Nec-1s

Neuroinflammation is a critical pathophysiological mechanism of depression. But the sources and processes involved remain unclear. Recent reports suggest that necroptosis with pro-inflammatory properties may facilitate inflammation. Therefore, we investigated the potential role of necroptosis-associated neuroinflammation in depression.

Depression model mice induced by intraperitoneal injection of lipopolysaccharide (LPS) were treated with RIPK1 inhibitor Necrostatin-1 s (Nec-1 s, 6 mg/kg), RIPK3 inhibitor GSK'872 (6 mg/kg) or intracerebroventricular injection of MLKL inhibitor GW806742X (5 μL of 200 μmol/L). Depressive-like behaviors were assessed using sucrose preference test and tail suspension test. Serum inflammatory cytokines were detected by ELISA, while glial biomarkers were determined by western blots. Hematoxylin & eosin and immunohistochemical staining were utilized to identify morphological characteristics of necroptotic cells in the hippocampus and prefrontal cortex. Further, specific molecules involved in necroptotic pathway were measured by immunoblots.

Mice treated with LPS exhibited depressive-like behaviors, as well as increased inflammatory cytokines, enhanced MLKL phosphorylation, and decreased cleaved Caspase-8 levels in hippocampus. GSK'872 rather than Nec-1 s exhibited significant antidepressant effects. Although necroptosis was present in both the hippocampus and prefrontal cortex, neuroinflammation was mainly manifested in the hippocampus. Additionally, GSK'872 restored the elevated levels of IL-1β, TNF-α, and HMGB1 in the serum and hippocampus of model mice, and simultaneously ameliorated necroptosis. However, neither GSK'872 nor Nec-1 s had sufficient effect on Caspase-8 and microgliosis. Furthermore, intracerebroventricular injection of GW806742X improved depressive-like behavior and neuroinflammation in hippocampus.

This study provides novel evidence that hippocampal RIPK3-MLKL-dependent necroptosis mediates depressive-like behavior induced by inflammatory stress. During this process, necroptosis may facilitate neuroinflammation by promoting the release of HMGB1. Interventions targeting this pathway may help treat depression with an inflammatory phenotype.

Human oral squamous cell carcinoma (OSCC) represents a significant global health challenge, with conventional treatments showing limited efficacy in improving patient survival rates. To investigate the therapeutic potential of acetylshikonin on OSCC, we conducted comprehensive analyses including cell viability assays, flow cytometry, and molecular pathway investigations. Our findings demonstrate that acetylshikonin significantly inhibits OSCC cell proliferation with IC50 values of 3.81 μM and 5.87 μM in HSC3 and SCC4 cells respectively. Flow cytometry analysis revealed that acetylshikonin treatment significantly increased reactive oxygen species (ROS) production and decreased mitochondrial membrane potential in OSCC cells. Additionally, Western blot analysis showed enhanced phosphorylation of RIPK1, RIPK3, and MLKL proteins, indicating activation of the necroptotic pathway. The critical role of necroptosis was further confirmed using specific inhibitors (GSK872, Necrostatin-1, and 7-CL-O Nec-1), which significantly attenuated acetylshikonin-induced cell death. Transmission electron microscopy revealed distinct ultrastructural changes in cellular organelles, while decreased GPX4 expression suggested potential cross-activation of ferroptotic pathways. These data demonstrate that acetylshikonin suppresses OSCC growth through selective activation of oxidative stress-mediated necroptosis and mitochondrial dysfunction, identifying it as a promising natural compound for OSCC therapy through its ability to activate alternative cell death pathways and overcome traditional therapy limitations.