Mechanisms Of GPR30 Agonist G1 In Reducing Neuro-Inflammation After Cerebral Ischemia

Objectives To investigate the role of Estrogen membrane receptor GPR30 activation on attenuating inflammatory using GPR30 specific agonist G1 and antagonist G36,and to elucidate the possible molecular mechanisms in hippocampal CA1 region of ovariectomized-female rats after global cerebral ischemia(GCI).Methods 1 SPF female SD rats(3-month)were used in the study.The animals were ovariectomized and randomly divided into sham group,ischemia/reperfusion 14d(IR14d)group,G1-treated group(IR14d+G1),and G36-treated group(IR14d+G1+G36).The GCI model was induced by four-vessel occlusion.2 Double-immunofluorescence staining was used to detect cell localization and protein expression of GPR30,Iba1,and NLRP3 inflammasome components,and so on;3 Immunofluorescence staining for Neu N and TUNEL analysis were used to detect hippocampal neuronal survival and apoptosis;4Western blot analysis was used to detect the protein expression;5 G1 or G36 was subcutaneously administrated using minipump beginning at OVX-surgery until the end of the experiment.Results 1 GPR 30 activation decreases GCI-induced neuroinflammation in the Hippocampal CA1 Region.Double immunofluorescence staining for Iba1 and GPR30 revealed that IR14 d induced a robust enhancement of Iba1 immunofluorescence intensity as well as higher co-localization of Iba1 with GPR30 than that in sham animals.G1 administration markedly attenuated the co-localization of Iba1 with GPER,while G36 reversed the effect of G1.Double-immunofluorescent staining of GPR30 and Neu N(a marker of survival neurons)indicated that G1-treatment enhanced GPR30 immunoreactive levels in neurons.Furthermore,TUNEL analysis revealed that the number of Neu Npositive cells was significant decreased by G1 treatment,while the number of apoptotic cells(TUNEL-positive cells)were markedly increased compared to IR group,and G36 administration significantly abolished the anti-apoptotic effects of G1(P<0.05).2 GPR30 activation reduced NLRP3 inflammasome components NLRP3 and ASC protein levels in hippocampal CA1 region after GCI.WB analysis showed that G1 significantly decreased NLRP3 and ASC protein expression compared to IR group(P<0.05),while G36 administration abolished the G1-induced effects(P<0.05).Furthermore,doubleimmunofluoresence staining for NLRP3 and Iba1 or ASC and CD11b(another microglia marker)revealed strongly co-localizatioin of both proteins in the IR group,as compared to sham animals,as well as higher all protein levels.G1 treatment significantly attenuated the IR-induced the enhancement and co-localization,whereas G36-treatment significantly reversed the G1 effects.3 GPR30 activation inhibited “Active” Caspase 1(Cle-Cas1)in hippocampal CA1 region after GCI.Western blot analysis revealed that G1 markedly suppressed caspase 1 activation(cle-cas 1)as compared with IR group,while G36 reversed the effect(P<0.05)without significant difference compared with Sham group(P>0.05).4GPR30 activation inhibited IL1? activation(Cle-IL1?)in hippocampal CA1 after GCI.WB results showed that G1 significantly decreased the Cle-IL1? protein level compared to IR gourp(P<0.05),while G36 treatment reversed the effect(P<0.05).Furthermore,double immunofluorescent staining for cle-IL1? and CD11 b confirmed the above results and showed a stronger co-localization of Cle-IL1? and CD11 b in IR and G36 groups than that of G1 group.5 GPR30 activation up-regulateed expression of endogenous IL1? receptor antagonist(IL1RA)in the hippocampal CA1 region after GCI.WB analysis revealed that G1 treatment significantly elevated IL1 RA protein level compared with IR group(P<0.05),and G36 reversed this effect of G1(P<0.05).Furthermore,immunofluorescence double staining of CD11 b and IL1 RA confirmed the WB results,showing that immunofluorescence intensity of IL1 RA strongly decreased in the IR group as compared to G1 groups,while G36 treatment prevented this effect of G1.Interestingly,in the IR group,the IL1 RA immunoreactive protein was found to be co-localized with CD11 b in microglia.In contrast,using Neu N as a survival neuronal marker,we found that G1 strongly upregulated IL1 RA immunoreactive protein levels in neurons,and this effect was blocked by G36.Conclusions 1 GPR30 activation decreases GCI-induced neuroinflammation in the hippocampal CA1 region following GCI.2 GPR30 activation inhibits NLRP3 inflammasome activity in hippocampal CA1 region after GCI.3 The mechanisms of GPR30 in attenuating neuro-inflammatory damage is closely related to up-regulating IL1 RA protein level in hippocampal CA1 region after GCI.Figure 19;Table 0;Reference 184...