Targeting macrophagic RasGRP1 with catechin hydrate ameliorates sepsis-induced multiorgan dysfunction.

BACKGROUND: The proinflammatory response induced by macrophages plays a crucial role in the development of sepsis and the resulting multiorgan dysfunction. Identifying new regulatory targets for macrophage homeostasis and devising effective treatment strategies remains a significant challenge in contemporary research. PURPOSE: This study aims to identify new regulatory targets for macrophage homeostasis and develop effective strategies for treating sepsis. STUDY DESIGN AND METHODS: Macrophage infiltration in septic patients and in lungs, kidneys, and brains of caecum ligation and puncture (CLP)-induced septic mice was observed using CIBERSORT and immunofluorescence (IF). Upon integrating the MSigDB database and GSE65682 dataset, differently expressed macrophage-associated genes (DEMAGs) were identified. Critical DEMAGs were confirmed through machine learning. The protein level of the critical DEMAG was detected in PBMCs of septic patients, RAW264.7 cells, and mice lungs, kidneys, and brains using ELISA, western blot, immunohistochemistry, and IF. siRNA was applied to investigate the effect of the critical DEMAG in RAW264.7 cells. A natural product library was screened to find a compound targeting the critical DEMAG protein. The binding of compounds and proteins was analyzed through molecular docking, molecular dynamics simulations, CETSA, and MST analysis. The therapeutic efficacy of the compounds against sepsis was then evaluated through in vitro and in vivo experiments. RESULTS: Macrophage infiltration was inversely correlated with survival in septic patients. The critical differentially expressed molecule RasGRP1 was frequently observed in the PBMCs of septic patients, LPS-induced RAW264.7 cells, and the lungs, kidneys, and brains of septic mice. Silencing RasGRP1 alleviated proinflammatory response and oxidative stress in LPS-treated RAW264.7 cells. Catechin Hydrate (CH) was identified as an inhibitor of RasGRP1, capable of maintaining macrophage homeostasis and mitigating lung, kidney, and brain damage during sepsis. CONCLUSION: This study demonstrates that RasGRP1, a novel activator of macrophage proinflammatory responses, plays a crucial role in the excessive inflammation and oxidative stress associated with sepsis. CH shows potential for treating sepsis by inhibiting RasGRP1.

Danlou tablet alleviates sepsis-induced acute lung and kidney injury by inhibiting the PARP1/HMGB1 pathway.

Background: Sepsis-associated acute lung injury (ALI) and acute kidney injury (AKI) are common complications that significantly impact patient prognosis. Danlou tablet (DLT) is a traditional herbal preparation with anti-inflammatory and antioxidant properties. However, its therapeutic potential in sepsis remains unknown. Methods: The impact of DLT on ALI and AKI was evaluated using the cecal ligation and puncture (CLP) experimental sepsis animal model. The effects of DLT on macrophages were observed through LPS-stimulated RAW264.7 cell line. Inflammatory cytokines, oxidative stress indicators, HE, PAS, and DHE staining, lung wet-to-dry weight ratio, and serum creatinine and urea nitrogen levels were used to assess tissue injury. Network pharmacology, molecular docking, and molecular dynamics simulations were used to explore the potential regulatory mechanisms of DLT in sepsis. Western blot and immunohistochemical staining were used to validate the expression of mechanism-related proteins. Results: DLT inhibited the inflammatory response and oxidative stress, improved structural and functional abnormalities in lung and kidney tissues in CLP mice, and alleviated pro-inflammatory responses of LPS-stimulated macrophages. PARP1 and HMGB1 were identified as key regulatory targets. The results of in vitro and in vivo experiments suggest that DLT can effectively inhibit PARP1/HMGB1 and improve sepsis-associated ALI and AKI. Conclusion: The present study demonstrated that DLT suppressed pro-inflammatory responses of macrophage and alleviated ALI and AKI in the CLP mice by inhibiting the transition activation of PARP1/HMGB1. These findings partially elucidate the mechanism of DLT in sepsis-associated ALI and AKI and further clarify the active components of DLT, thereby providing a scientific theoretical basis for treating sepsis with DLT.

