The Mechanisms Of Astrocyte GPR30 Involved In Neuroprotection

Background and objectiveThe incidence of central nervous system diseases is increasing gradually.It is difficult to cure and affect the quality of life.More than 2 million people suffer from stroke every year in China.Stroke is characterized by high morbidity,disability,and mortality.It is reported that more than 75% of stroke survivors have different sequelas due to the neuronal injury.In addition,China has entered an aging society,and the improvement of economic and medical level has greatly prolonged people's life span,but the incidence of neurodegenerative disease is gradually increased.These diseases seriously affect the life quality of patients and their families and also brings heavily financial burden to society.Neuronal injury is a common pathological phenomenon in stroke and neurodegenerative diseases.With the gradual aggravation of nerve injury,the patient's condition gets worse,facing the threat of permanent disability or even death.So it is a great significance to explore the molecular mechanism of nerve injury and find drug targets with neuroprotective effects.Estrogen is a steroid hormone,which not only plays a role in development,growth,and reproduction but also has neuroprotective effect.The effect of estrogen is mediated by classical nuclear receptors(ER?,ER?)and membrane receptors(GPR30,Gaq-ER,ER-X).G protein-coupled receptor 30(GPR30)is a novel estrogen membrane receptor uniquely localized to the endoplasmic reticulum,which was discovered in the 1990 s.Unlike classical estrogen nuclear receptors,GPR30 mediates rapid response and transcriptional regulation of estrogen.Our previous studies showed that activation of neuronal GPR30 protected neurons from excitotoxicity induced by N-methyl-D-aspartate(NMDA).Astrocytes,the largest glias,are the most widely distributed cell population in the brain.Many researches found that astrocytes not only provide the physical support and nutrition to neurons but also have cytotoxicity effects on neurons by releasing inflammatory cytokines after the activation in traumatic stress process.The neuronal loss induced by proliferation of AS in specific areas of brain is closely related to neurodegenerative diseases.It has been reported that GPR30 agonist could increase the expressions of glutamate transporters(GLAST and GLT-1)and the absorptive capacity of glutamate.Moreover,equol,a major isoflavone from Soybean,could mitigate LPS-induced neuroinflammation in astrocytes via the GPR30-mediated pathway.However,it is not clear whether astrocytic GPR30 protects neurons by regulating autophagy.In this study,we investigated the neuroprotective effect of astrocytic GPR30 in the GPR30 knockout mice,astrocytic-and neuronal-GPR30 conditional knockout mice with middle cerebral artery occlusion(MCAO)/reperfusion.The molecular mechanism of astrocyte GPR30 regulating autophagy and exerting neuroprotective effect was explored in primary cultured astrocytes.Methods1.GPR30 knockout mice,astrocytic-and neuronal-GPR30 conditional knockout mice were produced by CRISPR/Cas9 technology and Cre/lox P system.Genotyping was performed through PCR analysis and genetic sequencing.2.To investigate the neuroprotective effect of astrocytic GPR30,neurological deficit was scored in wild type(WT)and transgenic mice with G1(GPR30 agonist)treatment after MCAO.3.The infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride(TTC)staining in the brain slices of WT and transgenic mice after MCAO and G1 treatment.4.Nissl staining was performed in the frozen section of brain to investigate the morphological changes in WT and transgenic mic after MCAO and G1 treatment.5.Primary astrocytes were cultured and the purity of astrocytes was identified by immunofluorescence staining.Glutamate was used to mimic excitotoxic injury of stroke in vitro.6.The cell viability and proliferation were detected by 3-(4,5-Dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)in cultured primary astrocytes after Glu,G1,and/or G15 treatments.7.Astrocyte proliferation was further confirmed by 5-Bromo-2-deoxyuridine(Brd U)assay in cultured primary astrocytes after Glu,G1,and/or G15 treatments.8.Primary neurons were cultured and treated with astrocyte-conditioned medium(ACM),which was collected from cultured astrocytes after Glu,G1,and/or G15 treatments.The neuronal viability was determined by MTT.9.The levels of TNF-?,IL-1?,and IL-6 were measured by enzyme-linked immuno sorbent assay(ELISA)in astrocyte culture medium after treated with Glu,G1,and/or G15.10.The levels of proteins were quantified by Western blot in cultured primary astrocytes after Glu,G1,and/or G15 treatments.11.The numbers of