| BackgroudObstructive sleep apnea sysdrome is a highly prevalent and unrecognized entirely public health problem that can lead to more serious cardiovascular and neurocognitive morbidities in all age groups.In OSA,episodes of(partial)obstruction of the upper airway cause repeating intermittent hypoxemia and minor arousal or arousal during sleep,disrupting the sleep architecture and causing an overall poor quality of sleep.Moreover,OSA patients often report cognitive complaints in recent years[2-4].Many previous studies of experimental animals showed that these neurocognitive deficits are strictly associated with regional brain damage,particularly in the hippocampus,with apoptosis[9]likely mediated by the overproduction of reactive oxygen species(ROS)[5]under IH conditions.Recent studies have shown that the intermittent hypoxemia that is induced by sleep apnea activates orexin neurons and increases the level of orexin-A.[14,15].Orexinwhich is a multifunctional neuropeptideis involved in sleep disorders by activating their receptors(OX1R and OX2R),thus contributing to arousal,sleep.Orexin is also involved in learning,memory and neurons apoptosis of brain vital functional zone including hippocampus.At present the precise mechanisms through which OSA cause cognitive function impairment are still not complete clear and definite.Few studies carried on what are the signal pathway and molecular mechanisms of orexin-A in the process of cognition impairment caused by OSA.What is the definite mechanism by which OSA caused cognitive damage.In this study,the intermittent hypoxia model of Wistar rat hippocanpal neurons was established.We evaluate the effect of orexin-A on hippocampal neurons under intermittent hypoxia condition by detecting neuron's apoptosis rate.We explored the potential signal pathway and molecuar mechanism.ObjectiveTo establish IH model of Wistar rat hippocanpal neurons and observation the effect of orexin-A on hippocampal neurons under intermittent hypoxia condition by detecting neuron's apoptosis rate.To definite that orexin-A aggravates the impairment of hippocampal neurons caused by intermittent hypoxemia via phosphorylation ERK1/2 by detecting expression of p-ERK1/2,and verify by U0126.SiRNA-PLC?1 and siRNA-PLC?4 were used to explore the subtype by which orexin-A induced phosphorylation ERK1/2 in orexin-A-OX1R/OX2R-Gq-PLC-/PKC signal pathway.Methods1.Hippocampal tissue were separated from 24 h postnatal Wistar rats and obtain hippocampal neurons by trypsin digestion combined with mechanical blowing method to culture.The growth and morphological features were carefully observed through the process.The purity of neurons in primary cultures was detected by immunocytochemical technique using primary antibodies to NSE.2.Hippocampal neurons culturing 4d were exposed to normoxic or IH conditions.The normoxic condition consisted of 21%02,5%CO2,and 74%N2.The IH condition consisted of 64 cycles of 5 min IH(1%O2,5%CO2,and 74%N2)and 10 min normoxia.On this basis,the different concentrations of orexin-A(1 nM?3 nM?10 nM and 100 nM)were added to the cultured hippocampal neurons for 48 h.Then gather the neurons of each group to detect apoptosis rate by Flow Cytometer after staining by Annexin V-FITC/PI?3.The hippocampal neurons of rats were randomly divided into control,IH,IH+orexin-A(100Nm/L)and IH+orexin-A(100Nm/L)+U0126 groups.To extract total protein of each group hippocampal neuron and detect p-ERK1/2 by Western blot.4.The hippocampal neurons of rats were randomly divided into control,IH,IH+orexin-A(100Nm/L)and IH+orexin-A(100Nm/L)+U0126 groups.To gather the neurons of each group to detect apoptosis rate by Flow Cytometer after staining by Annexin V-FITC/PI.5.The hippocampal neurons of rats were randomly divided into control siRNA(-),control siRNA(+),siRNA-PLC?1(-),siRNA-PLC?1(?),siRNA-PLC?4(-)and siRNA-PLC?4(+)groups.