Supplementary file 1_A selective agonist of the prostacyclin receptor alleviates microglial and astroglial neuroinflammatory responses through P38 and NLRP3.docx

IntroductionThe selective prostacyclin (IP) receptor agonist selexipag is FDA-approved for the treatment of pulmonary arterial hypertension. Selexipag also has anti-inflammatory effects in peripheral tissues, but the ability of selexipag to modulate central neuroinflammation has not been comprehensively examined. Therefore, this study investigated the effect of selexipag on LPS-mediated neuroinflammatory responses in vitro and in vivo.
MethodsTo examine the effects of selexipag on LPS-mediated proinflammatory responses, BV2 or primary microglial cells were treated with vehicle (1% DMSO) or selexipag in dose (0.5, 1.0, or 5.0 μM) and time (3, 6, or 24 h)-dependent manner. For in vivo experiments, C57BL/6N mice were injected daily for 7 days with vehicle (1% DMSO) or selexipag (1 mg/kg, i.p.). On the 7th day, the mice were administered PBS or LPS (10 mg/kg, i.p.) and sacrificed 8 h later. Neuroinflammation-associated gene and protein expression were analyzed in vitro and in vivo by real-time PCR, ELISA, immunofluorescence staining, and/or western blotting.
Results and discussionWe investigated the effect of IP receptor agonist selexipag on LPS-mediated neuroinflammatory responses in vitro and in vivo. Here, we found that selexipag treatment significantly reduced LPS-induced proinflammatory mediator COX-2, IL-1β, IL-6, and TNF-α mRNA and/or protein levels in BV2 microglial cells and primary microglial cells. In LPS-treated C57BL/6N mice, selexipag administration significantly attenuated microgliosis/astrogliosis, proinflammatory mediator expression, and neuroinflammatory-associated dynamics molecules. In addition, selexipag treatment significantly inhibited LPS-induced NLRP3 inflammasome activation in BV2 microglial cells, primary microglial cells and C57BL/6N mice. Importantly, the anti-inflammatory effects of selexipag treatment in BV2 microglial cells were dependent on NLRP3. Moreover, selexipag administration significantly increased cAMP levels, decreased LPS-induced P38 phosphorylation, and suppressed LPS-induced proinflammatory responses via a P38-dependent manner in LPS-treated C57BL/6N mice and/or BV2 microglial cells. Taken altogether, the present results suggest that the selective IP receptor agonist selexipag may be a promising therapeutic candidate for mitigating neuroinflammation-associated neurological disorders.