Effects And Mechanism Of Angiotensin ? Type 2 Receptor Activation On PC12 Cells In Anoxia-reperfusion Injury

Ischemic cerebrovascular disease is a common type of cerebrovascular disease with a high rate of disability and mortality, and reperfusion injury of central nervous system can easily ooccurs during its treatment. Research finds that Angiotensin II type 2 receptor(AT2R) have an impact on the plasticity and cognitive function of normal nerves. It has a large amount of functions, including dilating vessels, regulating growth and development of cells, facilitating proliferation and differentiation,repairing and remolding tissues and promoting open of collateral circulation which can protect injury of ischemic cerebral by ameliorating cycle of cerebral blood. However, it is not clear till now that the specific mechanism of the impacts of AT2 R on hypoxia/reoxygenation injury of nerve cells. This research is focusing on the impacts and primary mechanism of it, providing new experiments evidences for more illustration.P12 cells is the cell line of adrenal gland chromaffin tumor cells which has general features of neuroendocrine cells,and it was widely used in neurophysiology and other related research. In our experiments, PC12 cells were cultured with DMEM(10% fetal bovine serum) in 37?,5%CO2 constant temperature incubator for 16~24 hours. When celldensity reaches to 80%~90%, extract medium, and rinse cells for three times by using PBS. Then utilizing no sugar free serum medium containing Na2S2O4 to cultivate PC12 cells. Lastly reusing fresh 10% fetal bovine serum with high glucose DMEM medium to cultivate cells one hour later so that models can be established. Dealing them with AT2 R antagonist(PD123319), AT2 R agonist(CGP42112) and P38inhibitor(SB203580) respectively. Then observing viability of cells after trypan blue staining through electron microscope, observing survival of cells through MTT method, detecting changes of expression of Bax and Bcl-2 mRNA through reverse Transcription PCR were carried out,analysing changes of expression of p-P38MAPK/Bax/Bcl-2 protein through western blot.Our experimental results show that: 1.It can be observed that the cell began swell and protuberances began contract even broken after dealing with Na2S2O4, and dead cells are stained blue. The survival rate of cellsis about 55~60% measured by MTT. Therefore, we established the hypoxia/reoxygenation injury model of PC12 cells successfully. 2.Comparing with control groups, the hypoxia/reoxygenation injury PC12 cells have a higher expression level of phosphorylate P38 MAPK protein and Bax mRNA, a reduction of cells survival rate as well as expression of Bcl-2 mRNA(P<0.05) when tackled with PD123319. 3.Comparing with control groups, the hypoxia/reoxygenation injury PC12 cells have a higher expression levelof phosphorylate P38 MAPK protein and Bax mRNA, a reduction of cells survival rate as well as expression of Bcl-2 mRNA(P<0.05) when tackled with CGP42112. 4.Comparing with control groups, the hypoxia/reoxygenation injury PC12 cells have an increase of cells survival rate and expression of Bcl-2 mRNA and protein, a reduction of expression level of phosphorylate P38 MAPK protein and Bax mRNA and protein(P<0.05) when tackled with SB203589. The results are prompted that: 1.the activation of AT2 R can promote the growth of PC12 cells, and alleviate injury caused by hypoxia/reoxygenation.2.AT2 R activation can promote the growth of PC12 cells by regulation of the expression of Bcl-2/Bax though inhibiting the activity of P38 MAPK signaling pathway.