The Effect Of Db-cAMP And Aminophylline Under Single Or Combined Administration On PC12 Cells With Oxygen-glucose Deprivation

BANCKGROUND AND OBJECTIVENeonatal hypoxic ischemic encephalopathy (Hypoxic-Ischemic Encephalopathy, HIE) is a variety of perinatal asphyxia caused by partial or complete lack of oxygen, reduction or suspension of cerebral blood flow cause fetal or neonatal brain damage. HIE is a leading cause of children's nervous system disability, about 2% to 4% of the fetus or newborn asphyxia occurs in the perinatal period, of which 15% to 20% will occur with hypoxic ischemic encephalopathy, in survivors of severe HIE,25% will have very serious consequences for the permanent neurological dysfunction. Since the pathogenesis of neonatal hypoxic ischemic encephalopathy is complex, involving multiple aspects.At the present, blocking a single sign of a disease mechanism, the effects is not ideal, many nerve protective treatment are still at exploratory stage. Therefore, there is no effective treatment which is abroad recognized,so to find more effective treatments have become a important topic of perinatology.The damages of HIE has been extended to the injury recovery period after reperfusion, the pathogenesis of disorders, including cerebral blood flow, increased anaerobic metabolism, ATP decreased, hypoglycemia, lactic acidosis, an increase of excitatory amino acids, intracellular calcium accumulation, activation of nitric oxide and oxygen free radical production, inflammation, delayed neuronal death (apoptosis), etc. These mechanisms are the foudation to develop neuroprotective therapy after brain injury. In recent years, domestic and international study found that cAMP (Cyclic Adenosine-3'5'-monophosphata, cAMP) involved in a variety of nervous system diseases, pathogenesis, plays an important role in injury repairing in the nervous system. cAMP is a cell transfer mediator hormones and neurotransmitters, it is also known as "second messenger." cAMP is made from ATP by catalytic action of adenylate cyclase (AC),and is hydrolyze by the phosphodiesterase (PDE), thus maintaining the stability of intracellular cAMP levels. The results show that cAMP in the nerve plays an important role in metabolic regulation. Changes in intracellular cAMP concentration can affect multiple intracellular signaling pathways, thereby regulating gene expression, protein activity and cell function, cell differentiation, growth, metabolism and transfer death process. Studise found that increased levels of intracellular cAMP, can promote the survival of newborn neurons and axonal regeneration after injury and recovery of neurological function. db-cAMP can promote axonal regeneration of retinal nerve cells, can induce nerve regeneration after spinal cord injury and induced neuronal axons and the cut off ends of dorsal column sensory fibers snout regeneration. Clinical study found that neonatal asphyxia decreased cerebrospinal fluid levels of cAMP, and cAMP levels and the severity of illness and prognosis, suggesting that cAMP may be involved in neonatal hypoxic-ischemic brain injury.But whether db-cAMP can improve neonatal hypoxic ischemic brain injury were rarely reported.Aminophylline, theophylline and ethylenediamine complex salt, is a non-selective PDE inhibitors which can delay the degradation of intracellular cAMP, including the inhibition of hydrolysis of cAMP of the major PDE3, PDE4, and indirectly to elevate intracellular cAMP. Studies found that PDE4 inhibitors by raising the concentration of intracellular cAMP could induce phrenic nerve after spinal cord injury recovery; PDE3 inhibitors can reduce the injury of cerebral ischemia in gerbils hippocampal CA1 area and improve learning and memory functions defect after cerebral ischemia. Animal experiments and clinical studies confirmed that aminophylline can significantly reduce the cerebral blood flow, cerebral edema, reduce the hypoxic-ischemic brain damage and mortality. But the mechanism of aminophylline on hypoxic ischemic brain damage is not yet in-depth.This study was to use rat pheochromocytoma (PC 12) cells in hypoxia glucose deprivation (oxygen-glucose deprivation, OGD) model, by inducing cytotoxicity, apoptosis to simulated ischemia on