The Antioxidative Effect Of PDE4 Inhibitor Rolipram On Alzheimer's Disease

Objective:To explore the antioxidative effect of Rolipram (Rol) on Alzheimer's diseasse(AD), by using hibateral AP25-35 injection into the hippocampuses of rats as AD rat model and hydrogen peroxide (H2O2)-induced PC 12 cells with oxidative-stress injury as the neuronal injured cell model.Methods:The present study investigated the antioxidative effects of Rolipram on AD from in vivo and in vitro.1 Experiment in vivo:(1) AD procedure, grouping and treatingAβ25-35 was injected into the hibateral hippocampuses of rats to establish Alzheimer's disease-like model, then the rats were randomly divided into five different groups as follows:sham-treated rats(saline was injected into hibateral hippocampuses of rats,AD-like group,rolipram(0.1mg/kg,ip),rolipram(0.5mg/kg, ip),rolipram(1.25mg/kg,ip).(2)The tests of spatial memory performance of ratsThe learning and memory capabilities of rats through step-through passive avoidance tests and Morris Water Maze behavioral test were recorded.(3) Micro-morphological examination by pathohistology observation by HE staining and Nissl's staining in AD ratsThe cerebrums of rats were fixed in 4% buffered paraformaldehyde to observe pathohistology change.(4) Measurement of the reactive oxygen species and antioxidases of AD ratsAfter the Morris Water Maze behavioral test, the rats were sacrificed, and the cerebrums were separated into hippocampuses and cortexes. The ability of clearing·OH and·O2-,the levels of MDA,GSH,NO and LDH, and the activity of SOD of hippocampus and cortex were measured by colorimetric Assay Kit. Effects of rolipram on Trx and iNOS expression levels of hippocampus and cortex were detected both by western blot and Real-time RT-PCR.2 Experiment in vitro:(1) PC12 cells culture and injury procedurePC 12 cells were cultured commonly. The culture media were changed every 24 h. The cells were subcultured every two or three days. The cells were synchronized by serum starvation before Rolipram and H2O2 treatment. Varying concentrations of H2O2 (final concentration 20～200μmol/L) were added to each well and co-incubated for varying times(4 h,6h,8h,12h,18h). The survival rate of PC 12 cells subject to the oxidative damage induced by H2O2 was determined by MTT assay.(2) The protective effects of rolipram on damaged PC 12 cellsThe culture media of PC 12 cells was collected after the treatment of rolipram and H2O2. The survival rate of PC 12 cells was determined by MTT assay. The level of LDH was determined by colorimetric Assay Kit. The PC 12 cells were stained with PI and the percentage of different phases in the cell cycle was analyzed by flow cytometry.(3) The antioxidative effects of rolipram on PC 12 cells injury induced by H2O2The ability of clearing·OH and·O2-, the content of MDA, GSH and NO and the activity of SOD were determined by colorimetric Assay Kit. Effects of rolipram on Trx and iNOS expression levels of damaged PC 12 cells induced by H2O2 were detected both by western blot and Real-time RT-PCR.Results:The present study investigated the effects of rolipram on AD from in vivo and in vitro.1 Experiment in vivo:Chronic administration of the PDE4 inhibitor rolipram reversed cognitive deficits in step-through passive avoidance test and Morris Water Maze behavioral testOn the 14th and 15th day after the injection of Aβ25-35, Aβ25-35-treated rats showed decreased retention tested 24 h after initial training; this was reversed by rolipram. Compared with vehicle-treated controls, rats treated with Aβ25-35 spent a longer time finding the hidden platform during the acquisition trials of the Morris water maze task; this was reversed by repeated treatment with rolipram. Similarly, rolipram reversed Aβ25-35-induced memory deficits, as evidenced by increased time in the target quadrant in the probe trial of the water maze test following rolipram treatment, relative to A025-35 alone. In visible platform-test, there were no differences among every groups of rats on the time they spent finding the visible platform, so all the rats have similar capability of vision and swimming.