NGR1 Protects Against Hypoxic-ischemic Injury In Rats And Its Mechanism

Hypoxic-ischemic encephalopathy(HIE)is the main reason for neonatal death,acute or chronic nervous system damage and brain retardation.The main pathology characteristic of HIE is cell apoptosis or death,so it is important to find a way to reduce the cell injury effectively.Notoginsenoside R1(NGR1)is a phytoestrogen that is isolated from Panax notoginseng.Panax notoginseng is believed to have anti-inflammatory,antioxidative,and antiapoptotic properties.However the neuroprotective properties and underlying mechanisms of NGR1 during HIE in newborns are largely unknown.As previous study,Endoplasmic reticulum(ER)stress is a cellular signaling response that is induced by various stimuli,such as oxidative stress and hypoxia-ischemia and it may cause apoptosis,the important is ER stress is regulated by the balance of endoplasmic reticulum calcium.When the body suffer undesirable stimulation,endoplasmic reticulum calcium were release from the endoplasmic reticulum through the inositol 1,4,5-trisphosphate(IP3)respector.Under ER stress,IP3 R and the IP3-induced Ca2+ release(IICR)from IP3 R activation in response to exterior stimulus.Activation of PLC will lead to the higher expression of IP3 and IP3 R.Most importantly,estrogen could activate the PLC-inositol phosphate signaling pathway.It indicated a probe into the relationship between NGR1 and PLC/IP3 R is significant to uncover the mechanism of NGR1 and to find out how it acts on ER stress.In this study,multiple approaches were employed to explore the neuroprotective effects and the underlying mechanisms of NGR1 against OGD/R injuries in primary cortical neuron cultures.It was shown for the first time,to the best of our knowledge,that the neuroprotective effects of NGR1 against HIE may suppress the activation of the PLC?/IP3 R pathway through the estrogen receptor to regulate ER stress.ObiectiveIn this study,primary cortical neurons and 7-dayold postnatal Sprague-Dawley rats were used to modelling HIE.Investigate the neuroprotective effect NGR1 on hypoxic-ischemic injury and its mechanism in vivo and in vitro.Methods:1.Oxygenglucose deprivation/reoxygenation(OGD/R)4h or 24 h in primary cortical neurons and unilateral ligation of the common carotid artery(CCL),followed by exposure to a hypoxic environment in 7-dayold postnatal Sprague-Dawley rats were used to mimic HIE episodes.2.Groups1: Control or sham group,OGD/R;HIE group,OGD/R+NGR1/HIE+NGR1 group,vehicleGroups2: Control or sham group,OGD/R;HIE group,OGD/R+NGR1;HIE+NGR1 group,OGD/R+NGR1+ICI-182780;HIE+NGR1+ICI-182780 group,vehicleGroups3: Control or sham group,OGD/R;OGD/R+NGR1;OGD/R+NGR1+m-3MFBS(m-3MFBS:agonist of PLC)3.Detection Indicator:(1)MTT assay used to test cell viability;LDH used to assess cell injury and Hochest33342 used to assay cell apoptosis(2)TTC stain used to test cerebral infarction,HE stain used to test pathological alteration,TUNEL stain used to test cell apoptosis(3)Western Blot was used to detected the expression of different protein.(4)Flou-3AM stain was used to detected the concentration of calcium in endoplasmic reticulum Results:1.Compared with OGD/R group,there were higher cell viability,less LDH leakage and less cell apoptosis in OGD/R+NGR1 groupCompared with HIE group,there were less cerebral infarction,pathological alteration,TUNEL positive cell.2.Compared with OGD/R+NGR1 group,there were lower cell viability,higher LDH leakage and more cell apoptosis in OGD/R+NGR1+ICI-182780 group.Compared with HIE+NGR1 group,there were more cerebral infarction,pathological alteration,TUNEL positive cell in HIE+NGR1+ICI-182780 group.3.OGD/R and HIE stimulated expression of ER stress related apoptotic proteins and treatment with NGR1 decreased levels of these,whereas ICI-182780 pretreatment significantly attenuated the effects of NGR14.NGR1 inhibit the activation of PLC/IP3 R at OGD case and compared with OGD/R group,NGR1 block the Ca2+ flow out from the endoplasmic reticulum ConclusionNGR1 Attenuated HIE-Induced cell Injury in vitro and vivo and NGR1 could have neuroprotective through two way.On the one hand,notoginsenoside R1 protects against neonatal cerebral hypoxic-ischemic injury through estrogen receptor–dependent activation of endoplasmic reticulum stress pathways,on the other hand Notoginsenoside R1 regulate the function of PLC/IP3 R and Ca2+ in endoplasmic reticulum.