GLP-1(9-36) Reduces Neuroinflammation From CIRI Via The Activation Of Insulin-like Growth Factor 1 Receptor In Astrocytes

AimIschemic stroke is a major cause of death and disability and endangers human life and health seriously.So far,timely recovery of blood supply after ischemia is the "gold scheme"for its treatment.However,reperfusion therapy after cerebral ischemia can cause more serious injury such as increased infarction area and neuronal apoptosis,that is cerebral ischemia-reperfusion injury(CIRI).To explore and find effective intervention drugs for CIRI is a hot and difficult scientific problem in this field,which is highly concerned and urgently needed to be solvedGlucagon-like peptide-1(GLP-1)is an incretin hormone that stimulates insulin secretion in a glucose level dependent manner.GLP-1 and its analogs have been reported to exert neuroprotective effects against central nervous system diseases such as CIRI.Binding with GLP-1 receptor(GLP-1R)and activating its downstream signaling pathways are recognized mechanisms of these agents.The search for highly active GLP-1 analogues and further study of their mechanism may provide new targets and drugs for the prevention and treatment of ischemic stroke.GLP-1(9-36)has been considered as merely a "bio-inactive" metabolite of GLP-1.However,accumulating evidence has shown that GLP-1(9-36)possesses critical biological functions,for instance,alleviating damage of cardiovascular system and promoting neuronal development.GLP-1(9-36)possesses functions distinct from its precursor GLP-1 because of its low affinity to the GLP-1R.Previous report demonstrates that GLP-1(9-36)exerts a protective effect in animal models of cardiac ischemia in a GLP-1R independent manner,but the exact mechanism is not clear.Little is known about the effects and mechanism of GLP-1(9-36)in CIRI,so addressing these issues will provide promising drugs for the prevention and treatment of CIRI and further update the current understanding of the pharmacology of GLP-1 analogs.Insulin-like growth factor 1 receptor(IGF-1R)belongs to the transmembrane receptor tyrosine kinase family.A previous study reported that GLP-1(9-36)had the potential to interact with and activate IGF-1R in cardiac fibroblasts.IGF-1R is expressed in astrocytes and has been identified as a critical player in the regulation of inflammation following CIRI.Thus,we hypothesize that GLP-1(9-36)may reduce inflammation in astrocytes via interaction with the IGF-1R and confer neuroprotective effect.By constructing GLP-1R knockout(Glp-1rKO)and IGF-1R knockdown(Igf-1rKD)mice and establishing the CIRI animal model and OGD/R cell model,this study aims to investigate the protective effects and clarify the mechanisms of GLP-1(9-36)against CIRI in multiple levels from the whole animal to the molecular levels using the methods in molecular cell biology,pharmacology and other research filed,Ultimately,it is expected that the new therapeutic strategies can be used to treat CIRI.Methods1.GLP-1(9-36)confers neuroprotective effect against CIRI via IGF-1RGLP-1(9-36)(250,500,1,000 ng/g/day i.p)was administrated in C57BL/6J mice for 7 days,then the CIRI animal model was established via middle cerebral artery occlusion/reperfusion(MCAO/R)injury.Behavior deficits were evaluated by using modified neurological severity score(mNSS),foot fault test and rotator test.Then the brains were removed and sliced,2,3,5-Triphenyltetrazolium chloride(TTC)staining was performed to calculate infarct volume.The neuroprotective effect of GLP-1(9-36)against CIRI was evaluated based on the results of above.Glp-1rKO mice were established,brain Igf-1rKD mice were established via stereotactic injection of lentivirus.GLP-1(9-36)(500 ng/g/day i.p.)was administrated in Glp-1rKO mice and Igf-1rKD mice for 7 days,then the MCAO/R injury was performed.Neurological deficit was evaluated via behavior test;infarct volume was calculated after TTC staining;astrogliosis was determined via GFAP immunofluorescence;neuronal apoptosis was detected via TUNEL staining.Then the impact of GLP-1R knockout or IGF-1R knock down on the neuroprotective effect of GLP-1(9-36)was determined.2.The effect and mechanism of GLP-1(9-36)on post-CIRI inflammatory responseRNA-Seq assay was performed to detect different genes caused by GLP-1(9-36);Primary culture of microglia and astrocytes,pretreated with GLP-1(9-36)and then oxygenglucose deprivation/reoxygenation(OGD/R)was performed,ELISA was