HAT regimen attenuates NF-κb-driven megakaryocyte apoptosis and neuronal cell death in sepsis: convergent mechanisms protecting thrombocytopenia and cognitive function.

Background: Sepsis-induced NF-κB hyperactivation drives two devastating cell death cascades: megakaryocyte apoptosis causing thrombocytopenia (incidence 35%-59%), and neuronal apoptosis with microglial-mediated neuroinflammation causing cognitive dysfunction in up to 70% of survivors. Mechanistically, NF-κB-driven upregulation of pro-apoptotic mediators (cleaved caspase-3, cytochrome c release) impairs megakaryopoiesis while simultaneously inducing hippocampal neuronal death and synaptic loss. The HAT regimen (hydrocortisone, ascorbic acid, and thiamine) modulates complementary nodes of this NF-κB/apoptosis axis, yet its cell-autonomous mechanisms of protecting megakaryocytes and neurons from sepsis-induced programmed cell death remain uncharacterized. Methods: We employed a multi-level translational approach to interrogate HAT-mediated cell survival mechanisms. A retrospective cohort of 184 propensity score-matched sepsis patients with thrombocytopenia provided clinical validation. Mechanistic studies used cecal ligation and puncture (CLP) in C57BL/6 mice, with cell death profiling (TUNEL, cleaved caspase-3, Annexin V), blood-brain barrier integrity assays, and synaptic protein quantification. Results: HAT synergistically suppressed NF-κB p65 nuclear translocation by 52% in megakaryoblasts and 54% in hippocampal tissue, reducing pro-inflammatory cytokines by 35%-42%. In megakaryocytes, HAT inhibited apoptosis by 48.6% and enhanced proplatelet formation by 52.4%, corresponding to accelerated platelet recovery (78.5% vs. 42.3% increase at day 7, P < 0.001) and reduced 28-day mortality (22.8% vs. 34.8%, P = 0.038) in patients. In the CLP model, HAT reduced hippocampal neuronal apoptosis (TUNEL+ cells -54.8%; cleaved caspase-3+ neurons -58.2%), attenuated microglial activation (Iba-1+ cells -48.6%), preserved blood-brain barrier integrity (Evans blue extravasation -62.4%), and maintained synaptic protein expression (PSD-95 + 42.6%; synaptophysin +38.4%), translating to significant cognitive and psychological benefit in survivors. Each HAT component contributed distinct anti-apoptotic mechanisms-hydrocortisone suppressed p65 translocation, ascorbic acid blocked ROS-mediated IKK activation, and thiamine restored mitochondrial membrane potential-producing formally synergistic (Chou-Talalay CI = 0.61 in megakaryocytes and CI = 0.58 in hippocampal neurons, both <1.0) pro-survival effects exceeding individual component efficacy. HAT group membership remained an independent predictor of preserved cognition on multivariable regression adjusting for ventilation duration and ICU stay (adjusted OR 0.38, 95% CI 0.18-0.79, P = 0.009). Conclusion: HAT therapy protects two distinct cell populations-megakaryocytes and hippocampal neurons-from sepsis-induced programmed cell death through convergent, component-specific suppression of NF-κB-driven apoptotic signaling. These mechanistic findings reframe HAT as a broad-spectrum anti-apoptotic intervention, providing a cellular and molecular rationale consistent with its dual clinical benefit against thrombocytopenia and cognitive dysfunction in sepsis. Given the null findings of major HAT RCTs in unselected populations, these mechanisms particularly support biomarker-enriched trial designs.