The Role And Mechanism Study Of LPA1 In Secondary Brain Injury After Intracerebral Hemorrhage In Mice

Background: Intracerebral hemorrhage(ICH)is a hemorrhage stroke with high mortality and disability rate.Secondary brain injury(SBI)after ICH,which caused by neuroinflammation,brain blood barrier(BBB)disruption,apoptosis and necrosis,lead to poor prognosis in patients.Lysophosphatidic acid receptor 1(LPA1)is activated by its ligand Lysophosphatidic acid(LPA)to performance many bioactivities.In the central nervous system LPA1 promote neuroinflammatory processes and cells damage by activating microglia.There is no report about the role of LPA1 in ICH.The aim of this research was to explore the anti-inflammatory effect and neurological function improvement of LPA1 inhibition after ICH in mice,and uncovered the mechanism.Methods: ICH models were induced by autologous whole blood injection.We detected LPA1,E-type prostaglandin receptor 2(EP2),NADPH oxidase 2(NOX2)expression after ICH in 6h,12 h,24h,72 h and7d by western blot(WB),and observed LAP1 colocalization with microglia,neuron and astrocyte and EP2 colocalization with microglia by immunofluorescence staining(IF).To evaluated the effect of LPA1 in short-term SBI and activation of microglia after ICH.AM966,a selective antagonist of LPA1,was administered by oral gavage 1h,12 h and 4h,16 h and LPA was administered by intraventricular injection 1h after ICH.Neurobehavior tests,brain water content and IF were performed.To evaluated the effect of LPA1 in long-term neurologic function after ICH,rotarod test,foot fault test and morris water maze test were performed.And we used Nissl staining to observed CA1 area of hippocampus.To elucidate potential mechanism of LPA1,we intracerebroventricular injected a selective EP2 activator,Butaprost,with either AM966 or LPA1 CRISPR knockout(KO)to asses neurologic function by neurobehavior test and expression of downstream protein by WB.Results:(1)The expression of LPA1,EP2,NOX2 increased at 6h,peaked at 24 h and decreased at 72 h post-ICH.(2)LPA1 colocalized with microglia,neurons and astrocytes,EP2 colocalized with microglia 24 h after ICH.(3)Inhibition of LPA1 improved neurologic function and reduced brain edema 24 h and 72 h after ICH by AM966,LPA aggravated neurologic function and brain edema 72 h after ICH.(4)AM966decreased the activation of microglia and infiltration of neutrophil perihematomal 24 h after ICH.(5)AM966 improved long-term neurobehavioral function after ICH.(6)AM966 reduced neurons loss of hippocampus CA1 region 28 days after ICH.(7)AM966 and LPA1 CRISPR KO attenuated neurology function deficit and decreased PGE2,EP2,NOX2,NF-κB,TNF-α,IL-6,IL-1βexpression 24 h after ICH.(8)Butaprost reversed the effects of AM966 and LPA1 CRISPR KO.Conclusions: This study demonstrated that inhibition of LPA1 attenuated neuroinflammation caused by ICH via PGE2/EP2/NOX2 signaling pathway in mice,which consequently improved neurobehavioral functions and alleviated brain edema.LPA1 may be a promising therapeutic target to attenuate ICH-induced SBI.