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Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats

  • Authors:
    • Risa Sakamoto
    • Hiroko Shimizu
    • Ryu Nakamura
    • Yifu Lu
    • Yaqiang Li
    • Emiko Omori
    • Toru Takahashi
    • Hiroshi Morimatsu
  • View Affiliations / Copyright

    Affiliations: Department of Anesthesiology and Resuscitology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama 700‑8558, Japan, Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Okayama 700‑8558, Japan, Department of Human Anatomy, Shantou University Medical College, Shantou, Guangdong 515041, P.R. China, Department of Anesthesiology, Okayama Saidaiji Hospital, Okayama, Okayama 704‑8194, Japan
    Copyright: © Sakamoto et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 22
    |
    Published online on: October 31, 2025
       https://doi.org/10.3892/mmr.2025.13732
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Abstract

Hemorrhagic shock and resuscitation (HSR) induces pulmonary inflammation, leading to acute lung injury (ALI). Notably, blocking β1 receptors can lead to organ protection through anti‑inflammatory and anti‑apoptotic effects. Additionally, although the β1 receptor pathway is blocked by the β1 blocker, the β2 receptor pathway may be preserved and activate the 5' adenosine monophosphate‑activated protein kinase (AMPK) pathway. The present study aimed to examine whether administration of the β1 blocker landiolol could achieve lung protection in a model of HSR‑ALI, alongside improvements in inflammation and apoptosis. Male Sprague‑Dawley rats underwent hemorrhage keeping their mean arterial pressure at 30 mmHg for 1 h. Resuscitation by reinfusion was initiated to restore blood pressure to pre‑hemorrhage levels for >15 min, followed by a 45‑min stabilization period to create the HSR‑ALI model. Landiolol (100 mg/kg/min) or saline was continuously administered after resuscitation. The lung tissues, which were collected for assessing inflammation and apoptosis‑related damage, underwent analyses, including histological examination, neutrophil count, assessment of lung wet/dry weight ratio, detection of the mRNA levels of tumor necrosis factor‑α (TNF‑α) and inducible nitric oxide synthase (iNOS), identification of terminal deoxynucleotidyl transferase dUTP nick‑end labeling (TUNEL)‑positive cells, and evaluation of caspase‑3 expression. In addition, phosphorylated AMPKα (pAMPKα) expression was tested via western blotting. Compared with the HSR/saline group, the HSR/landiolol group demonstrated a reduction in lung tissue damage score, and significant reductions in neutrophil count, lung wet/dry weight ratio, lung TNF‑α and iNOS mRNA levels, TUNEL‑positive cells and cleaved caspase‑3 expression. Furthermore, landiolol administration following HSR treatment increased pAMPKα expression. No significant hypotension occurred between the HSR/landiolol and HSR/saline groups; and blood loss did not differ significantly between the groups. In conclusion, landiolol administration after HSR reduced lung inflammation and apoptosis, suggesting a potential improvement in tissue damage. Furthermore, pAMPKα activation in the HSR/landiolol group may be the mechanism underlying the pulmonary protective effects of landiolol.
View Figures

Figure 1

Experimental design. Establishment of
the HSR rat model and the landiolol administration protocol.
Animals were assigned to four groups (n=10 per group). Sham/saline
group: administered saline after sham surgery; sham/landiolol
group: administered landiolol after sham surgery; HSR/saline group:
administered saline after HS procedure; HSR/landiolol group:
administered landiolol after HS procedure. Rats were euthanized 3
or 24 h after the start of resuscitation; thereafter, samples were
collected. Landiolol or saline was administered 15 min after the
start of resuscitation and continued until 3 h after resuscitation.
HS, hemorrhagic shock; HSR, hemorrhagic shock and resuscitation;
MAP, mean arterial blood pressure.

