Combined Exposure to High‐Cholesterol Diet and PM _2.5 : Brain Injury, Behavioral Alteration and Regulatory Mechanism of HIF‐1α in Female Mice

Both high-cholesterol diet (HCD) and fine particulate matter (PM_2.5) exposure are associated with increased stroke risk; however, their combined effects-particularly in females-remain poorly understood. This study aimed to investigate the synergistic effects of HCD and PM_2.5 exposure on brain injury, behavior, and underlying molecular mechanisms in female mice. Female mice were exposed to HCD, PM_2.5 inhalation, or both for 3 months (3M) or 6 months (6M), with control groups for comparison. We assessed brain-to-body weight ratio, behavioral performance, histopathological changes, inflammatory markers (IL-6, TNF-α), and expression of stroke- and blood-brain barrier (BBB)-related proteins. Additionally, we analyzed the epigenetic regulation of hypoxia-inducible factor-1α (HIF-1α) via chromatin immunoprecipitation (ChIP) assays. Co-exposure to HCD and PM_2.5 led to greater alterations in the brain-to-body weight ratio, exacerbated neuropathology, behavioral deficits, and increased neuroinflammation compared to exposure to either factor alone. Moreover, HCD+PM_2.5 significantly altered the expression of HIF-1α and genes involved in its downstream signaling pathway in the brain. ChIP assays revealed that HIF-1α was directly regulated by histone deacetylase 4 (HDAC4) and histone H3 lysine 9 acetylation (H3K9ac), indicating epigenetic modulation in response to dual exposure. HCD and PM_2.5 synergistically promote brain injury in female mice, potentially through HDAC4/H3K9ac-mediated epigenetic activation of the HIF-1α pathway. These findings highlight a novel molecular mechanism that may contribute to increased stroke risk in females exposed to both environmental and metabolic stressors.