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Article Open Access

CTSB mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury

  • Authors:
    • Shuang He
    • Lei Wang
    • Zhe Wang
    • Shiyong Yu
    • Qi Zhu
    • Yijun Xu
    • Yuhang Jiang
    • Yingxia Wu
    • Honggang Xiang
  • View Affiliations / Copyright

    Affiliations: Department of General Surgery, Shanghai Pudong New Area People's Hospital, Shanghai 201299, P.R. China, Department of General Surgery, Jinshazhou Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510168, P.R. China
    Copyright: © He et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 39
    |
    Published online on: November 12, 2025
       https://doi.org/10.3892/mmr.2025.13749
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Abstract

Intestinal ischemia‑reperfusion (I/R) injury is a clinical condition that leads to severe intestinal damage, inflammation and oxidative stress. While cathepsin B (CTSB) has been implicated in these pathophysiological processes, its precise role in mediating I/R‑induced injury remains poorly understood. The present study aimed to elucidate how CTSB knockdown influences oxidative stress, inflammatory responses and the integrity of the intestinal epithelial barrier in intestinal epithelial Caco‑2 cells subjected to I/R injury. To identify key genes implicated in I/R injury, a comprehensive analysis was conducted using differential expression profiling and protein‑protein interaction network analysis of the GSE37013 dataset. To simulate I/R damage in vitro, an oxygen‑glucose deprivation/reoxygenation (OGD/R) model was employed in Caco‑2 cells. Subsequently, inflammation was induced by stimulating the cells with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). To investigate the role of CTSB in this context, small interfering RNA was utilized to knock down CTSB expression. In vitro assays were then performed to evaluate NLR family pyrin domain‑containing 3 (NLRP3) inflammasome activation, oxidative stress levels, inflammatory cytokine production and cell survival. The results revealed that intestinal tissues from the I/R group in the GSE37013 dataset showed markedly higher CTSB expression, and the Caco‑2 cells subjected to OGD/R model resulted in a considerable increase in CTSB expression. However, the expression levels of tight junction proteins were enhanced, cell survival was improved and lactate dehydrogenase release was reduced by CTSB knockdown. This reduction in CTSB levels also reduced malondialdehyde levels, and alleviated oxidative stress by increasing the activities of glutathione peroxidase and superoxide dismutase. Furthermore, pro‑inflammatory cytokine production was reduced, and NLRP3 inflammasome activation was inhibited by CTSB knockdown, although a modest increase was still observed after LPS + ATP stimulation. Notably, although CTSB knockdown significantly reduced the inflammatory response, LPS + ATP stimulation still elicited a modest reversal in cytokine levels, suggesting that a CTSB‑independent pathway of inflammatory activation may exist. In conclusion, CTSB knockdown effectively mitigates I/R injury by reducing inflammation, preserving barrier integrity and alleviating oxidative stress, positioning CTSB as a promising therapeutic target. Future work should validate these findings in in vivo models and explore CTSB‑targeted therapies to improve clinical outcomes in I/R‑related diseases.
View Figures

Figure 1

Screening of DEGs, PPI network
construction and intersection analysis based on the GSE37013
dataset to identify hub genes. (A) Volcano plot of DEGs in the
GSE37013 dataset. Green represents 181 downregulated DEGs and blue
represents 224 upregulated DEGs. PPI network analysis of
upregulated DEGs using (B) degree, (C) MCC and (D) MNC algorithms,
and visualization of the top 15 genes in each network. (E) Venn
diagram of the intersection analysis of the top 15 genes of the
degree, MCC and MNC algorithms, where the overlapping genes
represent the intersection genes. (F) Box plot of the expression
levels of 13 intersection genes in control and case samples from
the GSE37013 dataset. *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001. CTSB, cathepsin B; DEG, differentially
expressed gene; MCC, maximal clique centrality; MNC, maximum
neighborhood component; PPI, protein-protein interaction.

