Protective Effect Of ICS ? On Hypoxic Axonal Injury In Rat Cortical Neurons And Its Mechanism

Objective: To investigate the protective effect and mechanism of Icariside ?(ICS?)on hypoxic axonal damage of cortical neurons in rats,and to provide experimental basis for the treatment of hypoxic axonal injury.Methods: Primary cultured cortical neurons of neonatal SD rats.To explore the effects of oxygen concentration and hypoxia time on axonal injury of neurons,neurons were randomly divided into normal group,5% hypoxia group,10% hypoxia group and 15% hypoxia group.To explore the protective effect and mechanism of ICS? on hypoxic axonal injury,neurons were randomly divided into normal group,10% hypoxia group,10% hypoxia+ICS?3?M group,10% hypoxia+ICS?6?M group,10% hypoxia+ ICS?9?M group,10% hypoxia + PI3 K inhibitor group,10% hypoxia + ICS?9?M + PI3 K inhibitor group.Neurons were cultured at 5% CO2 and 37 ?.The normal group was exposed to natural oxygen concentration.The hypoxic groups were cultured continuously in a sterile closed hypoxic chamber with N2 regulating oxygen concentration.Except LY294002 for 48 hours,the other groups were cultured for 24,48 and 72 hours respectively.PI3 K inhibitor group was pretreated with PI3 K inhibitor LY294002 20?gM for 1 hour before adding maintenance medium or ICS ?.Corresponding indexes were measured at each observation time point: MTT assay was used to determine neuronal cell viability,ELISA assay was used to determine lactate dehydrogenase(LDH)activity in supernatant of culture medium,immunofluorescence staining with beta-?I Tubulin was used to observe the morphological changes of axonal network,and the axonal degeneration index(DI)and the longest protuberance length were measured for quantitative analysis.Results: Compared with the normal group,the refraction of cortical neurons in SD rats cultured under hypoxic conditions decreased,the cell body swelled slightly,the axonal network gradually disintegrated,and the axons were broken,shortened or even disappeared.With the decrease of oxygen concentration,axon damage was aggravated gradually.At 15% oxygen concentration,axon network partially disintegrated,beads formed,axons fracture and shortened,cell survival rate and LDH activity in culture supernatant had no significant change(P>0.05,P>0.05);at 10% oxygen concentration,axon shortened significantly at 24 h and 48 h,while the survival rate of neurons did not change significantly(P<0.05,P>0.05).at 72 h,axon network disintegrated,the cell survival rate decreased and the LDH activity in the culture supernatant increased(P<0.05,P<0.05).Otherwise,At 5% oxygen concentration,the cell viability was decreased and the LDH activity in the supernatant of culture medium was increased,axonal network completely disintegrated or even disappeared(P<0.05,P<0.05).(2)After 72 hours of incubation at 10% oxygen concentration,compared with the 10% hypoxic group,ICS?3?M had no obvious protective effect.The axons of the middle and high(ICS?6?M,ICS?9?M)groups prolonged significantly and the cell survival rate increased,but it was still lower than that of the normal group.After pretreatment with PI3 K inhibitor,there was no significant change in axons of ICS? groups compared with 10% hypoxia group(P>0.05).Conclusion: Under hypoxic condition,axon damage occurred in cultured cortical neurons of SD rats in vitro.With the prolongation of hypoxic time and the aggravation of hypoxic degree,cell body injury gradually occurred,which indicated that axon damage under hypoxic condition was earlier than that of cell injury.(2)ICS ? has protective effect on axonal damage under 10% hypoxia,which may be through activating PI3 K signaling pathway.