Activation Of GPBAR1 By Bar501 Attenuates Neuroinflammation Through PKA/CREB Signaling Pathway After Intracerebral Hemorrhage In Mice

Part I Role for activation of GPBAR1 by Bar501 after ICH in miceAim:To look at short-term(1-3 days)neurological improvement and brain edema reduction after GPBAR1 activation by different dosage of Bar501 intranasal administration in collagenase-induced ICH mice model,and then choose the best dosage of Bar501 to look at the improvement of long-term(up to 28 days)neurological functions.Methods:1.Brain water content and neurobehavioral tests were performed at 24 and 72-h after ICH to demonstrate the anti-inflammatory effects of GPBAR1 selective agonist Bar501 by intranasal administration 1-h after ICH.To evaluate effects at 24-h after ICH,30 mice were randomly divided into five groups:sham(n=6),ICH+vehicle(n=6),ICH+Bar501 0.02 μg/g(n=6),ICH+Bar501 0.05 μg/g(n=6)and ICH+Bar501 0.15 μg/g(n=6).Best intranasal treatment dosage of Bar501 0.15 μg/g was chosen for 72-h drug effects evaluation based on the results of brain water content and neurobehavioral tests at 24-h after ICH.To evaluate the effect at 72-h after ICH,18 mice were randomly divided into three different groups:sham(n=6),ICH+vehicle(n=6),and ICH+Bar501 0.15 μg/g(n=6).2.To determine the effects of intranasal administration Bar501 on long-term(28 days)neurobehavioral functions after ICH,24 mice were randomly divided into three groups:sham(n=8),ICH+vehicle(n=8)and ICH+Bar501 0.15 μg/g(n=8).Foot fault and rotarod tests were both performed on day-1,8 and 15 after ICH.Morris water maze was performed from day-21 to day-25 after ICH.Results:1.Neurobehavioral functions and brain water content were tested at both 24 and 72-h after ICH.Mice in vehicle group performed worse in Garcia test(p<0.05 versus sham;Fig2.b),corner turn test(p<0.05 versus sham;Fig2.c)and forelimb placement test(p<0.05 versus sham;Fig2.d).It showed administration of Bar501 significantly improved performance in neurobehavioral function tests at 24-h after ICH(p<0.05 versus sham;Fig2.b,c,d).Brain water content in the ipsilateral basal ganglia was significantly increased in all ICH groups compared with sham group at 24-h after ICH(p<0.05 versus sham;Fig2.a).Based on the significant brain water content reduction and neurological outcomes improvement compared with the vehicle group,the high dosage of Bar501(0.15 μg/g)was chosen for 72-h outcomes,long-term outcomes and mechanism study.And in 72-h outcome evaluation,it still showed that the neurobehavioral functions and brain water content of treatment group were significantly different from vehicle group(p<0.05 versus sham;Fig2.e,f,g,h).2.Foot fault test and Rotarod test were performed to evaluate the movement coordination ability and spatial learning ability,and the Morris water maze were performed to assess animals’memory ability after ICH.In the foot fault test,animals in ICH+vehicle group had significantly more foot faults of left forelimb compared with animals in sham group at the second and third weeks(p<0.05 versus sham;Fig4.a).Meanwhile animals in ICH+vehicle group also had significantly shorter falling latency compared with animals in sham group at the second and third weeks(p<0.05 versus sham;Fig4.b).However,animals in ICH+BAR501 group had significantly decreased foot faults of left forelimb and increased falling latency compared with animals in ICH+vehicle group at the third week(p<0.05 versus sham;Fig4.a,b).As the Morris water maze data showed,the escape latency and swim distance(to find the submerged platform)of animals in ICH+vehicle group were significantly increased compared with in sham group on block 4 and 5(p<0.05 versus sham;Fig4.c,d),meanwhile the escape latency and swim distance(to find the submerged platform)of animals in ICH+vehicle group were also significantly increased compared with in ICH+BAR501 group on block 5(p<0.05 versus sham;Fig4.c,d).In the probe quadrant trail,the ICH+vehicle group spent less time in the target quadrant compared with sham group the escape latency and swim distance(to find the submerged platform)of animals in ICH+vehicle group were significantly increased compared with in sham group on block 4 and 5(p<0.05 versus sham;Fig4.f),while the ICH+BAR501 group spent significantly more time in the target quadrant compared with ICH+vehicle group(p<0.05 versus sham;Fig4.f).All these significant differences proved that administration of BAR501 increased animals’movement coordination ability,spatial learning ability and memory ability to some extent.Conclusion:1.Activation of GPBAR1 by Bar501 can reduce brain edema meanwhile to improve neurological outcomes at both 24 and 72 hours after ICH in mice.2.Activation of GPBAR1 by Bar501 can improve the movement coordination ability,spatial learning ability,and memory ability at 28 days after ICH in mice.Part Ⅱ Mechanism of neuroinflammation-attenuation effect byactivating GPBAR1 receptor with Bar501Aim:In the present study,we investigated the anti-inflammatory role of GPBAR1 by activation with its selective agonist BAR501 and its underlying mechanism in collagenase-induced ICH mouse model.Methods:1.MPO and Iba-1 in brain were detected via immunofluorescence staining to assess the levels of neutrophil infiltration and microglia/macrophage activation.Immunofluorescence staining were performed in sham,ICH+ vehicle and ICH+BAR501 groups at 24-h after ICH.2.Western Blot was performed as previously described.The proteins of ipsilateral/right hemispheres were extracted by cytoplasmic extraction reagents(Pierce Biotechnology,Rockford,IL,USA),then loaded(50 μg)and separated by SDS-PAGE gel electrophoresis.After blocking with 5%nonfat milk for 1.5-h,the membranes were incubated at 4℃ overnight with primary antibodies including rabbit anti-GPBAR1(1:1000,Abcam),rabbit anti-PKA(1:2000,Cell Signaling),rabbit anti-CREB(1:2000,Cell Signaling),rabbit anti-p-CREB(1:1000,Cell Signaling),rabbit anti-NF-κB(1:2000,Abcam),rabbit anti-p-NF-κB(1:1000,Abcam),rabbit anti-IL-10(1:1000,Abcam),rabbit anti-IL-6(1:1000,Abcam)and goat anti-β-actin(1:3000,Abcam).β-actin served as the loading control.The membranes were then incubated with appropriate secondary antibodies(1:3000,Abcam)for 2-h at room temperature.The bands were probed with an ECL Plus chemiluminescence regent Kit(Amersham Biosciences,Arlington Heights,PA,USA)and visualized with the image system(Bio-Rad,Versa Doc,model 4000),and the relative density of protein was analyzed by ImageJ software(ImageJ 1.4,NIH,USA).Results:1.GPBAR1 is expressed in microglia.Inflammatory stimulation can accelerate its transcription in microglia.2.Nasal administration of BAR501 decreased microglia/macrophage activation and neutrophil infiltration at 24-h after ICH.Conclusion:The cAMP/PKA/CREB pathway is one of the proposed pathway for GPBAR1 to promote IL-10 and decrease IL-6,NF-κB and TNF-α to attenuate neuroinflammation.