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Article Open Access

Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress

  • Authors:
    • Miao Xue
    • Junshuai Feng
    • Xiaorong Mao
  • View Affiliations / Copyright

    Affiliations: Department of Rheumatology and Immunology, The First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P.R. China, Department of Infectious Diseases, Gansu Provincial Hospital, Lanzhou, Gansu 730000, P.R. China, Department of Infectious Diseases, The First Hospital of Lanzhou University, Lanzhou, Gansu 730000, P.R. China
    Copyright: © Xue et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 214
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    Published online on: June 8, 2026
       https://doi.org/10.3892/ijmm.2026.5885
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Abstract

Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). It is characterized by autoantibody deposition and immune complex formation within the kidney, leading to progressive nephron loss and end‑stage renal disease. Suppression of immunology is a cornerstone for managing LN. The present study identified withaferin A (WA) as a promising therapeutic candidate for SLE via connectivity map analysis and validated its efficacy in ameliorating LN in an MRL/lpr mouse model. Candidate drugs for LN were identified via the Connectivity Map database by integrating transcriptomic signatures from GSE135779, GSE162577 and GSE142016. An animal model was established to evaluate the therapeutic efficacy of WA on LN‑associated inflammation and splenic dysfunction, followed by RNA‑sequencing analysis. To elucidate the mechanistic role of PON1 in WA‑mediated protection, siRNA‑mediated knockdown and plasmid‑driven overexpression were performed in HK‑2 cells. Furthermore, the direct impact of WA was assessed using isolated primary B cells. WA improved renal function by mitigating splenic immune cell dysregulation and attenuating renal inflammation. Mechanistically, RNA‑sequencing analysis and functional validation revealed that WA upregulated paraoxonase 1 (Pon1) expression, which in turn alleviates renal injury by decreasing reactive oxygen species via the peroxisome proliferator‑activated receptor signaling pathway. Pon1 activation enhanced cell viability, suppressed inflammatory responses and decreased oxidative stress in HK‑2 cells, underscoring its potential as a novel therapeutic target for LN. Collectively, the findings demonstrate WA is a viable candidate for SLE/LN treatment and Pon1 is a pivotal mediator of its protective effects, thereby providing a dual‑strategy insight for clinical intervention.
View Figures

Figure 1

WA is a potential therapeutic agent
for lupus nephritis. (A) CMAP query flow chart. DEGs in B cells in
GSE135779, GSE162577 and GSE142016 datasets from patients with SLE
and healthy donors were used to query the CMAP. (B) Top ten
compounds with positive connectivity scores identified by the CMAP
analysis. (C) Candidate rank according to the connectivity score.
CMAP, Connectivity Map; DEG, differentially expressed gene; IKK,
inhibitor of κB kinase; PLK, polo-like kinase; SLE, systemic lupus
erythematosus.

Figure 2

WA improves the renal function in
lupus nephritis. (A) Body weight change in lupus mice after WA
treatment. (B) Urinary protein concentrations. Serum (C) creatinine
and (D) BUN levels. (E) Spleen, (F) liver and (G) kidney weight.
*P<0.05, **P<0.01 vs. control. WA,
withaferin A; BUN, blood urea nitrogen.

Figure 3

WA attenuates splenic immune cell
dysfunction in lupus nephritis. (A) Representative spleen, lymph
node and kidneys from lupus mice in the presence or absence of WA
treatment. Splenic mRNA expression of (B) MCP-1, (C) TNF-α, (D)
IL-1β and (E) Foxp3. Serum (F) ANA and (G) dsDNA levels.
*P<0.05, **P<0.01,
***P<0.001. WA, withaferin A; MCP, monocyte
chemoattractant protein; KU, kilo unit; ANA, antinuclear antibody;
ds, double-stranded.

Figure 4

WA suppresses renal pathohistological
changes in lupus nephritis. (A) Representative histological images
of the kidney from lupus mice. Scale bar, 50 μm.
Quantitative analysis of (B) glomerular, (C) tubulointerstitial and
(D) vascular scores. (E) Representative glomerular expression of
(F) IgG and (G) C3 in the kidneys from lupus mice. Scale bar, 10
μm. ***P<0.001. WA, withaferin A; HE,
hematoxylin eosin; PAS, periodic acid Schiff.

Figure 5

WA alleviates renal inflammation in
lupus nephritis. (A) Representative immunohistochemical staining of
(B) α-SMA expression in the kidney of lupus mice after WA
treatment. Scale bar, 50 μm. Renal mRNA levels of (C) MCP-1,
(D) TNF-α and (E) IL-1β. (F) Western blotting analysis of renal
MCP-1, TNF-α, IL-1β and KIM-1 expression. **P<0.01,
***P<0.001. WA, withaferin A; SMA, smooth muscle
actin; MCP, monocyte chemoattractant protein; KIM, kidney injury
molecule.

