIM2, caspase-1, or
GSDMD protein levels in HBEpiCs following H1N1 infection but
increased expression of AIM2, caspase-1 and GSDMD in THP-1 cells
following H1N1 infection (E). (F) Flow cytometry analysis revealed
no significant cell death in HBEpiCs following infection, (G)
whereas THP-1 cells treated with the supernatant from H1N1-infected
HBEpiC cultures presented markedly increased cell death compared
with the control. The data are presented as the mean ± standard
deviation; *P<0.05, **P<0.01,
***P<0.001 vs. control. AIM2, absent in melanoma 2;
HBEpiCs, human bronchial epithelial cells; H1N1, influenza A;
RT-qPCR, reverse transcription-quantitative PCR; GSDMD, gasdermin
D; Con, control; MOI, multiplicity of infection.

Figure 3

TP modulates the inflammatory
response and immune cell activity in H1N1-infected HBEpiCs and
THP-1 cells. (A) No significant changes were observed in HBEpiCs
treated with various concentrations of TP (5, 10 and 20 nM)
following H1N1 infection compared with the control. (B) After TP
treatment, the levels of the inflammatory cytokines IL-1β, IL-6,
TNF-α and IL-8 in HBEpiCs were markedly lower than those in the
untreated group. (C) The viability of THP-1 cells pretreated with
H1N1-infected HBEpiC culture supernatant decreased after TP
treatment. (D) The levels of IL-1β, IL-6, TNF-α, and IL-8 in THP-1
cells were markedly lower after TP treatment. (E) The adhesion of
THP-1 cells to HBEpiCs induced by H1N1 infection decreased in a
dose-dependent manner with increasing TP concentration (scale bar,
10 μm). Arrow indicates THP-1 cells that remain attached to
the surface of HBEpiCs, highlighting the adhesion interaction
between the two cell types. (F) The migration capacity of THP-1
cells was markedly reduced when the supernatant from H1N1-infected
HBEpiC cultures was treated with TP (scale bar, 50 μm). The
data are presented as the mean ± standard deviation;
*P<0.05, **P<0.01,
***P<0.001 vs. control. TP, triptolide; H1N1,
influenza A; HBEpiCs, human bronchial epithelial cells; IL,
interleukin; TNF-α, tumor necrosis factor-α; Con, control.

Figure 4

Effects of TP on AIM2 signaling, LDH
leakage, and pyroptosis in HBEpiCs and THP-1 cells. (A) RT-qPCR
analysis revealed that TP treatment did not alter AIM2 mRNA
expression in HBEpiCs but markedly reduced AIM2 mRNA levels in
THP-1 cells. (B) TP did not affect the LDH leakage rate in HBEpiCs
but markedly reduced LDH leakage in THP-1 cells. (C)
Dual-fluorescence staining revealed that TP treatment decreased the
formation of ASC-AIM2 complexes in THP-1 cells (scale bar, 10
μm). (D) Western blot analysis indicated that TP treatment
did not markedly affect the expression of these proteins in
HBEpiCs. (E) TP treatment reduced the protein expression of AIM2,
caspase-1, and GSDMD in THP-1 cells. (F) Flow cytometry revealed no
difference in cell death between HBEpiCs treated with 20 nM TP and
the control. (G) In THP-1 cells infected with H1N1 and treated with
20 nM TP, a significant reduction in cell death was identified
compared with that in the H1N1 infection group. The data are
presented as the mean ± standard deviation; *P<0.05,
**P<0.01, ***P<0.001 vs. control. TP,
triptolide; AIM2, absent in melanoma 2; LDH, lactate dehydrogenase;
HBEpiCs, human bronchial epithelial cells; RT-qPCR, reverse
transcription-quantitative PCR; H1N1, influenza A; GSDMD, gasdermin
D; Con, control.

