The Protective Effect And Mechanism Study Of Heat Shock Protein 70 On Oxygen Glucose Deprivation Postconditioning In SK-N-SH Cells

Background: Cerebral ischemic postconditioning(RIPostC) was evolved on the basis of ischemic preconditioning, was an important way to start the endogenous brain protection mechanism, which had better application prospect and practical value than ischemic preconditioning, because of its implementation after ischemia, grasped easily and controllability strongly. RIPostC was defined as a series of rapid transient ischemia and reperfusion in the early phase of reperfusion, which induced the brain ischemic tolerance and provided obvious protective effect, reduced the ischemic/reperfusion injury. Despite the molecular mechanism of RIPostC has not yet been clear, but it has been focused on protective protein. Heat shock protein 70(HSP70), also called stress proteins, are generated by eukaryote and prokaryote, which can maintain normal cells physiological function, enhance the ability of cells against damage. It has become a hot spot and full of hope in the research of neurology. Its protective effect has been numerous researchs in the cerebral ischemic preconditioning, but its mechanism of RIPostC is unclear.RIPostC was proved in animal experiments, rarely involved in in-vitro model. Animal models were susceptible to a variety of uncontrollable factors in vivo, but the in-vitro studies could prevent the impact from strictly control experimental conditions, supple the shortage of animal experiments, and improve the integrity of the results. SK-N-SH cells were human neuroblastoma cell lines, not only bad the morphological characteristics of nerve cells, but also had the ability of tumor cells to proliferate indefinitely, and be easier culture than nerve cells, which had been widely applied in the research of ischemic injury. So, this research would culture the SK-N-SH cells in vitro, the cells were carried out oxygen glucose deprivation reperfusion(OGD/R) and then several times treatment with abbreviated oxygen glucose deprivation after re-oxygen-glucose to establish the oxygen glucose deprivation postconditioning(OGDP). From the cellular level, this research discussed the effect and mechenism of HSP70 in the OGDP, which could provide the mechanism of ischemic postconditioning with the reliable in vitro experimental basis, and offer new ideas to develop new drugs and prevent and treat the cerebrovascular disease in the clinical.Objective: 1. To observe the protective effect of OGDP on the OGD/R injury. 2. To investigate the expression of HSP70 in the OGDP and explore its influence on apoptosis. 3. To explore the possible mechanism of inhibiting apoptosis of HSP70 in the OGDP.Methods: 1.The SK-N-SH cells were cultured for 2-3 days in vitro, when were in logatithmic growth phases, the cells were randomly divided into:(1) The control group: the cells were cultured continuously under the DMEM high glucose medium;(2) OGD group: At first, the cells were treated by OGD 4h with sugar-free Earle’s liquid, which contained the final concentration of 5mmol/L Na2S2O4, and then replaced with the DMEM high glucose medium to 24h;(3) OGDP group: after 4h of OGD, the cells were carried out several brief alternate of oxygen glucose deprivation after re-oxygen-glucose(ROG), making oxygen glucose deprivation postconditioning. According to the processing time and the number of cycles, the OGDP groups were divided into three subgroups, as follow, all the subgroups were then recovered to 24h: ① The first OGDP subgroup(OGD4h + 3 cycles × 5 min group): after 4h of OGD, OGD was established for 5 min after re-oxygen-glucose(ROG) for 5 min(as one cycle), followed with another two cycles( total of three cycles); ② The second OGDP subgroup(OGD4h + 3 cycles × 10 min group): after 4h of OGD, OGD was established for 10 min after re-oxygen-glucose(ROG) for 10 min(as one cycle), followed with another two cycles( total of three cycles); ③ The third OGDP subgroup(OGD4h + 3 cycles × 15 min group): after 4h of OGD, OGD was established for 15 min after re-oxygen-glucose(ROG) for 15min(as one cycle), followed with another two cycles( total of three cycles). Detection index: MMT assay(3-(4, 5)-dimethylthiahiazo(-z-y1)-3, 5-di- phenytetrazoliumromide; MTT) was used to detect the survival rate of the cells, and the cell morphological change was directly