(2) The effect of rolipram on pathohistology in AD ratsHE staining:The neuronal pathohistology with HE staining was observed by the optical microscope. In sham group, the neurons neatly arranged and their conformations were integrated. In AD-like group, there were some malacia regions,Some neurons Showed ischemic morphology and the cell body reduced. Some nucleis of cells were compressed. There fewer neurons and the neurons mussily arranged. In rolipram 0.1-1.25mg/kg groups, the neuronal pathohistology above was improved.Nissl's staining:In the sham group, the neurons neatly arranged and the number of nissl body in neurons was large. In AD-like group, the conformations of neurons were indistinct, and the nissl body in the cell body and dendritic was dissolved. In rolipram 0.1-1.25mg/kg groups, the neurons neatly arranged and there were more nissl body in neurons comparing with the AD-like group.(3)The effects of rolipram on ROS and antioxidase of AD ratsAfter the injection of Aβ25-35, the ability of clearing·OH and·O2- of hippocampus and cortex significantly decreased (P<0.01 vs sham group). This was reversed by treatment with rolipram at the concentration dependent manner(P< 0.05 vs AD-like group). Meanwhile,the treatment of rolipram significantly decreased the levels of MDA,LDH and NO in hippocampus and cortex, while increased the level of GSH and the ability of SOD in hippocampus and cortex significantly(P< 0.05 vs AD-like group).What's more, the rats treated with rolipram expressed higher level of Trx and lower level of iNOS compared with those treated with the vehicle in the hippocampus (P<0.01 vs AD-like group).But only the dosages of 0.5mg/kg and 1.25mg/kg of rolipram treatment could significantly increase the expression of of Trx and decrease the expression of iNOS compared with those treated with the vehicle in the cortex (P <0.05vs AD-like group).2 Experiment in vitro:(1)H2O2-induced PC 12 cells damaged modelObserved by inverted microscope, the Blank group cells with full size attached solidly to the wall of culture flask, and stretched full. The damaged cells body shrinked back, accompaniment with enation disappearance, cell spaces augmentation and sparsate distribution. The obviously damaged morphous were showed in the damaged cells. The higher concentration of H2O2,the more severe the damage would be. The results of MTT showed that the survival rate of PC 12 cells which were incubated with H2O2 (40μmol/L) for 16h significantly decrease, and this concentration was mild enough.(2) The neuroprotective effect of rolipram on damaged PC 12 cellsThe results of MTT showed that rolipram significantly increased the survival rate of PC 12 cells injured by H2O2 at the concentration dependent manner (P<0.05 vs H2O2-treated group).Rolipram (20μmol-L"1 and 40μmol·L-1)could also increase the level of LDH signifcantly(P<0.01 vs H2O2-treated group). And rolipram rescued the mitosis of damaged PC 12 cell and stimulated the synthesis of DNA in S-phase of cell cycle, which were shown in rising level of cell ratio in different phases (P<0.01 vs H2O2-treated group)(3) The antioxidative effects of rolipram on H2O2-induced damaged PC 12 cellsThe biochemical assay data showed rolipram significantly improved the ability of clearing hydroxy radical and superoxide anion in impaired cells induced by oxidative stress with H2O2(P<0.01 vs H2O2-treated group). Meanwhile, inhibition of PDE4 increased the level of GSH and SOD in cell supernatant, while decreased the content of MDA and NO (P<0.05 vs H202-treated group). Furthermore, the impaired cells incubated with rolipram expressed higher level of Trx and lower level of iNOS compared with those treated with the vehicle (P<0.05 vs H2O2-treated group)Conclusion:In the present study we studied the antioxidative effects of rolipram on AD model from the aspects of biochemical and pathological indicator by using hibateral Aβ25-35 injection into the hippocampuses of rats as AD model in vivo and H2O2-induced PC 12 cells oxidative-stress injury as the neuronal damaga model in vitro. The conclusion as follows:(1)Rolipram played an important role in AD rats through antioxidatie mechanism. The neuroprotective effect attributed to several aspects:chronic administration of the PDE4 inhibitor rolipram reversed cognitive deficits,improved the pathological damage, increased the ability of antioxidase and the adjustment ability of trx, reduced ROS and depressed the activation of iNOS-NO pathway in hippocampus and cortex, especially in hippocampus.(2) The present study demonstrated that rolipram was able to protect H2O2-induced PC12 cells by decreasing ROS activity, balancing the oxidative and antioxidative systerms, increasing the adjustment ability of trx and depressing the activation of iNOS-NO pathway, which could maintain membrane integrity, increase the survival rate of cells,protect mitochondria, prolong the S phase in cell cycle and promote DNA synthesis of S phase.