Metformin Mitigates Sepsis-Induced Acute Lung Injury and Inflammation in Young Mice by Suppressing the S100A8/A9-NLRP3-IL-1β Signaling Pathway

Abstract

Background: Globally, the subsequent complications that accompany sepsis result in remarkable morbidity and mortality rates. The lung is among the vulnerable organs that incur the sepsis-linked inflammatory storm and frequently culminates into ARDS/ALI. The metformin-prescribed anti-diabetic drug has been revealed with anti-inflammatory effects in sepsis, but the underlying mechanisms remain unclear. This study aimed to ascertain metformin's effects and functions in a young mouse model of sepsis-induced ALI.

Methods: Mice were randomly divided into 4 groups: sham, sham+ Met, CLP, and CLP+ Met. CLP was established as the sepsis-induced ALI model accompanied by intraperitoneal metformin treatment. At day 7, the survival state of mice was noted, including survival rate, weight, and M-CASS. Lung histological pathology and injury scores were determined by hematoxylin-eosin staining. The pulmonary coefficient was used to evaluate pulmonary edema. Furthermore, IL-1β, CCL3, CXCL11, S100A8, S100A9 and NLRP3 expression in tissues collected from lungs were determined by qPCR, IL-1β, IL-18, TNF-α by ELISA, caspase-1, ASC, NLRP3, P65, p-P65, GSDMD-F, GSDMD-N, IL-1β and S100A8/A9 by Western blot.

Results: The data affirmed that metformin enhanced the survival rate, lessened lung tissue injury, and diminished the expression of inflammatory factors in young mice with sepsis induced by CLP. In contrast to sham mice, the CLP mice were affirmed to manifest ALI-linked pathologies following CLP-induced sepsis. The expressions of pro-inflammatory factors, for instance, IL-1β, IL-18, TNF-α, CXCL11, S100A8, and S100A9 are markedly enhanced by CLP, while metformin abolished this adverse effect. Western blot analyses indicated that metformin inhibited the sepsis-induced activation of GSDMD and the upregulation of S100A8/A9, NLRP3, and ASC.

Conclusion: Metformin could improve the survival rate, lessen lung tissue injury, and minimize the expression of inflammatory factors in young mice with sepsis induced by CLP. Metformin reduced sepsis-induced ALI via inhibiting the NF-κB signaling pathway and inhibiting pyroptosis by the S100A8/A9-NLRP3-IL-1β pathway.

Keywords: ALI; inflammation; pediatric; pyroptosis; sepsis.

Figure 1

The impact of metformin pre-treatment on survival state. (A) Kaplan–Meier curves for time for 7 days post-operation (n = 20). (B) The M-CASS score changes with time during the CLP-induced sepsis 7-day follow-up (mean ± S.E.M), (C–E). Changes of the weight between pre-operation and post-operation. **p<0.01, ***p<0.001, ****p<0.0001 (CLP+ Met vs CLP); ^###p<0.001 (sham vs CLP).

Figure 2

Metformin alleviated sepsis induced by CLP in mice lung injury. (A) At the end of 24 h following CLP, the histopathological lung injury was evaluated utilizing the HE staining (100x, bar = 200µm; 400x, bar = 50µm). (B) Histopathologic lung injury scores. (C) Lung wet weight/body weight within 24h CLP-induced sepsis. Met + CLP vs CLP, metformin treatment lowered the lung histopathological score in septic mice, and this suggests that this drug can improve the lung tissues with pathological injury. **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Metformin pre-treatment regulated inflammatory responses in sepsis-induced mice. (A–F) The levels of pro-inflammatory cytokines (IL-1β, CCL3, CXCL11 S100A8, S100A9), and the inflammasome NLRP3 (G–I) were measured by qPCR assay, (D–F) pro-inflammatory cytokines (IL-1β, TNF-α IL-18) were examined by ELISA assay in the lung tissues. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

Metformin exerts inhibitory effects on the NF -κB pathway by repressing the release of S00A8/A9 in inflammatory cells. (A–F) S00A8/A9, p65, and p-P65 protein quantification was performed through Western blot and was measured by densitometry. *p<0.05,**p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

Metformin inhibits pyroptosis via suppressing NLRP3 inflammasome activation and suppressing the cleavage of GSDMD. (A–H) NLRP3, ASC, caspase-1, GSDMD-F, GSDMD-N, and IL-1β, protein quantification was performed through Western blot and was measured by densitometry. *p<0.05,**p<0.01.

Figure 6

Presentation of metformin protecting sepsis-induced ALI in young mice by repressing S100A8/A9-NLRP3-IL-1β signaling pathway.