observed by using the inverted phase contrast microscope.2. The SK-N-SH cells were randomly divided into: The control group, OGD group, and OGDP group: according to the results of the first part, OGDP was selected the second subgroup: after 4h of OGD, OGD was established for 10 min after ROG for 10min(total of 3 cycles), then the cells were recovered to 24 h. The immunochemistry was detected the expression of HSP70 and counted the positive cells. AO/EB double staining was observed the diversification of cell fluorescence and counted the cell apoptotic rate in every group.3. The SK-N-SH cells were randomly divided into: The control group, OGD group, and OGDP group. The immunochemistry was detected the expression of Bcl-2 and counted the positive cells.The Results:1.1 The survival rate changes of each group of OGDP cells:The survival rate of the control group and OGD group was 100% and(53.94±6.66) %. The survival rate of the first、second、third OGDP subgroup respectively was(58.20±6.86)%、(70.72±7.64)% and(58.43±6.83)%. Compared with the control group, the cell survival rate of OGD and OGDP group decreased significantly(P<0.01). Relative to the OGD group, though the cell survival rate of the first and third OGDP subgroup was improved(P<0.05), the second OGDP subgroup could improve most obviously in all OGDP subgroups, increased by 16.78%, which was statistically significant(P<0.01), suggested that the second OGDP subgroup had the strongest protection.1.2 The cell morphological observation:The cells of the control group were cultivated normal conditions, so the cell growth state was excellent, the number was more, each cell was adjacent to each other closely and grew rapidly, long spindle or irregular, the boundary was clearly and obvious halo, and highly refractive. Compared with normal culture, the growth condition of cells was worse in OGD group, the intercellular tightness was poor, the number was less, the growth rate was slower, and the number of death was increased. Cell population of OGDP group significantly increased, compared to OGD group, the majority morphological of cells was normal, adherence was better and refraction rise, only a few became shrinkage and round.2.1 The expression HSP70 of immunocytochemistry:The number of HSP70 positive cells respectively was(4.53±0.77)/HP,(21.56±1.75)/HP,(30.71±3.13)/HP in the control group, OGD and OGDP group. Compared with the control group, the protein expression level of HSP70 was significantly higher in the OGD and OGDP group; and it was significantly higher in OGDP group than OGD group(P<0.01).2.2 The apoptosis result of AO/EB double staining:Most cells in the control group showed normal structure, emerged uniform green fluorescence, but the fluorescence of only a few cell was lighter(only a few cell was in the early stages of apoptosis); the cells of OGD group were substantial in the early apoptosis, the fluorescence were brighter and the nucleus gathered together, and dyeing deepen. Compared with OGD group, the fluorescent of nucleus was fainter, and the shape tended to rule, early apoptotic cells were significantly reduced in the OGDP group.The result of apoptosis count showed that the apoptosis rate of the control group, OGD and OGDP group respectively was(5.25 + 5.25) %,(43.35 + 4.88) % and(35.25 + 3.44) %. Compared with normal control group, the apoptosis rate in the OGD group and OGDP group was significantly higher(P<0.01), but the OGDP group was significantly lower than the OGD group(P<0.01).3. The expression Bcl-2 of immunocytochemistry:The number of Bcl-2 positive cells respectively was(4.8±0.837)/HP,(22.2±2.86)/HP,(46.2±2.38)/HP in the control group, OGD and OGDP group. Compared with the control group, the protein expression level of Bcl-2 was significantly higher in the OGD and OGDP group(P<0.01); and it was significantly higher in OGDP group than the OGD group(P<0.01).Conclusions: 1. By comparing the survival rate with the different processing time of OGDP, it had the obvious protective effect on the OGD/R injury, and the three cycles of OGD 10 min after re-oxygen-glucose(ROG) for 10 min was the most significant. 2. The over-expression of HSP70 has a significantly effect on anti-OGD/R injury in the OGDP, and it may be achieved through inhibition of apoptosis. 3. Conclusions: The anti-apoptosis effect of HSP70 achieved possibly by promoting the expression of Bcl-2 in the OGDP.