Effects Of CXC Chemokine Receptor 7 On Oxygen-glucose Deprivation/Reoxygenation-injured Neurons

Background: Stroke is one of the top neurological diseases in terms of morbidity and mortality worldwide,accounting for approximately 10% of global deaths.Studies have shown that the incidence of stroke is gradually increasing and the lifetime risk of stroke can reach 24.9% globally,with the risk of ischemic stroke being significantly higher than that of hemorrhagic stroke.Ischemic stroke(IS)is a disease caused by a sudden decrease in blood supply to the brain,which leads to a series of pathological changes and ultimately causes brain dysfunction.CXC chemokine receptor 7(CXCR7)is a member of the family of atypical chemokine receptors(ACKRs)with seven transmembrane structures.It is one of the receptors for chemokine 12(CXCL12)and has a significantly higher affinity for CXCL12 than CXC chemokine receptor 4(CXCR4),another CXCL12 receptor.Although,CXCR7 has a 7-times transmembrane structure with homology to the conserved structural domains of typical G Protein-Coupled Receptors(GPCRs),it differs from typical GPCRs in that CXCR7 lacks functional domains that can be used for coupling to Gi proteins and for signaling,so it is unable to signal through Gi proteins induces cellular signaling,but functions as a scavenger receptor for ligands.Brain neurons are highly specialized cells involved in intercellular signaling in the brain and are among the most abundant and functionally important cell types in the Central Nervous System(CNS).Studies have demonstrated that CXCR7 expression can be detected in neurons of the brain.However,in the case of ischemic stroke,the role and mechanisms of CXCR7 in neurons are not fully understood,and the signaling pathways through which CXCR7 acts need to be further investigated.Objective: To investigate the effect of oxygen glucose deprivation/re-oxygenation(OGD/R)on CXCR7 expression in human neuroblastoma cells(SH-SY5Y),to elucidate the effects of CXCR7 on OGD/R-induced SH-SY5 Y cell cycle,apoptosis,toxicity and proliferative capacity and the relationship with AKT signaling pathway.It provides an experimental basis to further investigate the role and mechanism of CXCR7 in the development of ischemic stroke.Materials and methods: 1.Recombinant lentivirus expressing exogenous CXCR7 gene and control group were constructed and infected with SH-SY5 Y cells cultured in vitro at logarithmic growth stage,puromycin screening of cell line SH-SY5Y-CXCR7 and recombinant lentiviral control cell line SH-SY5Y-NC for stable expression of CXCR7,total cell protein and RNA were extracted,CXCR7 protein expression was detected by Western Blot and CXCR7 mRNA expression was detected by real-time quantitative PCR(RT-qPCR).2.The OGD/R model was constructed in SH-SY5 Y cells,and CXCR7 protein expression was detected by flow cytometry and Western Blot,and cell cycle alteration was detected by flow cytometry.3.The OGD/R model was constructed again in successfully lentivirally infected cells,and Western Blot was used to detect AKT signaling pathway expression,cell cycle and apoptosis-related protein expression,flow cytometry was used to detect cell cycle and apoptosis,lactate dehydrogenase(LDH)assay kit was used to detect cellular LDH release,and cell proliferation and activity assay kit(Cell Counting Kit 8(CCK-8))was used to detect cell proliferation.4.Small interfering RNA targeting the CXCR7-encoding gene was synthesized and SH-SY5 Y cells cultured in vitro at logarithmic growth stage were transfected after 48 h,and the expression of CXCR7 protein in the target cells was detected by Western Blot.The OGD/R model was constructed again after si-RNA interference with CXCR7 expression,Western Blot was used to detect AKT signaling pathway expression,cell cycle and apoptosis-related protein expression,flow cytometry was used to detect cell cycle and apoptosis,LDH kit was used to detect LDH release,and CCK-8 was used to detect cell proliferation ability.The LSD-t test was used for the comparison of two pairs.Results: 1.After CXCR7 overexpression lentivirus successfully infected SH-SY5 Y cells,the increased CXCR7 expression was successfully detected in SH-SY5Y-CXCR7 cells.2.CXCR7 protein expression was reduced in SH-SY5 Y cells after 6 h hypoxia/reoxygenation 24 h treatment,and cell cycle block was increased in the G0/G1 phase.3.After lentiviral overexpression of CXCR7 and reconstitution of the hypoxic 6h/reoxygenation 24 h model,cell cycle block was reduced in the G0/G1 phase.The cell cycle block was reduced in G0/G1 phase,apoptosis was not significantly altered,LDH release was reduced,cell proliferation capacity was significantly increased on day 3,and AKT signaling pathway was activated.4.After si-RNA interference with CXCR7 expression,the hypoxic 6 h/reoxygenated 24 h model was constructed again,cell cycle block was increased in G0/G1 phase,apoptosis was not significantly altered,and LDH release was increased.The apoptosis was not significantly changed,LDH release was increased,cell proliferation capacity was significantly reduced on both day 3 and day 4,and AKT signaling pathway was inhibited.Conclusion 1.OGD/R decreased CXCR7 expression in SH-SY5 Y cells.2.CXCR7 may affect SH-SY5 Y cell cycle and proliferation capacity after OGD/R through phosphorylation expression of AKT signaling pathway,which is involved in regulating neuronal function and exerts some degree of protection against hypoxia-induced neuronal cell injury.