performed to determine the inflammatory factors.Primary culture of WT and Glp-1rKO astrocytes,Igf1rKO astrocytes were generated by using CRISPR Cas9,pretreated with GLP-1(9-36)and then OGD/R was performed,ELISA was performed to determine the inflammatory factors.WT,Glp-1rKO,and Igf-1rKO astrocytes were treated with GLP-1(9-36)then OGD/R was performed,the supernatant was added into the neuron culture medium after OGD/R injury,TUNEL assay was used to determine the percentage of neuronal apoptosis,these experiments were performed to evaluate whether GLP-1(9-36)alleviates OGD/R-induced neuronal cell injury through astrocyte IGF-1R.Astrocytes were cultured,interaction between GLP-1(9-36)and IGF-1R was determined by immunoprecipitation,impact of GLP-1(9-36)on downstream of IGF-1R signaling was evaluated by Western Blot assay,these experiments were performed to verify whether GLP-1(9-36)interacts and activates IGF-1R in astrocytes.Results1.GLP-1(9-36)confers neuroprotective effect against CIRI via IGF-1RBehavioral experiments and TTC staining were performed to evaluate the protective effect of GLP-1(9-36)on MCAO/R injury.mNSS neurological deficit score,foot fault and rotation test indicated that mice displayed serious behavioral deficits 24 h after MCAO/R,the behavior deficits of GLP-1(9-36)group(500,1,000 ng/g/day)were significantly improved compared with the model group.TTC staining results showed that MCAO/R caused large infarct area in brain,the infarct volume in GLP-1(9-36)group(500,1,000 ng/g/day)were significantly reduced compared with the model group.These results indicate GLP-1(9-36)confers neuroprotective effect against MCAO/R.To determine whether GLP-1R or IGF-1R participates in the neuroprotective effect of GLP-1(9-36),the Glp-1rKO and Igf-1rKD mice were constructed.Results of behavior tests indicated that,in Glp-1rKO mice,GLP-1(9-36)improved neurological deficit and reduced infarct volume caused by MCAO/R.However,these effects were partially reversed in Igf1rKD mice.GFAP immunofluorescence and TUNEL results showed that,in WT and GlpIrKO mice,GLP-1(9-36)reduced GFAP positive cells(astrocytes)and TUNEL positive cells(apoptosis cells)caused by MCAO/R.These effects,however,were partially reversed in Igf-1rKD mice.These results indicate IGF-1R but not GLP-1R mediates the neuroprotective effects of GLP-1(9-36)on MCAO/R.2.GLP-1(9-36)reduces neuroinflammation from CIRI via interaction with and activation of IGF-1R in astrocytesRNA-Seq results showed that GLP-1(9-36)treatment causes large number of gene expression difference in peri-infarct area.DAVID database analysis showed that the difference gene clustered in inflammatory response.These results showed that GLP-1(9-36)reduced inflammatory after MCAO/R.ELISA results showed that GLP-1(9-36)reduced the TNF-α,IL-1β,and IL-6 in cultured astrocytes but not microglia upon OGD/R condition.These effects were not impacted in Glp-1rKO astrocytes,however,was reversed in Igf-1rKO astrocytes,indicating GLP-1(9-36)conferred anti-inflammatory effect via IGF-1R.TUNEL results showed that GLP-1(9-36)treatment of astrocytes reduced neuronal apoptosis in co-cultured system,this effect was diminished in Igf-1rKO astrocytes,these results showed that GLP-1(9-36)reduced neuronal apoptosis in co-cultured system caused by OGD/R via IGF-1R in astrocytes.Western Blot assay results showed that GLP-1(9-36)reduced p-NF-KB level in astrocytes upon OGD/R condition.GLP-1R knockout did not impact this effect,IGF-1R knockout reversed this trend.ELISA results showed that NF-κB overexpression reversed the anti-inflammation effect of GLP-1(9-36).These results indicate GLP-1(9-36)reduced inflammation in astrocytes upon OGD/R condition via IGF-1R-NF-κB signaling.Co-immunoprecipitation results showed that GLP-1(9-36)was pulled down from astrocytes together with anti-IGF-1 receptor antibodies.Western Blot results showed that treatment with GLP-1(9-36)induced tyrosine phosphorylation of the IGF-1R and PI3KAKT signaling in astrocytes upon OGD/R injury,these effects were reversed by PPP.These results showed that GLP-1(9-36)interacts with and directly activates the IGF-1R in astrocytes.Conclusion1.GLP-1(9-36)confers neuroprotective effect against CIRI,and this effect may be mediated via IGF-1R but not GLP-1R.2.GLP-1(9-36)interacts with and directly activates the IGF-1R in astrocytes,subsequently activates downsteam PI3K-AKT signaling pathway,ultimately reducing inflammatory caused by CIRI.