Figure 6

RNA sequencing analysis of kidney in
SLE mice after WA treatment. (A) Hierarchical clustered heatmap of
gene expression profiles for WA- or vehicle-treated SLE mice. (B)
GO enrichment analysis of DEGs. (C) KEGG pathway analysis of DEGs.
(D) Volcano plot of gene expression profiles. (E) Number of DEGs
(q<0.05). The top seven genes are presented. SLE, systemic lupus
erythematosus; WA, withaferin A; GO, gene ontology; DEG,
differentially expressed genes; KEGG, Kyoto encyclopedia of genes
and genomes; TPM, transcripts per kilobase; Dmkn, dermokine; Pon,
paraoxonase; Ankef, ankyrin repeat and EF-hand domain containing;
Mup, major urinary protein; Gm, predicted gene; Car, carbonic
anhydrase; Klb, klotho β.

Figure 7

WA alleviates inflammation and
eliminates reactive oxygen species in HK-2 cells by regulating
Pon1/PPAR signaling under inflammatory conditions. (A) Viability of
HK-2 cells after WA treatment in the presence or absence of si-Pon1
transfection. All cells were pretreated with LPS. mRNA expression
of (B) MCP-1, (C) TNF-α and (D) IL-1β and (E) western blotting
analysis of the expression of MCP-1, TNF-α, IL-1β and KIM-1 in HK-2
cells. (F) Pon1 expression levels. (G) Protein expression of PPAR-α
and PPAR-γ. (H) LDH and (I) SOD activities. (J) MDA levels in HK-2
cells. (K) Expression of Bax and BCl-2. **P<0.01,
***P<0.001. WA, withaferin A; Pon, paraoxonase; si,
small interfering; LPS, lipopolysaccharide; MCP, monocyte
chemoattractant protein; LDH, lactate dehydrogenase; SOD,
superoxide dismutase; MDA, malondialdehyde.

Figure 8

WA suppresses activation of B cells
from systemic lupus erythematosus mice. (A) IgG levels in B cell
culture medium. mRNA levels of genes related to B cell
differentiation, including (B) CD19, (C) CD20, (D) CD21 and (E)
CD22. (F) mRNA levels of CD24. ***P<0.001. WA,
withaferin A.

Figure 9

Pon1 activation improves the
inflammation and eliminates ROS in HK-2 cells. (A) Pon1 expression
in HK-2 cells following ZnPA treatment. (B) Viability of HK-2 cells
following ZnPA treatment. All cells were pretreated with
lipopolysaccharide. (C) Western blotting analysis of MCP-1, TNF-α,
IL-1β and KIM-1 expression. (D) Protein expression of PPAR-α and
PPAR-γ. (E) Expression of Bax and BCl-2. (F) ROS levels.
***P<0.001. ZnPA, (Z)-2,3-bis
(4-nitrophenyl)-acrylonitrile; Pon, paraoxonase; MCP, monocyte
chemoattractant protein; KIM, kidney injury molecule; ROS, reactive
oxygen species.

Figure 10

Pon1 overexpression attenuates
inflammation and eliminates ROS in HK-2 cells. (A) Pon1 expression
in HK-2 cells following plasmid transfection. (B) Viability of HK-2
cells following plasmid transfection. All cells were pretreated
with lipopolysaccharide. ELISA of (C) TNF-α, (D) IL-6 and (E)
IL-1β. (F) ROS levels. *P<0.05,
**P<0.01, ***P<0.001. OE,
overexpression; Pon, paraoxonase; ROS, reactive oxygen species.
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Copy and paste a formatted citation
Spandidos Publications style
Xue M, Feng J and Mao X: Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress. Int J Mol Med 58: 214, 2026.
APA
Xue, M., Feng, J., & Mao, X. (2026). Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress. International Journal of Molecular Medicine, 58, 214. https://doi.org/10.3892/ijmm.2026.5885
MLA
Xue, M., Feng, J., Mao, X."Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress". International Journal of Molecular Medicine 58.2 (2026): 214.
Chicago
Xue, M., Feng, J., Mao, X."Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress". International Journal of Molecular Medicine 58, no. 2 (2026): 214. https://doi.org/10.3892/ijmm.2026.5885
Copy and paste a formatted citation
x
Spandidos Publications style
Xue M, Feng J and Mao X: Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress. Int J Mol Med 58: 214, 2026.
APA
Xue, M., Feng, J., & Mao, X. (2026). Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress. International Journal of Molecular Medicine, 58, 214. https://doi.org/10.3892/ijmm.2026.5885
MLA
Xue, M., Feng, J., Mao, X."Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress". International Journal of Molecular Medicine 58.2 (2026): 214.
Chicago
Xue, M., Feng, J., Mao, X."Withaferin A, a candidate drug for lupus nephritis, confers renal protection via Pon1‑mediated attenuation of oxidative stress". International Journal of Molecular Medicine 58, no. 2 (2026): 214. https://doi.org/10.3892/ijmm.2026.5885
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