Figure 2

Landiolol administration did not
affect vital signs during or after HSR. Landiolol or vehicle
(normal saline) was administered through the tail vein immediately
following resuscitation. The MAP and HR were recorded at 5-min
intervals during and after the HSR procedure. (A) MAP measured at
5-min intervals throughout and following HSR. The x-axis represents
the time elapsed since the onset of hemorrhagic shock while the
y-axis shows the MAP values. The gray band at the bottom of the
graph represents the duration of drug or vehicle administration (B)
HR recorded at 5-min intervals during and after HSR. The x-axis
shows the time from the start of hemorrhagic shock, and the y-axis
displays the heart rate. The gray band at the bottom of the graph
represents the duration of drug or vehicle administration. Data are
presented as mean ± standard error of the mean (n=10 per group).HR,
heart rate; HSR, hemorrhagic shock and resuscitation; MAP, mean
arterial blood pressure.

Figure 3

Histological assessment of lung
injury following HSR. Lungs were collected from HSR model rats,
treated with or without landiolol, 24 h after resuscitation for
histological analysis. (A) Representative images from four
independent experiments (H&E staining; original magnification,
×200; scale bar, 100 µm). (B) The severity of histopathological
alterations in the lungs was scored for congestion, edema,
inflammation, and hemorrhage. Ten areas of lung parenchyma per rat
were rated on a scale from 0 (normal) to 3 (severe) for each
parameter, resulting in a maximum of 30 points per parameter.
Scoring was performed independently by five blinded observers, and
data are presented as median (interquartile range) and analyzed
using the Kruskal-Wallis test followed by Dunn's post hoc test. (C)
The total histopathological score was calculated by summing scores
across the four parameters, with a maximum possible score of 120.
Again, scoring was performed by five blinded observers, and the
median values (interquartile range) were analyzed using the
Kruskal-Wallis test followed by Dunn's post hoc test. The sum score
reflects the extent of lung tissue injury for each group.
*P<0.05 vs. sham/saline. Sham/saline and sham/landiolol groups
received sham surgery with saline or landiolol; HSR/saline and
HSR/landiolol groups underwent HSR with saline or landiolol
administration. HSR, hemorrhagic shock and resuscitation.

Figure 4

Neutrophil accumulation in the lungs
following HSR. Lungs from HSR model rats treated with or without
landiolol were collected 24 h post-resuscitation. Neutrophils were
stained using a naphthol AS-D chloroacetate esterase assay. (A)
Representative images from four independent experiments. Black
arrows point to neutrophils stained positively (original
magnification, ×400; scale bar, 50 µm). (B) Neutrophil counts in
five non-consecutive lung sections per rat at ×400 magnification
(n=5 per group). Data are shown as mean ± standard error of the
mean and analyzed using analysis of variance followed by the
Tukey-Kramer test. *P<0.0001 vs. sham/saline;
#P<0.0001 vs. HSR/saline. Sham/saline and
sham/landiolol groups received sham surgery with saline or
landiolol; HSR/saline and HSR/landiolol groups underwent HSR with
saline or landiolol administration. HSR, hemorrhagic shock and
resuscitation.

Figure 5

Lung wet/dry weight ratio following
HSR. Lungs from HSR model rats, treated with or without landiolol,
were collected 24 h post-resuscitation, and the wet/dry weight
ratio was measured. Data are presented as mean ± standard error of
the mean (n=5 per group). Statistical analysis was conducted using
analysis of variance, followed by the Tukey-Kramer test.
*P<0.0001 vs. sham/saline; #P<0.005 vs.
HSR/saline. Sham/saline and sham/landiolol groups received sham
surgery with saline or landiolol; HSR/saline and HSR/landiolol
groups underwent HSR with saline or landiolol administration. HSR,
hemorrhagic shock and resuscitation.

Figure 6

mRNA levels of inflammatory mediators
in the lungs following HSR, measured by RT-qPCR. Lungs from HSR
model rats treated with either saline or landiolol were collected 3
h post-resuscitation, and mRNA levels of (A) TNF-α and (B) iNOS
were measured by RT-qPCR. Data are presented as mean ± standard
error of the mean and analyzed using analysis of variance followed
by the Tukey-Kramer test (n=5 per group). *P<0.05 vs.
sham/saline; #P<0.05 vs. HSR/saline. Sham/saline and
sham/landiolol groups received sham surgery with saline or
landiolol; HSR/saline and HSR/landiolol groups underwent HSR with
saline or landiolol administration. HSR, hemorrhagic shock and
resuscitation; RT-qPCR, reverse transcription-quantitative
polymerase chain reaction; TNF-α, tumor necrosis factor-α; iNOS,
inducible nitric oxide synthase.

