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Updated: Jun 28, 2026
Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation
Published on: July 6, 2019
Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury
Yingkai Feng1, Qinghua Yang, Jiancheng Xu
1Institute of Pathology, Southwest Hospital Affiliated to Third Military Medical University, Chongqing 400038, China.
Objective:
To observe the effects of intravenous injection of HMGB1 inhibitor sodium butyrate on changes in apoptosis of PMN during LPS-induced acute lung injury in rats and HMGB1 in vitro on human circulating PMN apoptosis, in order to clarify the role of HMGB1 in the pathogenesis of acute lung injury.
Methods:
(1) LPS-induced acute lung injury rat model was developed by LPS infusion. At different time-points after LPS challenge in the presence or absence of sodium butyrate (SB), the rat tissue sample, peripheral blood PMNs and BALF were collected. RT-PCR was applied to examining rat lung tissue HMGB1 mRNA expression level, and Western blotting analysis was adopted to determine expression of rat lung tissue HMGB1 protein. PMN apoptotic changes were determined by flow cytometric (FCM) analysis, Giemsa staining and TdT-mediated dUTP nick end labeling (TUNEL) method. (2) Separated and purified human circulating PMN were coincubated for 24 h with different doses of HMGB1 (0, 10, 100, 1000 ng/ml, respectively) at 37 degrees C in 5% CO(2). PMN apoptosis rate was determined by flow cytometric (FCM) analysis and by TdT-mediated dUTP nick end labeling (TUNEL) method.
Results:
(1) The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreased as compared with that of normal control. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged significantly as compared with that of normal control. The injured rat lung tissue HMGB1 mRNA and protein expression was upregulated 6-24 h after LPS exposure; SB intervention significantly ameliorated the upregulation. In addition, the morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGB1 expression level, the above changes indicate that the pathological changes of lung tissue were related to the injured lung HMGB1 expression, as well as apoptotic changes in PMN. (2) After coincubation of HMGB1 with human circulating PMNs, TUNEL and flow cytometry were performed. The study revealed that PMN apoptosis ratios was (40.53+/-4.12) % in control group (PMNs+RPMI 1640 medium), (19.05+/-2.44) % in LPS group (PMNs+RPMI 1640 medium+10 microg/ml LPS), (40.52+/-2.73) % in HMGB1-1 group (PMNs+RPMI 1640 medium+10 ng/ml HMGB1), (34.89+/-1.15) % in HMGB1-2 group (PMNs+RPMI 1640 medium+100 ng/ml HMGB1), and (18.77+/-3.02) % in HMGB1-3 group (PMNs+RPMI 1640 medium+1 000 ng/ml HMGB1). There was statistical significance. Meanwhile, PMN TUNEL positive rate was (31.42+/-4.40) %, (31.39+/-3.80) %, (25.62+/-2.46) %, and (17.98+/-3.20) % in control group, HMGB1-1 group, HMGB1-2 group and HMGB1-3 group, respectively. The inhibitory effect was HMGB1 dose-depended as compared with that of control group.
Conclusion:
After LPS challenge, high expression of rats' lung HMGB1 mRNA occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and down-regulated HMGB1 mRNA expression of injured lung. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly, and the inhibitory effect was HMGB1 dose-depended. The results demonstrated that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALI. It concludes that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.
Insights
HMGB1 inhibition by sodium butyrate (SB) reduced apoptosis delay in neutrophils during LPS-induced acute lung injury in rats. In vitro, HMGB1 dose-dependently inhibited human neutrophil apoptosis, suggesting HMGB1’s role in ALI pathogenesis.
Area of Science:
- Immunology
- Cell Biology
- Pathology
Background:
- Neutrophil apoptosis plays a crucial role in resolving inflammation during acute lung injury (ALI).
- High-mobility group box 1 (HMGB1) is implicated in the pathogenesis of ALI, but its specific role in neutrophil apoptosis during this condition requires further clarification.
Purpose of the Study:
- To investigate the effect of the HMGB1 inhibitor sodium butyrate (SB) on neutrophil apoptosis in a rat model of lipopolysaccharide (LPS)-induced ALI.
- To examine the in vitro effect of HMGB1 on human circulating neutrophil apoptosis.
Main Methods:
- Established an LPS-induced ALI rat model, administering SB and collecting samples for analysis of HMGB1 expression and neutrophil apoptosis via RT-PCR, Western blotting, flow cytometry, Giemsa staining, and TUNEL assay.
- Co-incubated isolated human neutrophils with varying doses of HMGB1 in vitro and assessed apoptosis rates using flow cytometry and TUNEL assay.
Main Results:
- LPS challenge in rats led to delayed neutrophil apoptosis and increased HMGB1 expression in lung tissue, which was ameliorated by SB treatment.
- SB treatment reduced lung tissue damage and HMGB1 upregulation in the ALI rat model.
- In vitro, HMGB1 significantly inhibited human neutrophil apoptosis in a dose-dependent manner.
Conclusions:
- HMGB1 expression is upregulated in LPS-induced ALI and contributes to delayed neutrophil apoptosis.
- Sodium butyrate effectively attenuates LPS-induced neutrophil apoptosis delay and reduces HMGB1 expression.
- HMGB1 acts as a significant inhibitor of human neutrophil apoptosis, suggesting its critical role in the development of ALI.
