Sepsis-associated acute liver injury (SAALI) is a common and severe complication in intensive care unit patients, serving as a significant risk factor for mortality in sepsis. Its pathogenesis is intricate, involving dysregulated systemic inflammatory response, oxidative stress, abnormal activation of immune cells, among other processes. However, specific and effective clinical treatments for SAALI are currently lacking. Recent studies indicate that macrophage pyroptosis is a key driver of excessive inflammation and organ damage in sepsis. This process is precisely regulated by oxidative stress and the NLRP3 inflammasome pathway, making it a potential therapeutic target. Angiotensin-(1-7) [Ang-(1-7)], as a protective component of the renin-angiotensin system, exhibits anti-inflammatory, antioxidant, and organ-protective effects in various disease models. Nevertheless, whether it functions in SAALI by regulating macrophage pyroptosis, and its specific cellular targets and upstream molecular mechanisms, remain unclear. Therefore, this study aims to systematically elucidate the protective role of Ang-(1-7) in SAALI and investigate whether it alleviates oxidative stress by activating the SIRT1/Nrf2/HO-1 endogenous antioxidant signaling axis, thereby inhibiting NLRP3 inflammasome-mediated macrophage pyroptosis and ultimately mitigating liver injury.

This study systematically elucidated the protective effect and molecular mechanism of Ang-(1-7) against sepsis-associated acute liver injury by integrating clinical sample analysis, in vivo animal experiments, and in vitro cellular experiments. First, a cross-sectional clinical study was conducted, enrolling 58 subjects (28 sepsis patients and 30 healthy controls). Plasma levels of Ang II, Ang-(1-7), IL-1β, and IL-18 were measured by ELISA, and intergroup comparisons and correlation analyses were performed in conjunction with liver function markers. Second, at the animal level, a sepsis model was established in C57BL/6 mice via intraperitoneal injection of LPS. The protective effects of Ang-(1-7) were evaluated by detecting serum inflammatory factors, liver function markers, liver histopathology, the expression of pyroptosis-related proteins (NLRP3, cleaved caspase-1, and GSDMD-N), and in situ labeling of macrophage pyroptosis using techniques including ELISA, biochemical analysis, H&E staining with semi-quantitative scoring, Western blot, qPCR, and multiplex immunofluorescence staining. At the cellular level, an in vitro pyroptosis model was established by stimulating THP-1-derived macrophages with LPS combined with Nigericin. The effects of Ang-(1-7) on cell viability, inflammatory cytokine secretion, cellular pyroptosis, and the NLRP3/caspase-1/GSDMD signaling pathway were assessed using CCK-8 assay, ELISA, LDH release assay, PI/Hoechst staining, Western blot, and qPCR. Finally, at the mechanistic level, transcriptome sequencing was initially employed to screen the key pathways regulated by Ang-(1-7). Subsequently, its effects on reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), mitochondrial membrane potential (JC-1 staining), and the SIRT1/Nrf2/HO-1 pathway proteins were examined in the cellular model to systematically elucidate its antioxidant effects and upstream molecular mechanisms. These findings were then validated in the animal model by assessing its regulatory effects on hepatic MDA content, SOD activity, and the SIRT1/Nrf2/HO-1 pathway. Finally, loss-of-function experiments using the SIRT1-specific inhibitor EX527 were conducted to confirm the necessity of SIRT1 in the Ang-(1-7)-mediated protective cascade.

Clinical studies revealed that plasma Ang-(1-7) levels were significantly decreased (P < 0.001), and the Ang II/Ang-(1-7) ratio was significantly increased (P < 0.001) in sepsis patients. Furthermore, Ang-(1-7) levels showed a significant negative correlation with the liver injury marker ALP (rho = -0.473, P = 0.013). Concurrently, plasma levels of the pyroptosis-characteristic cytokines IL-1β and IL-18 were significantly elevated in patients compared to healthy controls (P < 0.001). These findings suggest that the imbalance of the RAS in septic patients is closely associated with liver injury and the activation of a systemic pyroptotic state.In vivo experiments demonstrated that Ang-(1-7) pretreatment significantly inhibited the release of serum pro-inflammatory cytokines (TNF-α, IL-1β, IL-18), improved liver function in septic mice (P < 0.05), alleviated liver histopathological damage, and significantly downregulated the expression of pyroptosis-related proteins NLRP3, cleaved caspase-1, and GSDMD-N in liver tissue. Multiplex immunofluorescence further showed that Ang-(1-7) significantly reduced GSDMD aggregation and activation in liver macrophages (F4/80⁺), confirming its inhibitory effect on hepatic macrophage pyroptosis in situ. This indicates that the hepatoprotective effect of Ang-(1-7) is closely associated with its inhibition of NLRP3 inflammasome-mediated pyroptosis in liver macrophages.In vitro experiments confirmed that Ang-(1-7) pretreatment significantly suppressed the gene transcription and protein secretion of inflammatory cytokines (TNF-α, IL-1β, IL-18) induced by LPS/Nig in THP-1 macrophages, improved cell viability, reduced LDH release and the proportion of PI-positive cells, and inhibited NLRP3 inflammasome activation as well as downstream caspase-1-mediated GSDMD cleavage. These results demonstrate that Ang-(1-7) effectively inhibits the NLRP3-mediated pyroptosis signaling pathway at the cellular level.Mechanistic studies using transcriptome sequencing analysis revealed that, compared to the LPS/Nig model group, a total of 285 differentially expressed genes were identified in the Ang-(1-7) intervention group. Subsequent KEGG pathway enrichment analysis suggested that Ang-(1-7) intervention significantly affected oxidative phosphorylation, p53, and NOD-like receptor signaling pathways, indicating that oxidative stress plays a crucial role in macrophage pyroptosis. Functional validation showed that Ang-(1-7) significantly reduced intracellular ROS levels and MDA content, increased SOD activity, improved mitochondrial membrane potential (elevated red/green fluorescence ratio), and coordinately upregulated the protein expression of SIRT1, Nrf2, and HO-1. Moreover, Ang-(1-7) pretreatment also significantly reduced hepatic MDA content, increased SOD activity, and upregulated the expression of SIRT1, Nrf2, and HO-1 proteins in the animal model. These in vivo and in vitro data collectively demonstrate that Ang-(1-7) exerts its antioxidant effects by activating the SIRT1/Nrf2/HO-1 signaling pathway. Key loss-of-function experiments confirmed that the SIRT1-specific inhibitor EX527 partially reversed the aforementioned antioxidant, mitochondrial protective, and anti-pyroptotic effects of Ang-(1-7), as well as its inhibition of the NLRP3/caspase-1/GSDMD signaling pathway, establishing the central role of SIRT1 in this protective cascade.

Ang-(1-7) effectively alleviates oxidative stress and mitochondrial dysfunction by activating the SIRT1/Nrf2/HO-1 endogenous antioxidant signaling axis within macrophages, thereby inhibiting the assembly and activation of the NLRP3 inflammasome and blocking caspase-1/GSDMD-mediated macrophage pyroptosis, ultimately mitigating sepsis-induced liver injury. This not only deepens our understanding of the role of the RAS system in immune regulation but also links it to anti-pyroptosis and oxidative stress, providing a novel theoretical foundation and potential therapeutic targets for sepsis and its associated liver injury.