Mechanism of Artesunate Regulating NLRP3/ASC/Caspase-1 Signaling Pathway to Reduce Inflammation and Protect Neurological Function in Mice with Intracerebral Hemorrhage

Abstract

Background The inflammatory response is a major factor in the progression of intracerebral hemorrhage (ICH). Artesunate (ART) has antibacterial and anti-inflammatory pharmacological activity with high concentrations in the brain, but its neuroprotective effect on cerebral hemorrhage injury remains unclear. Objective To observe the effect of ART on inflammatory response after intracerebral hemorrhage and explore its mechanism. Methods From March 2022 to February 2023, 108 male C57BL/6 mice aged 8 to 10 weeks were selected and randomly divided into the sham-operated group (Sham group, n=36), ICH control group (ICH+Vehicle group, n=36) and ART treatment group (ICH+ART group, n=36). ICH model was established. The mice in the ICH+ART group were intraperitoneally injected with 150 mg·kg-1·d-1 2 h after modeling, and the mice in the ICH+Vehicle group were intraperitoneally injected with 5% sodium bicarbonate solution for 3 consecutive days. The behavioral indicators of mice were observed. The brain tissue damage of mice in each group was observed by HE staining; the number of positive cells per unit area of interleukin (IL) 6, IL-1β and myeloperoxidase (MPO) were detected by immunohistochemical staining; the activation of microglia/macrophages was observed by IBA1 immunofluorescence staining. TUNEL/NeuN immunofluorescence double staining was performed to observe neuronal death. The levels of MPO, IL-1β, tumor necrosis factor α (TNF-α), Nod-like receptor protein 3 (NLRP3), apoptosis-related granuloid protein (ASC) and cysteine aspartic protease 1 (Caspase-1) were compared by western blotting. Results The modified neurological severity scores and percentages of right turn of mice were higher in the ICH+Vehicle group than the Sham group, and lower in the ICH+ART group than the ICH+Vehicle group (P<0.05). HE staining results showed that there was a small amount of blood around the striatum in the Sham group and negligible edema. In the ICH+Vehicle group, brain tissue was seriously damaged, with increased intercellular space, perivascular hematoma and inflammatory cell infiltration. All of the above symptoms were significantly improved in the ICH+ART group. The numbers of IL-6, IL-1β and MPO positive cells in the ICH+Vehicle group were higher than the Sham group, the numbers of IL-6, IL-1β and MPO positive cells in the ICH+ART group were lower than the ICH+Vehicle group (P<0.05). The number of activated microglia/macrophages in the ICH+Vehicle group was higher than the Sham group, the number of activated microglia/macrophages in the ICH+ART group was lower than the ICH+Vehicle group (P<0.05). The number of neuron death in the ICH+Vehicle group was higher than the Sham group, and lower in the ICH+ART group than the ICH+Vehicle group (P<0.05). The levels of MPO, IL-1β, TNF-α, NLRP3, ASC and Caspase-1 in the ICH+Vehicle group were higher than the Sham group, the levels of MPO, IL-1β, TNF-α, NLRP3, ASC and Caspase-1 in the ICH+ART group were lower than the ICH+Vehicle group (P<0.05) . Conclusion ART therapy after ICH attenuates the inflammatory response of striatum and the activation of microglia in mice through targeted regulation of NLRP3/ASC/Caspase-1 signaling pathway, and ultimately reduces striatal neuronal apoptosis and improves brain edema.

Keywords

• cerebral hemorrhage|inflammation|artesunate|nod-like receptor 3|caspase-1|mouse