S Study On Protective Effect And Mechanism Of Icam-5 On Paju Cells Challeaged By Serum Deprivation And Hypoxia

Backgroud And ObjectiveIschemic stroke is a frequent and serious disease with limited treatment options. Ischemia accompanied by hypoxia and hypoxic-ischemic brain injury in pathophysiological mechanisms is extremely complex and involve a number of related gene and protein changes and complexity of the link has not been understood thoroughly. There was discovered by cerebral ischemic preconditioning in experimental cerebral ischemia which may stimulate endogenous neural protection and promotion of new protein synthesis, thereby preventing or reduce cerebral ischemia caused by ischemic cascade. In view of the current treatment for ischemic stroke is limited, thrombolytic therapy by the time constraints, many neuroprotective agents have no definite effect, therefore, to find new therapeutic targets is our goal. In our previous work found in intercellular adhesion molecule 5 (ICAM-5) can prevent Aβinduced PAJU cell apoptosis, therefore, we further study whether ICAM-5 also has a neuroprotective effect and its mechanism on PAJU cells in the serum-deprivation and hypoxia medium.Methods1. To use a stable expression of human ICAM-5 protein PAJU-TLN cell lines and PAJU-NEO cell line which has a NEO resistance but does not express ICAM-5 as a neuronal cell model of ischemia and hypoxia. Firstly, we established a serum-free PAJU cell model and observe whether there is an effect of expression of ICAM-5 protein on PAJU cells; and then we established a PAJU cell model culturing in serum-deprivation and hypoxia medium and verify the validity of the model, to further study the effect of ICAM-5 protein expression on PAJU cells.2. With serum-deprivation and hypoxia medium as a stimulus source, both PAJU-TLN and PAJU-NEO cells were stimulated in different time stages at the same time respectively.Using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) colorimetric assay for examming cell viability, Hoechst 33258 nuclear staining for observing the nuclear apoptosisf and detection of apoptosis by flow cytometry, so that we can understand whether ICAM-5 with a protective effect on the PAJU cells culturing in serum-deprivation and hypoxia medium.3. Western blot was used to detecte effect of serum-deprivation and hypoxia on expression of ICAM-5, as well as effect of ICAM-5 on phosphorylation of Ezrin (Thr567), Radixin (Thr564) and Moesin (Thr558), PI3K (Tyr508, p85a) and Akt (Ser473) which is ERM/PI3K/Akt pathway in serum-deprivation and hypoxia medium for understand whether or not the cell survival signaling pathway is activated. Results1. PAJU-TLN cells due to the expression of ICAM-5 with higher survival rate and lower apoptosis rate than the PAJU-NEO cells’in serum-deprivation and hypoxia medium. It showed increased cell survival and reduced apoptosis rate in PAJU cells and the differences were statistically significant.2. Due to long time expose to serum-deprivation and hypoxia medium, the expression of ICAM-5 protein in PAJU cells decreased gradually and to complete cessation of expression in the absence of serum and 2% of the anoxic medium culturing for 72 hours.3. Western blot analysis showed that the expression of ICAM-5 ICAM-5 interacted with ezrin-radixin-moesin (ERM) protein family in PAJU cells, and active phosphorylation of Ezrin (Thr567), Radixin (Thr564) and Moesin (Thr558). Phosphorylated ERM activate phosphorylation of PI3K (Tyr508, p85α), which made activation of Akt (Ser473) directly.4. Western blot analysis also showed that 30μM of the LY294002 markedly inhibited ICAM-5, phospho-ERM, phospho-PI3K and phospho-Akt in PAJU cells culturing in serum-deprivation and hypoxia medium, while the total ERM, PI3K and Akt expression in both PAJU-ICAM-5 and PAJU-NEO cell lines was identical in nontreated and treated cells. It suggested that the activation of ERM/PI3K/Akt signal pathway.Conclusions1. Here we demonstrate for the first time that nomalexpression of ICAM-5 in human neuroblastoma PAJU cells can protect the neurons from absence of serum and 2% of the anoxic medium as indicated by reducing serum-deprivation and hypoxia medium induced apoptosis and promote cell survival.2. Long time expose to absence of serum and 2% of the anoxic medium may reduce or complete cessation of expression of ICAM-5.3. We also demonstrate for the first time that ICAM-5 protects PAJU cells in serum-deprivation and hypoxia medium by activating the ERM/PI3K/Akt signaling pathway. And therefore itn may contr(?)bute to neurons survival and reduce apoptosis in hypoxia-ischemia.