the nerve cells injury. By using MTT test cell activity, measuring with Hoechst33342 fluorescent dye to detect apoptosis, using calcium fluorescent probe Fura-2 AM to detect intracellular calcium and caspase-3 apoptosis reagent to detected with caspase-3 activity. Aim to screen the best concentrations of single or combined administeation of db-cAMP and aminophylline, and the protection time of db-cAMP. Preliminary study if administe exogenous cAMP new drugs db-cAMP which can increas intracellular cAMP concentration, as well as,non-selective phosphodiesterase (PDE) inhibitor aminophylline which can slow down the degradation of intracellular cAMP and wether the two drugs in combination administeation can block nerve cell injury in hypoxic ischemic and the possible mechanisms.METHEOD1.PC12 model of oxygen glucose deprivation:Cells were cultured 48 hours for OGD treatment:first to blot cells medium, and use sugar-free balanced salt solution (EBSS) gently to wash the cell for twice, add sugar-free balanced salt solution, and then placed the cell in hypoxia incubator N2/CO2/O2 (94%/5%/1%) under the conditions of OGD injury for 4h.At last, replace EBSS for DMEM medium and reoxygenation 24h. Used EBSS containing glucose in the control group,it was placed it in culture CO2 incubator for 4h and then replaced EBSS for DMEM medium.2. PC 12 cell activityPC 12 cells in OGD for 4 hours and reoxygenation for 24 hours, adding MTT, 37℃incubation 4h, removed the culture medium, and added dimethyl sulfoxide (DMSO) low-speed oscillation to fully dissolve crystals, read the absorbance (OD) which were measured by microplate reader at wavelength of 570nm, and set the control holes, the absorbance of each hole cell minus the absorbance of the hole shall be the absorbance of cells. Absorbance of cells directly reactive the viability of cells.Each group had 6 holes.3. Use MTT to screen the optimal concentration single or combined administeation of db-cAMP and aminophylline3.1 the best concentration of aminophylline screening:Trial groups were divided into normal control group and OGD groups, OGD groups were divided into the untreated group and aminophylline treatment groups, according to the different concentration of aminophylline into 1×10-2mg/L,3×1010-2mg/L,5×10-2mg/L, 8×10-2mg/L Land 10×10-2mg/L groups.3.2 db-cAMP optimal concentration of screening:Trial groups were divided into normal control group and OGD groups, OGD groups was divided into the untreated group and db-cAMP treatment groups, depending on the drug concentration the db-cAMP treatment groups were divided into 1×10-6mol/L,1×10-5ol/L,1×10-4ml/L and 1×10-3mol/L groups.3.3 combined administeation of db-cAMP and aminophylline screening:Trial groups were divided into normal control group and OGD groups, OGD groups was divided into the untreated group and the combined treatment groups which were divided intoⅠGroup:aminophylline 1×10-2mg/L+db-cAMP1×10-6mol/L,Ⅱgroup:aminophylline 3×10-2mg/ L+db-cAP1×10-5mol/L,ⅢGroup: aminophylline 5×10-2mg/L+db-cAMP1×10-4mol/L according to different combination of drug concentrations.4. The cell viability under single or combined administeation of db-cAMP and aminophyllineTrial groups were divided into control group and OGD groups, OGD groups were divided into untreated group and drugs treated groups which wre divided into aminophylline group, db-cAMP group and the combined administeation group. 5. The rate of apoptosis under single or combined administeation of db-cAMP and aminophylline5.1 Trial groups were divided into control group and OGD groups, OGD groups were divided into untreated group and drugs treated groups which wre divided into aminophylline group, db-cAMP group and the combined administeation group.5.2 Use Hoechst33342 fluorescent dye to determination cell apoptosis, PC 12 cells reoxygenated for 24h after OGD injury for 4h,discard culture medium, rinsed with PBS, after adding fixative fixed cells, and then use PBS to rinse liquid. Natural drying, added Hoechst33342 fluorescent dye to the cell away from light staining at the room temperature. Observed under fluorescence microscope and counted form the nucleus, that cell has been infected with fluorescent apoptosis. Counting cells, cell apoptosis rate with