Figure 7

Apoptotic cell death in the lungs
following HSR. Lung tissue was collected 24 h post-resuscitation
and assessed for apoptosis using the TUNEL assay and cleaved
caspase-3 protein expression via western blot. (A) Representative
TUNEL-stained images of lung sections 24 h after HSR (original
magnification, ×400). White arrows point to TUNEL-positive cells.
(B) Quantification of TUNEL-positive cells in lung sections at 24 h
post-HSR. Data are presented as mean ± SEM (n=5 per group). (C)
Western blot analysis for caspase-3 and cleaved caspase-3, with
GAPDH as the loading control. (D) Densitometric analysis of cleaved
caspase-3 expression, normalized to caspase-3 levels. Data are
presented as mean ± SEM (n=3 per group). Data were analyzed with
analysis of variance followed by the Tukey-Kramer test. *P<0.05
vs. sham/saline; #P<0.05 vs. HSR/saline. Sham/saline
and sham/landiolol groups received sham surgery with saline or
landiolol; HSR/saline and HSR/landiolol groups underwent HSR with
saline or landiolol administration. HSR, hemorrhagic shock and
resuscitation; SEM, standard error of the mean; TUNEL, terminal
deoxynucleotidyl transferase dUTP nick-end labeling.

Figure 8

Expression of AMPK in the lungs
following HSR. Lung samples were collected 24 h post-resuscitation
and analyzed for AMPK expression by western blotting. (A) Western
blot analysis using an antibody for pAMPKα and AMPKα with GAPDH as
the loading control. (B) Densitometric analysis of pAMPKα levels,
normalized to AMPKα. Data are shown as mean ± SEM and analyzed
using analysis of variance followed by the Tukey-Kramer test (n=3
per group). *P<0.005 vs. sham/landiolol; #P<0.005
vs. HSR/saline. Sham/saline and sham/landiolol groups received sham
surgery with saline or landiolol; HSR/saline and HSR/landiolol
groups underwent HSR with saline or landiolol administration. AMPK,
5′ adenosine monophosphate-activated protein kinase; HSR,
hemorrhagic shock and resuscitation; pAMPK, phosphorylated
AMPK.
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Copy and paste a formatted citation
Spandidos Publications style
Sakamoto R, Shimizu H, Nakamura R, Lu Y, Li Y, Omori E, Takahashi T and Morimatsu H: Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats. Mol Med Rep 33: 22, 2026.
APA
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E. ... Morimatsu, H. (2026). Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats. Molecular Medicine Reports, 33, 22. https://doi.org/10.3892/mmr.2025.13732
MLA
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E., Takahashi, T., Morimatsu, H."Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats". Molecular Medicine Reports 33.1 (2026): 22.
Chicago
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E., Takahashi, T., Morimatsu, H."Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats". Molecular Medicine Reports 33, no. 1 (2026): 22. https://doi.org/10.3892/mmr.2025.13732
Copy and paste a formatted citation
x
Spandidos Publications style
Sakamoto R, Shimizu H, Nakamura R, Lu Y, Li Y, Omori E, Takahashi T and Morimatsu H: Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats. Mol Med Rep 33: 22, 2026.
APA
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E. ... Morimatsu, H. (2026). Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats. Molecular Medicine Reports, 33, 22. https://doi.org/10.3892/mmr.2025.13732
MLA
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E., Takahashi, T., Morimatsu, H."Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats". Molecular Medicine Reports 33.1 (2026): 22.
Chicago
Sakamoto, R., Shimizu, H., Nakamura, R., Lu, Y., Li, Y., Omori, E., Takahashi, T., Morimatsu, H."Protective impact of landiolol against acute lung injury following hemorrhagic shock and resuscitation in rats". Molecular Medicine Reports 33, no. 1 (2026): 22. https://doi.org/10.3892/mmr.2025.13732
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