The Role And Mechanism Of SDF1 And Its Related Signaling Pathways In The Angiogenesis Of Degenerated Intervertebral Discs

Objective To investigate the effects of SDF1 on angiogenesis induced by degenerated nucleus pulposus cells(NPCs)in the coculture system by regulating the expression of SDF1 and its related receptors and explore the intrinsic mechanism.Methord The primary NPCs were isolated and cultured,the expression of SDF1 in NPCs and CXCR7 in vascular endothelial cells(VECs)were respectively regulated by virus transfection.The expression of SDF1 and CXCR7 was verified by real time PCR,WB and immunofluorescence.In the coculture system,NPCs(or conditioned medium)with different SDF1 expression levels were cocultured with VECs with different CXCR4 or CXCR7 expression levels,and the effects on the cell viability,cell migration ability and tube formation ability were observed.After coculture,the expression of associated pathway proteins of VECs was detected.Subsequently,the key proteins in the pathway were inhibited,and the effects on the cell viability,cell migration ability and tubeformation ability were observed.Thereby,the underlying mechanism through which SDF1 and its related receptors take effect can be derived.Results In the first part,The SDF1 expression in NPCs was up-regulated by adenovirus transfection.The mRNA and protein expression of SDF1 in the UP group were significantly higher than those in the D group and NC group(P<0.05).The fluorescence intensity was significantly strengthened.The SDF1 in NPCs was successfully up-regulatd.After coculture of degenerated NPCs and VECs,the cell viability,cell migration ability and tube formation ability in the UP group were significantly enhanced compared with those in the D group(P<0.05).The cell viability,cell migration ability and tube formation ability in the UP+AMD3100group were significantly weakened than those in the UP group(P<0.05).After coculture,the expression of pAKT of VECs in the UP group was increased(P<0.05)and the expression of PTEN was decreased by contrary(P<0.05).After respective pretreatment of VECs with pAKT or PTEN inhibitors,the cell viability,cell migration ability and tube formation ability in the UP+MK2206 group were significantly decreased than those in the UP group(P<0.05),while the cell viability,cell migration ability and tube formation ability in the UP+SF1670 group were significantly increased than those in the UP group(P< 0.05).In the second part,the expression of SDF1 in NPCs was down-regulated by siRNA of lentivirus transfection.The mRNA andprotein expression of SDF1 in the DOWN group were significantly lower than those in the D group and NC group(P<0.05).The expression of SDF1 was also detected by immunofluorescence.The green fluorescence was the strongest in the UP group and the weakest in the DOWN group.According to the expression level of SDF1,the NPCs were defined as the DOWN,D,and UP groups.The expression of CXCR7 in VECs was also down-regulated by siRNA of lentivirus transfection.The expression of CXCR7 in the DOWN group was significantly decreased in the mRNA and protein levels(P<0.05),and the fluorescence intensity was also significantly decreased.The expression of CXCR7 in VECs was successfully down-regulated.Three groups of NPCs were respectively cocultured with VECs,and the cell viability,cell migration ability and tube formation ability were enhanced gradually with the increase of SDF1 in the DOWN,D and UP groups(P<0.05).When the CXCR7 expression was down-regulated,the cell viability,cell migration ability and tube formation ability in the D and UP group were significantly decreased compared with the previous(P<0.05).After coculture,the expression of pAKT in VECs was increased as SDF1 increased,and conversely,the expression of PTEN was decreased as SDF1 increased.After MK-2206 pretreatment,the cell viability,migration ability and tube formation ability in the D and UP groups decreased significantly compared with the previous(P<0.05);After SF1670 pretreatment,the cell viability,migration ability and tubeformation ability in the DOWN,D and UP groups was significantly increased compared with the previous(P<0.05).Conclusion Degenerated NPCs can promote the angiogenesis by SDF1 binding to its receptor CXCR4 or CXCR7,and this activity,regulated by whether the SDF1/CXCR4 signaling or the SDF1/CXCR7 signaling,can be conducted through the PI3K/AKT pathway in VECs.