(-)groups were treated with IH;(?)groups were treated with IH+orexin-A.To extract total protein of each group hippocampal neuron and detect p-ERK1/2 by Western blot.6.The hippocampal neurons of rats were randomly divided into control siRNA(-)?control siRNA(+),siRNA-PLC?1(-),siRNA-PLC?1(+),siRNA-PLC?4(-)and siRNA-PLC?4(+)groups.(-)groups were treated with IH;(+)groups were treated with IH+orexin-A.To gather the neurons of each group to detect apoptosis rate by Flow Cytometer after staining by Annexin V-PE/7AAD.Result1.Morphological characteristics of neuron culture:The isolated hippocampal neurons are round,bright and similar size and suspended in planting liquid.The hippocampal neurons adhered to the plate surface with growing neuritis and the shape grew bigger and irregular or fusiform after 24h.Neurons became more stereoscopic with classic axons and dendrites and synaptic connections were observed.The synaptic connected closely and the neurons intensively distributed.2.Purity of neuron culture:The cytoplasm and neuritis of neurons were stained by NSE antibody on the 8th day.The purity of neuron culture was 90.5%.3.Apoptosis rate detection:?After IH,the apoptosis of hippocampal neurons were observed in the different time course(6 h,24 h and 48 h).The results show that neuronal apoptosis rate gradually ascended from 6.73 ± 1.22%to 10.80±0.92%,15.5±1.51%and 25.8±2.01%.?After IH+orexin-A,the apoptosis of hippocampal neurons were detected after 48h.The apoptosis rate of IH+orexin-A was higher than IH group and depended on concentration of orexin-A.Orexin-A increase in neuronal apoptosis rate was significant at concentrations of 10 nM or greater.(P<0.05)4.Detection of lentiviral transfection efficiency The lentiviral transfection efficiencies of control siRNA group?siRNA-PLC?1 group and siRNA-PLC?4 group were all above 90%.5.Orexin-A aggravated injury of hippocampal neurons under intermittent hypoxiavia phosphorylation of ERK1/2.?The expression of p-ERK1/2 of rat hippocampal neurons increased after treatment with intermittent hypoxia and further elevated when orexin-A was added in after intermittent hypoxia.The expression of p-ERK1/2 in IH+orexin-A+U0126 group was lower than in IH+orexin-A group.?The apoptosis rateof rat hippocampal neurons increased after treatment with intermittent hypoxia and further elevated when orexin-A was added in after intermittent hypoxia.The apoptosis ratein IH+orexin-A+U0126 group was lower than in IH+orexin-A group.6.Orexin-A induced phosphorylation of ERK1/2 via PLC?1 signal pathway to aggravated injury of hippocampal neurons under intermittent hypoxia.? The expression of p-ERK1/2 of rat hippocampal neurons in control siRNA(?)group and siRNA-PLC?4(+)group elevated significantly compared with respective control group.However The expression of p-ERK1/2 of rat hippocampal neurons in siRNA-PLC?1(?)group changed little compare with siRNA-PLC?1(-)group.?The apoptosis rate of rat hippocampal neurons in siRNA-PLC?1(+)group decreased significantly compare with siRNA-PLC?1(-)group.But the apoptosis ratein control siRNA(+)group and in siRNA-PLC?4(+)group altered little compared with respective control group.Conclusion1.The hippocampal neurons of rats in this model were good at quantity,purity and survival ability.2.Neuronal apoptosis process was gradual and time-dependent caused by intermittent hypoxia.3.Orexin-A has injury effect on hippocampal neuron treated with intermittent hypoxia.The effect of orexin-A was dose-dependent and was significant at concentrations of 10 nM or greater.4.Orexin-A aggravated injury of hippocampal neurons under intermittent hypoxiavia phosphorylation of ERK1/2.U0126 blocked phosphorylation ERK1/2 induced by orexin-A to alleviate the injury of orexin-A to hippocampal neurons under anoxia and to decrease apoptosis rate.5,Orexin-A induced phosphorylation of ERK1/2 via PLC?1 signal pathway to aggravated injury of hippocampal neurons under intermittent hypoxia,not PLC?4. |
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