L-menthol mediates cGAS-STING signaling in microglia to decrease neuroinflammation and pyroptosis, thereby alleviating brain injury in intracerebral hemorrhage mice

L-menthol (LM), as the primary active component of peppermint, has been demonstrated to exhibit pharmacological effects like anti-inflammatory and antioxidant properties. However, its neuroprotective effects following intracerebral hemorrhage (ICH) and the underlying mechanisms remain unclear. In vivo experiments employed collagenase injection to establish an ICH mouse model, with mice divided into sham, ICH, and LM (5, 10, 20 mg/kg) treatment groups. Following neurological and behavioral assessments, pathological staining was performed to examine brain tissue lesions in the mice. Inflammatory cytokines, microglial polarization, the cGAS-STING signaling, and pyroptosis-related protein levels were assessed using ELISA, immunofluorescence, and Western blot. Pathway specificity was validated via intracerebroventricular injection of cGAMP (a STING agonist). In vitro experiments established a damage model by stimulating BV2 microglia with hemin, establishing treatment groups with different concentrations of LM (0.5, 1, 2 μM) and a cGAMP intervention group to verify pathway specificity. Cell viability, apoptosis, pyroptosis, and related pathway protein expression were assessed through Cell Counting Kit-8, flow cytometry, lactate dehydrogenase (LDH) assays, and Western blot. LM improved neurological deficits in ICH mice, reduced brain water content and pathological brain tissue damage, and diminished neuronal degeneration and apoptosis. Simultaneously, LM inhibited microglia activation, neutrophil infiltration, and the release of pro-inflammatory mediators, while downregulating the cGAS-STING pathway and NLRP3 inflammasome protein expression. cGAMP intervention reduced the aforementioned neuroprotective effects of LM. In vitro experiments demonstrated that LM at 0.5–2 μM did not affect BV2 cell viability. Furthermore, LM enhanced cell viability following hemin treatment while reducing both apoptosis and pyroptosis rates. Additionally, LM reduced inflammatory cytokine levels, and suppressed the cGAS-STING pathway, but cGAMP intervention attenuated these effects of LM. LM mitigates brain injury in ICH mice by suppressing the cGAS-STING signaling in microglia, thereby inhibiting neuroinflammation and pyroptosis.