Dl-3-n-butylphthalide Alleviates Cardiac Dysfunction and Injury Possibly by Inhibiting Cell Pyroptosis and Inflammation via the cGAS-STING-TBK1 Pathway in a Porcine Model of Hemorrhage-Induced Cardiac Arrest
Authors: Zhou Ting , Liu Yong , Zhu Lijun , Jiang Jian , Chen Qijiang , Li Lulu , Wu Xianlong , Xu Jiefeng , Fang Jianjiang 
Published: January 2025

ABSTRACT

Introduction
Dl-3-n-butylphthalide (NBP), a small molecular compound extracted from celery seeds, has been shown to exhibit diverse pharmacological activities, including anti-inflammatory, antioxidative, and anti-apoptotic effects. Recent studies have highlighted its efficacy in treating various cardiovascular conditions, such as myocardial infarction, hypertrophy, heart failure, and cardiotoxicity. This study aimed to investigate whether NBP could alleviate cardiac dysfunction and injury following hemorrhage-induced cardiac arrest (HCA) in a porcine model and elucidate its potential mechanisms.

Methods
Seventeen pigs were randomized into three groups: Sham (n = 5), HCA + vehicle (n = 5), and HCA + NBP (n = 7). In the HCA + vehicle and HCA + NBP groups, the HCA model was established by continuous bleeding at a rate of 2 mL/kg/min to induce cardiac arrest. Cardiac arrest was maintained for 7 min, followed by the reinfusion of 50% of the shed blood at a rate of 5 mL/kg/min. After successful resuscitation, the HCA + NBP group received an intravenous dose of 2.5 mg/kg of NBP within 120 min. Post-resuscitation cardiac function (stroke volume, global ejection fraction) and injury biomarkers (cardiac troponin I, creatine kinase-MB) were assessed at regular intervals. At the end of the post-resuscitation observation, cardiac tissue samples were collected to assess: 1) histopathological injury, 2) cellular apoptosis, 3) levels of pro-inflammatory cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-18 (IL-18), 4) the expression levels of NOD-like receptor pyrin domain 3 (NLRP3), caspase 1, gasdermin D (GSDMD), cyclic-GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and tank-binding kinase 1 (TBK1), and 5) the integrated optical density (IOD) of GSDMD N-terminal (GSDMD-N), phosphorylated STING (p-STING), and phosphorylated TBK1 (p-TBK1).

Results
Following resuscitation, both stroke volume and global ejection fraction were significantly reduced, while serum levels of cardiac troponin I and creatine kinase-MB were markedly elevated in the HCA + vehicle and HCA + NBP groups compared with the Sham group. However, the extent of cardiac dysfunction and injury was significantly attenuated in the HCA + NBP group relative to the HCA + vehicle group. At 24 h post-resuscitation, substantial cardiac pathological injury and apoptosis were observed. Additionally, pyroptosis-related proteins (NLRP3, caspase-1, GSDMD, GSDMD-N) were upregulated, inflammatory markers (TNF-α, IL-1β, IL-6, IL-18) were elevated, and the activation of the cGAS-STING-TBK1 pathway (cGAS, STING, TBK1, p-STING, p-TBK1) were noted in both the HCA + vehicle and HCA + NBP groups compared to the Sham group. Notably, these pathological changes were significantly attenuated in the HCA + NBP group compared to the HCA + vehicle group.

Conclusions
NBP provided substantial cardiac protection following HCA and resuscitation in pigs. This protective effect was likely mediated through the inhibition of cell pyroptosis and inflammation by suppressing the cGAS-STING-TBK1 signaling pathway.