autophages were detected by immunofluorescence staining in cultured primary astrocytes after Glu,G1,and/or G15 treatments.12.The numbers of acidic autophagic lysosome were checked by monodansylcadaverine(MDC)staining in cultured primary astrocytes after Glu,G1,and/or G15 treatments.Results1.Construction,reproduction,and identification of GPR30 transgenic miceThe results of genomic PCR,RT-PCR,and genetic sequencing confirmed that GPR30 KO mice,astrocytic-and neuronal-GPR30 conditional knockout mice were successfully constructed and reproduced.2.Astrocytic GPR30 was involved in neuroprotection in vivoThe neuroprotective effect of G1(GPR30 specific agonist)was totally abolished in GPR30 KO mice and partially attenuated in astrocytic or neuronal GPR30 conditional KO mice after MCAO.3.The activation of astrocytic GPR30 prevented glutamate-induced reactive astrogliosisThe results of MTT assay showed that astrocyte viability markedly increased in a concentration-dependent(100,300,and 500 ?M)and time-dependent(15,30,60,and 120 min)manner after glutamate exposure in primary cultured astrocytes.G1 pretreatment(1 and 10 n M)significantly inhibited glutamate-induced astrogliosis in MTT and Brd U assay.The effect of G1 was completely abolished by GPR30 antagonist G15(100 n M).The results of western blot and immunofluorescence staining indicated that G1 treatment significantly reversed increased expression of GFAP and decreased ratio of fibrous astrocytes induced by glutamate(300?M,30 min).4.The activation of astrocytic GPR30 inhibited inflammatory-cytokine release from astrocytesG1(1 n M)pretreatment reduced the levels of TNF-? and IL-6 induced by glutamate,and the effect of G1 was abolished by G15(100 n M).Neuronal viability was decreased in the ACM from glutamate-treated astrocytes but not ACM from astrocytes pretreated with G1(1 n M)before glutamate exposure.5.Glutamate-induced reactive astrogliosis was associated with autophagic inhibitionGlutamate exposure decreased the level of Beclin-1 and the ratio of LC3-II/I but increased the level of p62 in a concentration-dependent(100,300,and 500 ?M)and timedependent(15,30,60,and 120 min)manner.Bafilomycin A1(Baf A1),a vacuolar-type H+-ATPase inhibitor,was reported to block autophagosome-lysosome fusion and prevent LC3-II degradation.The presence of Baf A1 did not affect the decrease of glutamateinduced autophagy in cultured primary astrocytes.6.The activation of astrocytic GPR30 reversed autophagic imbalance in glutamatetreated astrocytesG1(1 n M)pretreatment reversed the decreased ratio of LC3-II/I induced by glutamate.This effect was completely blocked by pretreatment with G15(100 n M).Immunofluorescence staining of LC3 and MDC staining further revealed that G1 pretreatment reversed the decrease of autophagy in glutamate-treated astrocytes.7.Astrocytic GPR30-mediated autophagic balance was not dependent on the AKT/m TOR signaling pathwayThe results of Western blot showed that glutamate(300?M,30 min)exposure increased the levels of p-AKT,p-m TOR,p-p70,and p-S6 RP.However,G1(1 n M)pretreatment did not affect the phosphorylations of these proteins.Glu,G1,and/or G15 did not change the total protein levels of AKT,m TOR,p70,and S6 RP.8.Astrocytic GPR30-mediated autophagic balance was not dependent on the ERK or JNK pathwaysGlutamate(300?M,30 min)exposure drastically increased the levels of p-ERK and pJNK but did not change total ERK and JNK levels in astrocytes.G1(1 n M)pretreatment did not affect the phosphorylated and total protein levels of ERK and JNK.9.Astrocytic GPR30-mediated autophagic balance was modulated through the p38 MAPK signaling pathwayGlutamate(300?M,30 min)inhibited phosphorylated p38(p-p38),and G1(1 n M)pretreatment restored the p-p38 level with no effect on total p38 expression.The effect of G1(1 n M)on autophagy was completely blocked by p38 MAPK inhibitors SB203580(SB,10 ?M)and LY2228820(LY,20 ?M).The inhibitory effect of G1 on glutamate-induced astrogliosis was completely blocked by LY.Meanwhile,the neuroprotection of ACM from G1-treated astrocytes was also abolished in the ACM pretreated with LY.Conclusion1.In vivo experiment,astrocytic GPR30 was involved in the neuroprotection in mice after MCAO.2.The activation of astrocytic GPR30 inhibited glutamate-induced reactive astrogliosis and inflammatory cytokine release from astrocytes in vitro experiment.3.Excitotoxicity induced reactive astrogliosis and inflammatory cytokine release through autophagic inhibition,and neuroprotective effect of astrocytic GPR30 was related to inducing protective autophagy in astrocytes.4.Activation of astrocytic GPR30 restored autophagy balance by regulating the p38 MAPK pathway and produced neuroprotective effect.