Figure 2

CTSB expression is elevated in
intestinal barrier damage induced by ischemia-reperfusion. (A) Cell
Counting Kit-8 detected the viability of Caco-2 cells in the
control and OGD/R-treated (2, 4 and 6 h) groups. (B) LDH activity
detection in Caco-2 cells from the control and OGD/R-treated (2, 4
and 6 h) groups. Reverse transcription-quantitative PCR was used to
detect the relative mRNA expression levels of (C) ZO-1 and
(D) CLDN1 in Caco-2 cells in the control and OGD/R-treated
(2, 4 and 6 h) groups. (E) Enzyme-linked immunosorbent assay
detected CTSB activity in Caco-2 cells in the control and
OGD/R-treated (4 h) groups. (F) Western blot analysis was used to
detect the protein expression levels of CTSB and active CTSB in
Caco-2 cells in the control and OGD/R-treated (4 h) groups, and (G)
semi-quantitative analysis was performed. All data were obtained
from at least three independent biological experiments (n=3).
*P<0.05, **P<0.01 vs. control. CLDN1, claudin-1; CTSB,
cathepsin B; LDH, lactate dehydrogenase; OGD/R, oxygen-glucose
deprivation/reoxygenation.

Figure 3

Effects of CTSB knockdown on
cell viability, LDH activity and tight junction protein expression
in OGD/R-treated Caco-2 cells. (A) Reverse
transcription-quantitative PCR analysis of the relative expression
levels of CTSB mRNA in the control group, the OGD/R + si-NC
group and the OGD/R + si-CTSB group. (B) Representative
western blot of CTSB protein in the Control, OGD/R + si-NC and
OGD/R + si-CTSB groups. GAPDH was used as the loading
control. (C) Densitometric semi-quantification of CTSB protein
expression (normalized to GAPDH). (D) Cell Counting Kit-8 detected
the viability of Caco-2 cells in the control, OGD/R (4 h) + si-NC
and OGD/R (4 h) + si-CTSB groups. (E) LDH activity of Caco-2
cells was detected in the control, OGD/R (4 h) + si-NC and OGD/R (4
h) + si-CTSB groups. (F) WB analysis of the protein
expression of CTSB, ZO-1 and claudin-1 in control, OGD/R (4 h) +
si-NC and OGD/R (4 h) + si-CTSB Caco-2 cell groups, and (G)
semi-quantitative analysis. Immunofluorescence analysis of (H) ZO-1
and (I) claudin-1 in Caco-2 cells in the control, OGD/R (4 h) +
si-NC, and OGD/R (4 h) + si-CTSB groups. Magnification, ×40.
All data were obtained from at least three independent biological
experiments (n=3). *P<0.05, **P<0.01 vs. control;
#P<0.05 vs. OGD/R (4 h) + si-NC. CTSB, cathepsin B;
LDH, lactate dehydrogenase; NC, negative control; OGD/R,
oxygen-glucose deprivation/reoxygenation; si, small interfering;
WB, western blot.

Figure 4

CTSB knockdown mitigates
ischemia-reperfusion-induced oxidative stress and inflammation.
Detection of (A) ROS levels, (B) MDA levels, (C) SOD activity and
(D) GSH-Px activity in Caco-2 cells in the control, OGD/R (4 h) +
si-NC and OGD/R (4 h) + si-CTSB groups. Enzyme-linked
immunosorbent assay detected the activities of the cytokines (E)
TNF-α, (F) IL-6 and (G) IL-1β in Caco-2 cells in the control, OGD/R
(4 h) + si-NC and OGD/R (4 h) + si-CTSB groups. All data
were obtained from at least three independent biological
experiments (n=3). **P<0.01 vs. control; #P<0.05
vs. OGD/R (4 h) + si-NC. CTSB, cathepsin B; GSH-Px,
glutathione peroxidase; MDA, malondialdehyde; NC, negative control;
OGD/R, oxygen-glucose deprivation/reoxygenation; ROS, reactive
oxygen species; si, small interfering; SOD, superoxide
dismutase.