QiShenYiQi pills preserve endothelial barrier integrity to mitigate sepsis-induced acute lung injury by inhibiting ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The QiShengYiQi pill (QSYQ) is a traditional Chinese medicinal formulation. The effectiveness and safety of QSYQ in treating respiratory system disorders have been confirmed. Its pharmacological actions include anti-inflammation, antioxidative stress, and improving energy metabolism. However, the mechanism of QSYQ in treating sepsis-induced acute lung injury (si-ALI) remains unclear. AIM OF THE STUDY: Si-ALI presents a clinical challenge with high incidence and mortality rates. This study aims to confirm the efficacy of QSYQ in si-ALI and to explore the potential mechanisms, providing a scientific foundation for its application and insights for optimizing treatment strategies and identifying potential active components. MATERIALS AND METHODS: The impact of QSYQ on si-ALI was evaluated using the cecal ligation and puncture (CLP) experimental sepsis animal model. The effects of QSYQ on endothelial cells were observed through coculturing with LPS-stimulated macrophage-conditioned medium. Inflammatory cytokine levels, HE staining, Evans blue staining, lung wet/dry ratio, and cell count and protein content in bronchoalveolar lavage fluid were used to assess the degree of lung injury. Network pharmacology was utilized to investigate the potential mechanisms of QSYQ in treating si-ALI. Western blot and immunofluorescence analyses were used to evaluate barrier integrity and validate mechanistically relevant proteins. RESULTS: QSYQ reduced the inflammation and alleviated pulmonary vascular barrier damage in CLP mice (all P < 0.05). A total of 127 potential targets through which QSYQ regulates si-ALI were identified, predominantly enriched in the RAGE pathway. The results of protein-protein interaction analysis suggest that COX2, a well-established critical marker of ferroptosis, is among the key targets. In vitro and in vivo studies demonstrated that QSYQ mitigated ferroptosis and vascular barrier damage in sepsis (all P < 0.05), accompanied by a reduction in oxidative stress and the inhibition of the COX2 and RAGE (all P < 0.05). CONCLUSIONS: This study demonstrated that QSYQ maintains pulmonary vascular barrier integrity by inhibiting ferroptosis in CLP mice. These findings partially elucidate the mechanism of QSYQ in si-ALI and further clarify the active components of QSYQ, thereby providing a scientific theoretical basis for treating si-ALI with QSYQ.

Identification of the key ferroptosis-related genes involved in sepsis progression and experimental validation in vivo.

Background: Ferroptosis has a vital role in sepsis, but the mechanism is not known. Understanding the mechanism of ferroptosis during sepsis will aid in developing improved therapeutic strategies. Methods: We used the Gene Expression Omnibus database and FerrDb database to obtain ferroptosis-related differentially expressed genes (DEGs) between sepsis patients and healthy volunteers (HVs). Analyses of PPI networks, functional enrichment, as well as use of the MCODE algorithm were used to identify key ferroptosis-related DEGs. Expression of key ferroptosis-related DEGs was verified using: GSE57065 and GSE65682 datasets; rats in which ferroptosis was induced with erastin; sepsis-induced acute lung injury (siALI) rats. The effects of acupoint catgut embedding (ACE) on ferroptosis and expression of key ferroptosis-related DEGs in the lungs of siALI rats were also observed. A Cox proportional hazard model was used to verify the effect of key ferroptosis-related DEGs on the survival of sepsis patients. Cytoscape was used to construct ceRNA networks and gene-transcription factor networks. Results: Between sepsis patients and HVs, we identified 33 ferroptosis-related DEGs. According to analyses of PPI networks and the MCODE algorithm, we obtained four modules, of which the most significant module contained nine ferroptosis-related DEGs. Functional-enrichment analyses showed that four of the nine DEGs were enriched in the MAPK signaling pathway: MAPK14, VEGFA, TGFBR1, and DUSP1. We verified expression of these four genes in GSE57065 and GSE65682 datasets and ferroptosis rats. In addition, expression of these four genes and that of the oxidative-stress indicators GSSG and MDA was upregulated, and glutathione peroxidase-4 (GPX4) expression was downregulated, in siALI rats, but ACE reversed these changes. The Cox proportional hazard model showed that survival of sepsis patients in the high-risk group was shorter than that in the low-risk group. We found that the XIST-hsa-let-7b-5p-TGFBR1/DUSP1 ceRNA network and transcription factor E2F1 may be important regulators of these four DEGs. Conclusion: Our results suggest that MAPK14, VEGFA, TGFBR1, and DUSP1 may be key regulatory targets of ferroptosis in sepsis, and that ACE pretreatment may be antioxidant treatment for sepsis and alleviate ferroptosis. These findings provide a basis for further ferroptosis-related study in sepsis and provide new targets for its treatment.

Exogenous ghrelin ameliorates acute lung injury by modulating the nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB pathway after hemorrhagic shock.

Previous studies have shown that ghrelin, a peptide produced in the stomach, attenuates acute lung injury (ALI) in various animal models, and that some of these effects are associated with inhibition of the nuclear factor κB signaling pathway. This study investigated whether ghrelin exerts beneficial effects on hemorrhagic shock (HS)-induced ALI by modulating nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB (IKK/IκBα/NF-κB) pathway activity. HS was induced in male SD rats by withdrawing blood to a mean arterial pressure (MAP) of 40 mm Hg for 1 h; rats then received ghrelin (10 nmol/kg) or vehicle intravenously and were resuscitated with the shed blood and an equal volume of Ringer lactate solution followed by observation for 2 h. After resuscitation, samples were collected and analyzed for lung histopathology, wet to dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, neutrophil infiltration, plasma inflammatory cytokines (TNF-α and IL-6), and cytoplasmic phosphorylated IKKß, IκBα, phosphorylated IκBα and nuclear NF-κB expression. Compared to those in the two sham groups, lung injury, W/D, BALF protein, neutrophil infiltration, plasma TNF-α and IL-6 levels, and IKK/IκBα/NF-κB pathway activation were significantly increased in HS rats. After ghrelin administration, all parameters analyzed were decreased compared to those without ghrelin in HS rats. Moreover, ghrelin alleviated the decreased MAP after resuscitation compared to that in HS rats. Exogenous ghrelin attenuates the inflammatory response and acute lung injury after HS. These beneficial effects appear to be mediated through inhibition of IKK/IκBα/NF-κB signaling.