Protective Effects And Mechanisms Of Cyclophilin A Against Neurotoxicity Induced By β-amyloid Protein

ObjectiveAlzheimer's disease(AD) is a common central neurodegenerative disease.The clinical manifestation of AD include inreversible progressive failure of memory, disorder of recongnition and language and change of personality.A lot of study show that the pathological feature of AD is atrophy of brain,senile plaque(SP) appeared out of cells,neurofibrillary tangles and loss of neurons at hippocampus and cortex.β-amyloid protein(Aβ) is the main component of SP,and it has been an important factor of genesis of AD.Experiments in vivo and in vitro demonstrate Aβcan induce apoptosis and death of neurons,but the mechanism of Aβinduced-neurotoxity is complex.Recently,study has shown Aβcould incuce overproduction of free radical by different pathway and cause oxidative stress induced-injury in cells.Several studies suggest that the oxidative stress play a key role in Aβ-mediated neuronal cytotoxicity by triggering or facilitating neurodegeneration through a wide range of molecular events which eventually lead to neuronal cell loss.Aβ-induced reactive oxidative species(ROS) overproduction can increas the vulnerability of cells and in turn increase apoptotic cell death.cyclophilin A(CyPA) is a member of cyclophilin family,which has the activity of peptidyl-prolyl cis-trans isomerase.The molecular weight of CyPA is 18000,so it is also named CyP18.CyPA occurs cytosolically,within the nucleus and extracellularly.It is a highly conservative protein,appears in prokaryotic organism and mammalian.It is widely distributed in many tissues.In the brain,CyPA displays the high expression, where it is predominantly localized to neurons.CyPA is implicated in neuronal differentiation and adult cortical plasticity.Moreover,CyPA over-expression increases growth rates of human embryonic brain cells.Recently,study showed that increase in expression of CyPA was occurred in neural cells and cells from other tissues which were suffered oxidative stress,ischemic or hypoxemic injury.Moreover, overexpression of CyPA and exogenic CyPA can protect neurons agaist oxidative stressinjury and ischemic and hypoxemic injury.But CyPA down-regulation renders cells more susceptible to the injury of noxious substance.In one hand,it is a endogenicly protective mechnism that CyPA is up-regulated after cells injury as response to cells injury.In the other hand,overexpression of CyPA and exogenic CyPA can protect cells agaist oxidative stress-induced injury.That is to say,CyPA has the antioxidative effect.In our expriments,we set up a rat AD model by injection Aβinto bilateral hippocampus of rat and a cell model through using PC12 cells incubated by Aβwhich is pretreated by CyPA.In one hand,we observe the morphological feature of hippocampus,the number of neuron and apoptotic cells in CA1 area and the change of CypAmRNA and protein in hippocampus are detected after Aβ_25-35 was injected.In the other hand,we observe the effect of CyPA on Aβ_25-35-induced survival rate, morphological feature of cells and apoptotic rate and transmembrane potential,the lever of cellular ROS,activity of SOD and GSH-Px,apoptosis relative gene Bcl-2,Bax and p53 as well as p38MAPK pathway are studied.MethodsAnimal experiment:60 healthy Wistar rats were divided into experimental group and control group randomly.In each group there were three subgroup -1d subgroup,7d sungroup and 14d subgroup-according to the time after injection of Aβ_25-35.In every sungroup there were 10 rats.The model was established through injection into bilateral hippocampus,injection of 2μl(10μg) Aβ_25-35(Aβ_25-35 was aggregated at 37℃for 2 days) in the experimental group and injection of NS in the control group.The brains of 5 rats in every group were perfused and fixed at 1d,7d and 14d after injection,and the samples of brain were stained with HE,Nissel and TUNEL.The other 5 rats were killed at 1d,7d and 14d after injection.The brains were taken quickly,the hippocampi were separated and preserved in refrigerator at -70℃,then were detected the expression of CyPA by RT-PCR and Western Blot.Cell culture:The PC12 cells were pretreated with CyPA(0.1,1,10 and 100nmol/L) for 30 minutes,then incubated with Aβ_25-35(Aβ_25-35 was aggregated at 37℃for 2 days). In every experiment there were normal control group(0μmol/L Aβ_25-35),treatment group(10μmol/L Aβ_25-35) and drug-protective group(CyPA+Aβ_25-35).After incubation with Aβ_25-35 for 48 hours,the PC12 cells were detected the survival rate by MTT method,observed the morphological feature by HE stain and apoptosis by PI stain and Hoechst 33258 stain,detected the expression of mRNA and protein of Bcl-2,Bax as well as p53 by RT-PCR and Western Blot.After incubation with Aβ_25-35 for 24 hours, the PC12 cells were detected transmembrane potential by Rh123 stain,evaluated the level of ROS in cells by DCF-DA stain as well as the activities of SOD and GSH-Px, detected the activation of p38MAPK pathway by observation the protein's expression of p38MAPK and p-p38MAPK through Western Blot.Results1.HE stain of rat hippocampusIn control group,the cell zone was complete in CA1 area of rat hippocampus.The loss of neurons and the proliferation of microglia cells were not seen and the arrangement of cells were regular.In 1d subgroup of experimental group,the number of cells was decreased compared with control group and the arrangement of cells appeared alteration slightly.In 7d subgroup of experimental group,the decrease of cells was more obviois and the cells were arranged unregular.In 14d subgroup of experimental group,the above-mentioned changes were more significant than that in 7d subgroup.2.Nissel stain of rat hippocampus In control group,the cell zone was not injured in CA1 area of rat hippocampus. We could see the close neurons and complete arrangement.In 1d subgroup of experimental group,arrangement of the cells was tmregular slightly,the decreas of cells appeared.In 7d subgroup of experimental group,the arrangement of