-Humulene Improves Cerebral Ischemia/Reperfusion Injury by Blocking KCNA3 to Inhibit Microglia Activation

Background: alpha-Humulene (Hum) can alleviate cerebral ischemia/reperfusion (I/R) injury. However, the mechanism of Hum in microglial activation induced by cerebral I/R injury remains unclear. This study aims to investigate the role of Hum in microglial activation resulting from cerebral I/R injury. Methods: Several cerebral I/R injury models were established in adult Sprague-Dawley (SD) rats treated with 25 and 50 mg/kg Hum, and in primary microglia extracted from newborn SD rats treated with 0.25, 0.5, and 1.0 mM Hum. Neurological deficits were assessed using a modified neurological severity score. Cerebral infarction was assessed by triphenyltetrazolium chloride staining. Immunofluorescence assay was applied to measure the levels of potassium voltage-gated channel subfamily A member 3 (KCNA3) and ionized calcium-binding adapter molecule 1 (Iba-1). Gene expression levels of KCNA3, NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), and brain-derived neurotrophic factor (BDNF) were measured by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were measured using enzyme-linked immune sorbent assay (ELISA). Additionally, a rescue assay was used to investigate whether the inhibitory effect of Hum on microglial activation was mediated through KCNA3 regulation. Results: Hum significantly reduced neurological deficits and cerebral infarction in rats with cerebral I/R injury (p < 0.001). Both in vivo and in vitro, Hum reduced the levels of Iba-1, KCNA3, and NLRP3, yet promoted the level of BDNF in cerebral I/R injury models (p < 0.01). Moreover, Hum suppressed the levels of TNF-alpha and IL-6 in oxygen-glucose deprivation/reperfusion (OGD/R)-exposed microglia (p < 0.001). Notably, KCNA3 overexpression reversed the effects of Hum on Iba-1, TNF-alpha, IL-6, NLRP3, and BDNF in OGD/R-exposed microglia (p < 0.001). Conclusion: Hum improves cerebral I/R injury by blocking KCNA3 to inhibit microglial activation.