Figure 5

Knockdown of CTSB inhibits
activation of the NLRP3 inflammasome in Caco-2 cells injured by
OGD/R. (A) Representative WB analysis of inflammasome-related
proteins in Caco-2 cells under control conditions, OGD/R (4 h) +
si-NC and OGD/R (4 h) + si-CTSB. Each GAPDH blot shown
corresponds to the target protein immediately above it.
Semi-quantitative analysis of protein expression levels: (B) NLRP3,
CTSB, ASC, IL-1β and IL-18; (C) cleaved caspase-1/caspase-1 ratio;
and (D) GSDMD-N/GSDMD ratio. (E) Co-IP of CTSB and NLRP3 (IP: CTSB;
IB: NLRP3 and CTSB). Inputs and IgG controls are shown. (F)
Reciprocal co-IP (IP: NLRP3; IB: CTSB and NLRP3) confirming the
interaction between CTSB and NLRP3 under OGD/R conditions. Reverse
transcription-quantitative PCR detection of the relative mRNA
expression levels of the cytokines (G) TNF-α, (H)
IL-6 and (I) IL-1β in Caco-2 cells in the control,
OGD/R (4 h) + si-NC, OGD/R (4 h) + si-CTSB and OGD/R (4 h) +
si-CTSB + LPS + ATP groups. All data were obtained from at
least three independent biological experiments (n=3). *P<0.05,
**P<0.01, ***P<0.001 vs. control; #P<0.05 vs.
OGD/R (4 h) + si-NC; &P<0.05 vs. OGD/R (4 h) +
si-CTSB. ATP, adenosine triphosphate; CTSB, cathepsin B;
GSDMD, gasdermin D; GSDMD-N, cleaved N-terminal GSDMD; IB,
immunoblot; IP, immunoprecipitation; LPS, lipopolysaccharide; NC,
negative control; NLRP3, NLR family pyrin domain-containing 3;
OGD/R, oxygen-glucose deprivation/reoxygenation; si, small
interfering; WB, western blot.

Figure 6

Effects of CTSB knockdown and
LPS + ATP treatment on cell viability, oxidative stress and tight
junction protein expression in OGD/R-treated Caco-2 cells. (A)
Western blot analysis of the protein expression of CTSB, ZO-1 and
claudin-1 in Caco-2 cells in the control, OGD/R (4 h) + si-NC,
OGD/R (4 h) + si-CTSB and OGD/R (4 h) + si-CTSB + LPS
+ ATP groups, and (B) semi-quantitative analysis. (C) Cell Counting
Kit-8 detected the viability of Caco-2 cells in the control, OGD/R
(4 h) + si-NC, OGD/R (4 h) + si-CTSB and OGD/R (4 h) +
si-CTSB + LPS + ATP groups. Detection of (D) MDA levels and
(E) SOD activity in Caco-2 cells in the control, OGD/R (4 h) +
si-NC, OGD/R (4 h) + si-CTSB and OGD/R (4 h) +
si-CTSB + LPS + ATP groups. All data were obtained from at
least three independent biological experiments (n=3). *P<0.05,
**P<0.01, ***P<0.001 vs. Control. #P<0.05,
##P<0.01 vs. OGD/R (4 h) + si-NC.
&P<0.05 vs. OGD/R (4 h) + si-CTSB + LPS +
ATP. ATP, adenosine triphosphate; CTSB, cathepsin B; LPS,
lipopolysaccharide; MDA, malondialdehyde; NC, negative control;
OGD/R, oxygen-glucose deprivation/reoxygenation; si, small
interfering; SOD, superoxide dismutase.
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Copy and paste a formatted citation
Spandidos Publications style
He S, Wang L, Wang Z, Yu S, Zhu Q, Xu Y, Jiang Y, Wu Y and Xiang H: <em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury. Mol Med Rep 33: 39, 2026.
APA
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y. ... Xiang, H. (2026). <em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury. Molecular Medicine Reports, 33, 39. https://doi.org/10.3892/mmr.2025.13749
MLA
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y., Jiang, Y., Wu, Y., Xiang, H."<em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury". Molecular Medicine Reports 33.1 (2026): 39.
Chicago
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y., Jiang, Y., Wu, Y., Xiang, H."<em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury". Molecular Medicine Reports 33, no. 1 (2026): 39. https://doi.org/10.3892/mmr.2025.13749
Copy and paste a formatted citation
x
Spandidos Publications style
He S, Wang L, Wang Z, Yu S, Zhu Q, Xu Y, Jiang Y, Wu Y and Xiang H: <em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury. Mol Med Rep 33: 39, 2026.
APA
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y. ... Xiang, H. (2026). <em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury. Molecular Medicine Reports, 33, 39. https://doi.org/10.3892/mmr.2025.13749
MLA
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y., Jiang, Y., Wu, Y., Xiang, H."<em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury". Molecular Medicine Reports 33.1 (2026): 39.
Chicago
He, S., Wang, L., Wang, Z., Yu, S., Zhu, Q., Xu, Y., Jiang, Y., Wu, Y., Xiang, H."<em>CTSB</em> mediates oxidative stress and intestinal epithelial barrier disruption in intestinal ischemia‑reperfusion injury". Molecular Medicine Reports 33, no. 1 (2026): 39. https://doi.org/10.3892/mmr.2025.13749
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