the number of apoptotic cells in a percentage of total cells.6. The intracellular calcium concentration under single or combined administeation of db-cAMP and aminophylline6.1 Trial groups were divided into control group and OGD groups, OGD groups-were divided into untreated group and drugs treated groups which wre divided into aminophylline group, db-cAMP group and the combined administeation group.6.2 PC 12 cells were treated 24h after OGD and reoxygenation, cells were collected and washed with D-Hanks solution, trypan blue staining, and counting living cells above-95%,adding calcium fluorescent indicator Fura-2/AM,37℃load, and then washed by D-Hanks solution, using a dual wavelength fluorescence spectrophotometer, the excitation wavelength is 340nm, the emission wavelength is 500nm, slit is 10nm, and measured sample fluorescence value F, then add Triton-100 (TritonX-100) measured fluorescence value Fmax, at last,adding calcium chelator ethylene glycol-bis-2-aminoethyl tetraacetic acid (EGTA) fluorescence to the cell to measurements Fmin.Calculated [Ca2+] i by the following formula. Kd is 224 at 37℃nmol/L. [Ca2+]i(umol/L)=Kd(F-Fmin)/Fmax-F 7. The cell caspase-3 enzyme activity under single or combined administeation of db-cAMP and aminophylline7.1 Trial groups were divided into control group and OGD groups, OGD groups were divided into untreated group and drugs treated groups which wre divided into aminophylline group, db-cAMP group and the combined administeation group.7.2 Using Caspase-3 activity kit to the detection the cell caspase-3 activity. Steps the followed:(1) Determination of 4-nitroaniline (pNA) and total protein standard curve; (2) sample collection:cells were treated with OGD injury and drug treated cells were collected, adding with lysate, cells were centrifuged, and the supernatant was cllected; (3) caspase-3 activity detection of apoptosis: Set the control bore holes and sample, incubated at 37℃for 4h, the A405 under the detection microplate reader sample minit blank hole shall be the A405 generated by pNA. pNA standard curve to calculate the sample catalyzed the amount of pNA; (4) Bradford assay Samples of total protein concentration; (5) Calculate the caspase-3 activity unit.8.MTT detect time window of protective effects of db-cAMP on PC12 cellExperiment were divided into normal control group and OGD group, OGD group was divided into the untreated group and drug administration groups, drug administration group s were divided into two Oh, 1h group,2h group,4h group,8h group,16h group,according to different administration time.9. Statistical methodsEach set of data application SPSS 13.0 statistical software for analysis. Experimental data with mean±standard error ((?)±s) said that all measurement data to compare the homogeneity of variance in determining the post (P> 0.05), all groups were analyzed using single factor analysis of variance (One-Way ANOVA), the Multiple groups were used to compare the number of LSD method (Least-significant difference test) or SNK method. If the the homogeneity of variance out determining the post (P< 0.05),using the Welch method, multiple comparisons between groups using dunnett's T3 test. The two treatment groups were compared using two sample t number of test, P<0.05 was considered statistically significant difference.RESULTS1. Successfully established PC 12 model of oxygen glucose deprivationReoxygenation after oxygen glucose deprivation, compared with the cells cultured in the normal condition, OGD decreased neuronal activity by 35.71%, increased apoptosis rate by 31.57 %, increased intracellular calcium concentration to 286.182nmol/L, cell caspase-3 activity increased to 4.444 units.2.The optimum concentration of single or combined administeation of db-cAMP and aminophyllineThrough the screening of drug concentrations, the concentration of aminophylline is 5×10-2mg/L, the concentration of db-cAMP is 1×10-4mol/L, the concentration of combined administeation is 3×10-2mg/L + db-cAMP1×10-5mol/L, respectively.3. The cell activity under single or combined administeation of db-cAMP and aminophyllineAminophylline group compared with untreated group the cells viability was no significant increase(P> 0.05), db-cAMP group and the combined