cells was disordered,the contruction of cell zone was disappeared and the number of cells was diminished.In 14d subgroup of experimental group,the above-mentioned changes were more significant than that in 7d subgroup.3.TUNEL stain of rat hippocampusIn the control group,we saw few TUNEL-positive cells.In 1d subgroup of experimental group,there was several TUNEL-positive cells.In 7d and 14d subgroup of experimental group,there were many apoptotic cells.With the prolong of time,the number of apoptotic cells was increased.We could see the significant difference between experimental group and control group.4.The expression of CyPAmRNA and proteinThe expression of CyPAmRNA and protein were appeared in every subgroup of control group,and there was no significant difference among 3 subgroups.In 1d subgroup of experimental group,the expression of CyPAmRNA and protein were increased dramaticly and there was significant difference compared with control group. In 7d subgroup of experimental group,the expression of CyPAmRNA and protein were lower than that in 1d subgroup,but they were higher than that in control group and there was a significant difference compared with control group.In 14d subgroup of experimental group,the expression of CyPAmRNA and protein decreased,and they were lower than control group.There was no significant difference compared the expression of CyPAmRNA with that in control group,but there was a significant difference compared the expression of CyPA protein with that in control group.5.Cell viability detected by MTT methodThe cell viability in Aβ_25-35 treatment group was lower than that in control group. The cell viability in drug-protective group was increased compared with Aβ_25-35 treatment group.Except o.1nmol/L CyPA,other CyPA with concentration of lnmol/L-100nmol/L could rise the cell viability significantly and it was concentration-dependent.6.HE stain of cellsWe could see full cell body,tight intercellular junction and blue-purple neucli in cultured PC12 cells.In Aβ_25-35 treatment group,decrease in the number of cells, shrinkage or swell of cytoplasm,condense of cytoplasma,karyopyknosis,karyorrhexis and blue-black neucli could been seen.Pretreatment with 10nmol/L and 100nmol/L CyPA could protect PC12 cells and make the morphological feature get close to normal.7.Apoptosis was detected by flow cytometry with PI stainThere were slight apoptotic cells in normal control group.In Aβ_25-35 treatment group,the apoptotic cells were increased obviously,which could been diminished by treatment with 1,10 and 100nmol/L CyPA.But 0.1 nmol/L CyPA could not prevent the increase of apoptotic cells induced by Aβ_25-35.8.Hoechst33258 stainNormal PC12 cells appeared diffusedly and uniformly blue flourescence.In Aβ_25-35 treatment group,there were significant apoptotic cells appearing sapphirine and condensed granular flourescence and the neucli appeared condensed,aggregated and disrupted.Except 0.1nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could decrease apoptosis of cells.9.Transmembrane potentialThe transmembrane potential in Aβ_25-35 treatment group was decreased compared with control group,which manifestated that the intensity of flourescence decreased. CyPA with concentration of 1,10 and 100nmol/L could increased the transmembrane potential,but 0.1 nmol/L CyPA play no role. 10.Intracellular ROSThe level of ROS in Aβ_25-35 treatment group was increased compared with control group.CyPA with concentration of 1,10 and 100nmol/L could inhibit the increase of ROS induced by Aβ_25-35.There was no significant change in 0.1nmol/L CyPA treatment group.11.Activities of SOD and GSH-PxActivities of SOD and GSH-Px were decreased by treatment with Aβ_25-35.CyPA with concentration of 10 and 100nmol/L could increase the activities of SOD and GSH-Px and there was a significant difference compared with Aβ_25-35 treatment group. We could see it is no statistical significance that,activity of SOD in 1nmol/L CyPA treatment group compared with that in Aβ_25-35 treatment group,but there was a siginificant difference in the activity of GSH-Px.0.1nmol/L CyPA play no role.12.The expression of mRNA and protein about Bcl-2 and BaxWhen the cells were incubated with Aβ_25-35,the expression of mRNA and protein of Bcl-2 decreased and the expression of mRNA and protein of Bax increased.Except 0.1 nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could inhibit Aβ_25-35-induced the decrease of Bcl-2 and the increase of Bax concentration-dependently.The ratio of Bcl-2 and Bax in mRNA and protein was reduced when the PC12 cells were incubated with Aβ_25-35.Except 0.1nmol/L CyPA,other CyPA with concentration of 1,10 and 100nmol/L could prevent Aβ_25-35-induced the decrease of the ratio concentration-dependently.13.The expression of mRNA and protein about p53In Aβ_25-35 treatment group,the expression of p53mRNA and protein increased which could been inhibited by CyPA with concentration of 1,10 and 100nmol/L concentration-dependently,but could not been prevented by 0.1nmol/L CyPA.14.The activation of p38MAPK pathway There was no activation of p38MAPK pathway in control group.Aβ_25-35 could activat p38MAPK pathway,which could been inhibited by CyPA with concentration of 1,10 and 100nmol/L,not 0.1 nmol/L CyPA.Conclusions1.After injection Aβ_25-35 into hippocampus of rat,the neurons in hippocampus of rat were injuried and occurred apoptosis.With the prolong of time,the injury enhanced.2.After injection Aβ_25-35 into hippocampus of rat,the expression of CyPAmRNA and protein were changed.In the early stage the expression increase and in turn,the expression decreased gradually.3.CyPA protect PC12 cells agaist Aβ_25-35-induced decrease of viability and increase of apoptotic rate through increase of the transmembrane potential,decrease of ROS and increase of activities of SOD and GSH-Px.4.CyPA protect PC12 cells agaist Aβ_25-35-induced apoptosis through increase of the expression of Bcl-2,decrease of the expression of Bax and p53 and preventation of activation of p38MAPK pathway.