NRG-1 Rescues Cortical Neurons From Oxygen-Glucose Deprivation Via GABAergic Mechanism

Stroke is the rapidly developing loss of brain functions due to a disturbance in the blood vessels supplying blood to the brain. In china, stroke has been rising to the first leading cause of death. Even the survivor may be a disability. And in the developed nation, stroke is the third leading cause of death, only surpassed heart disease and cancer. The best clinical therapy to the stroke is thrombolytics. But it is limited by the strict time window (<3h), and may lead to intracranial hemorrhage and so on. So, to extend the time window, decrease the incidence rate of intracranial hemorrhage, and make patients better, it is more important to study the neuroprotectant. Neuregulin-1(NRG-1) is a member of growth factor family known to be involved in the survial and function of neurocytes.The neuregulins (NRGs) are cell-cell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB family. The neuregulin family of genes has four members:NRG1, NRG2, NRG3, and NRG4. Relatively little is known about the biological functions of the NRG2,3, and 4 proteins. Our experimental results show that NRG-1 has significant neuropretective effects to the cortical neurons. However, according to our knowledge, there have been no reports about concerning that how the NRG-1 works. After explaining the mechanism, we can not only further know the function of NRG, but also provide a new target to therapy the stroke.In 2007, our lab has reported that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, and enhances depolarization-Induced GABA release. Our experimental results show that the GABA receptor agonists also rescued cortical neurons from oxygen-glucose deprivation. Therefore, we consider that when NRG-1 bind to the receptor erbB4 which on the membrane surface of GABAergic neuron, the GABA release, and then the GABA bind to its own receptors to give full play to the neuroprotective effects. We do experiments as follows:First, we get the cortical tissue from the neonate Sprague Dawley rats, and primary culture the cortical neurons in vitro. After 12 days, the matured neurons are taken to get immunochemical staining of GAD67, and then we can get the proporty of GABAergic neurons, that is 20%. Second, we build the model of oxygen-glucose deprivation/reoxygenation on the matured cortical neurons. We divided the matured neurons into two groups:the control and the OGD. In the control group, we change the Neurobasal-A Medium for the EBSS(containing the glucose)and then put them in the normal incubator; in the OGD group, we change the Neurobasal-A Medium for the EBSS(not containing the glucose) and then put them in the incubator without oxygen. After 4 hours, we change the two groups for normal Neurobasal-A medium, and 24 hours later, stain Hoechst33342/PI to count the rate of PI positive cells to evaluate the condition of the cell death. The results tell us that the cell death of the control and the OGD group is 5.0478%±3.9862% and 30.3178%±1.6481% (P<0.01).So the cell death of OGD/R group is higher than the control group, and the model is succeeding. On the base of the right model, we administrate some drops to the model. The drugs contain that NRG-1, NRG-1 and ecto-erbB4, NRG-1 and CGP52432 and BMI, NRG-1 and baclofen and muscimol. After administrating NRG-1, the result is that the rate of cell death is 14.5508%±1.1598%(compared to the OGD group, P<0.01).The result shows that NRG-1 can deeply decrease the cell death of the OGD group. So NRG-1 has neuroprotective effects to the cortical neurons. The result is the same as Byron D. Ford experimental group's result in vivo. After administrating NRG-l+ecto-erbB4, the result is that the rate of cell death is 25.4851%±2.3788% (compared to the NRG-1 group, P<0.01).So we can see that ecto-erbB4 can partly reduce the neuroprotective effects of NRG-1. We can get the conclusion that the neuroprotective effects of NRG-1 is via the NRG-1-erbB4 signal.There have been many literatures show that the GABA receptor agonists have the neuroprotective effects to the hippocampus neurons through some in vivo experiments. After administrating baclofen+muscimol, the result is that the rate of cell death is 19.9773%±0.7476%(compared to the OGD group, P<0.01).The result shows that the GABA receptor agonists also have the neuroprotective effects to the cortical neurons, we administrate NRG-1+CGP52432+BMI to the model, and the result is that the rate of cell death is 23.6560%±1.6259%(compared to the NRG-1 group, P<0.01), The result shows that the GABA receptor antogons can partly blocked the neuroprotective effects of NRG-1. T To deeply test the hypothesis that NRG-1 rescued cortical neurons from oxygen-glucose deprivation:via increasing GABA release, we administrate NRG-1+baclofen+muscimol to the model, and the result is that the rate of cell death is 13.3528%±1.6691%(compared to the NRG-1 group, P>0.05).The result shows that the rate of cell death between NRG-1 and NRG-1+baclofen+muscimol group don't have cooperation. The results indicate that NRG-1 rescued cortical neurons from oxygen-glucose deprivation via GABAergic mechanism. GABA.