treatment group compared with untreated group the viability was significantly increased (P= 0.001, P = 0.000), the use of db-cAMP single and combine with aminophylline after OGD can increase the activity of PC 12 cells.4. The rate of apoptosis under single or combined administeation of db-cAMP and aminophyllineAminophylline group compared with the untreated group, apoptosis rate was not significantly lower (P> 0.05), db-cAMP group and the combined administeation group compared with the untreated group,the rate of apoptosis was significantly lower, the difference has statistically significant (P= 0.004, P= 0.002), the use of db-cAMP single and combine with aminophylline after OGD can reduces PC 12 cell apoptosis.5.The intracellular calcium under single or combined administeation of db-cAMP and aminophyllineAminophylline group compared with untreated group,the intracellular calcium levels was no significantly lower (P> 0.05); the db-cAMP group and the combination group compared with untreatment group the concentration of intracellular calcium significantly decreased,the difference statistically significant (P <0.05), the use of db-cAMP single and combine with aminophylline after OGD can decrease the calcium concentration in PC 12 cells.6.The cell caspase-3 activity under single or combined administeation of db-cAMP and aminophyllineControl group cells have not detected activity in the apoptosis caspase-3.The activity of cell caspase-3 enzyme was increased in each OGD group. The Theophylline group, db-cAMP group and the combination group compared with the untreated group the Caspase-3 activity was not significant decreased (P> 0.05).7.The protective time window of db-cAMP on PC 12Oh, 1h,2h,4h groups compared with untreated group, the cell viability was increased significantly (P<0.05), predicted that administration db-cAMP before 4h after OGD could improve the survival of cells, There was no significant difference within the four groups (P> 0.05).The 8h group and the 16h group compared with untreated cells the cell viability was no significant difference (P> 0.05), db-cAMP administrated within 4h ater OGD has a protective effects.CONCLUSION1. This experiment show that cell viability of injured PC 12 cells significantly been increased after adding db-cAMP, and the rate of apoptosis also been reduced. It is suggested that the increased c-AMP concentration by adding exogenous c-AMP can protect ischemic cells.2. Using aminophylline alone can not increase cell activity and decreased apoptosis significantly. It is suggested that the level of c-AMP in is very low, although using aminophylline alone can delay the hydrolysis of intracellular cAMP, but can not increase the intracellular cAMP to high level, so the protection to hypoxic-ischemic injured cells of cells is limited.3.The study found combination of small dose of aminophylline and small doses of db-cAMP can single use of get same protective effect as single use large dose of db-cAMP. It is suggested that after cAMP level been increased by a certain concentration of exogenous cAMP, low-dose theophylline play a assistant role to anti cell death and apoptosis.4. As to the timing of using drug after hypoxic-ischemic injury, it is studied that db-cAMP can increase cell activity withing 4h after hypoxic-ischemic injury, there are no significant improvement of cell activity when 8h after hypoxic-ischemic injury. This study can provide an experimental basis to the timing of using drug in clinical situation.5. By detecting of the concentration of intracellular and activity of caspase-3 apoptotic enzymes, we found that that influx of Ca2+ and caspase-3 apoptosis may be involved in apoptosis of PC 12 cell injury after OGD. db-cAMP group and combined group can decrease the concentration of intracellular calcium after OGD injury, it is suggested that exogenous db-cAMP can block intracellular calcium influx, which may be the possible mechanism of protecting nerve cells by db-cAMP, but db-cAMP group and combined group can not reduced apoptotic activity of caspase-3 significantly, which indicated that caspase-3 pathway might not the mainly mechanism of anti-apoptosis by db-cAMP, while the detail mechanism of anti-apoptosis by db-cAMP need further study.