Figure 5

AIM2 overexpression reverses the
immunosuppressive effects of TP in THP-1 cells. (A) Transfection
with Ad-AIM2 upregulated the expression of AIM2 compared with that
of Ad-NC in THP-1 cells. (B) AIM2 protein levels were elevated in
THP-1 cells overexpressing AIM2 compared with control THP-1 cells.
(C) In THP-1 cells, AIM2 overexpression increased the LDH leakage
rate. (D) AIM2 overexpression reversed the TP-induced reduction in
adhesion between THP-1 cells and HBEpiCs (scale bar, 10 μm).
(E) AIM2 overexpression enhanced the migration ability of THP-1
cells compared with that of H1N1-treated control cells and reversed
the TP-induced decrease in THP-1 cell migration (scale bar, 50
μm). (F) AIM2 overexpression reversed the TP-induced
reduction in inflammatory cytokine levels. The data are presented
as the mean ± standard deviation; *P<0.05,
**P<0.01, ***P<0.001 vs. Con;
#P<0.05, ##P<0.01,
###P<0.001 vs. Ad-AIM2. AIM2, absent in melanoma 2;
TP, triptolide; LDH, lactate dehydrogenase; HBEpiCs, human
bronchial epithelial cells; H1N1, influenza A; GSDMD, gasdermin D;
IL, interleukin; TNF-α, tumor necrosis factor-α; Con, control.

Figure 6

TP modulates the immune response and
alleviates lung injury in a murine H1N1 pneumonia model. (A)
Representative images of hematoxylin and eosin stained sections
(magnification, ×20; red arrows demarcate regions of pulmonary
consolidation; scale bar, 100 μm). (B) H1N1 infection led to
increased lung weight compared with that in the control group,
whereas lung weight was markedly reduced in the Tamiflu, L-TP,
M-TP, and H-TP groups. (C) No significant changes in body weight
were observed among the treatment groups. (D) The lung index was
decreased in the H1N1 group and did not fully recover in the four
treatment groups. (E) The Tamiflu, L-TP, M-TP, and H-TP treatment
groups presented markedly reduced viral loads compared with the
H1N1 group, with the Tamiflu group exhibiting the most significant
reduction. (F-I) Following H1N1 infection, inflammatory cytokine
levels were elevated, but treatment with Tamiflu, L-TP, M-TP, and
H-TP markedly reduced these levels in a dose-dependent manner. The
data are expressed as the mean ± standard deviation;
*P<0.05, **P<0.01,
***P<0.001, vs. Con; #P<0.05,
##P<0.01, ###P<0.001 vs. H1N1;
$$P<0.01 vs. H1N1+L-TP. TP, triptolide; H1N1,
influenza A; Con, control.
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Copy and paste a formatted citation
Spandidos Publications style
Chen Y, Wu H, Wu X, Che L, Fu X, Qi R, Jia D, Li M, Xie W, Zhu W, Zhu W, et al: Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells. Int J Mol Med 57: 158, 2026.
APA
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R. ... Zhu, W. (2026). Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells. International Journal of Molecular Medicine, 57, 158. https://doi.org/10.3892/ijmm.2026.5829
MLA
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R., Jia, D., Li, M., Xie, W., Zhu, W."Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells". International Journal of Molecular Medicine 57.6 (2026): 158.
Chicago
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R., Jia, D., Li, M., Xie, W., Zhu, W."Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells". International Journal of Molecular Medicine 57, no. 6 (2026): 158. https://doi.org/10.3892/ijmm.2026.5829
Copy and paste a formatted citation
x
Spandidos Publications style
Chen Y, Wu H, Wu X, Che L, Fu X, Qi R, Jia D, Li M, Xie W, Zhu W, Zhu W, et al: Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells. Int J Mol Med 57: 158, 2026.
APA
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R. ... Zhu, W. (2026). Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells. International Journal of Molecular Medicine, 57, 158. https://doi.org/10.3892/ijmm.2026.5829
MLA
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R., Jia, D., Li, M., Xie, W., Zhu, W."Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells". International Journal of Molecular Medicine 57.6 (2026): 158.
Chicago
Chen, Y., Wu, H., Wu, X., Che, L., Fu, X., Qi, R., Jia, D., Li, M., Xie, W., Zhu, W."Triptolide exerts antiviral effects and alleviates influenza A‑induced pneumonia by inhibiting the overactivation of absent in melanoma 2 signaling in immune cells". International Journal of Molecular Medicine 57, no. 6 (2026): 158. https://doi.org/10.3892/ijmm.2026.5829
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