Neuronal Apoptosis Of Hippocampus And Dynamic Imbalances Of Mitochondria And Protective Effects Of SS31in SAMP8Mice

Alzheimer’s disease (AD) is a kind of age-related neurodegenerativedisease that mainly shows up as progressive decline of memory and cognitivefunction with abnormal personality and behavior. AD includes familial AD(FAD) and sporadic AD (SAD). The pathologic changes of AD include Aβplaque, neurofibrillary tangles formed of Tau protein that is excessivelyphosphorylated, structure and function changes of mitochondria/synaptic andreduction of neurons. Hypotheses about pathogenesis of AD are Energymetabolism hypothesis, Oxidative stress hypothesis, Amyloid protein (amyloid)cascade hypothesis, Cholinergic hypothesis and so on. However, concretepathogenesis is not yet clear. The incidence of AD is increasing that causesinconvenience to life and work of more and more AD patients and theirrelatives.Experiments show that AD is related to abnormity of oxidative stress andfunction of mitochondria. ROS produces mainly in mitochondria and affectsmainly on mitochondria. ROS affects structure and function of mitochondriaand causes a series of changes in cells promoting cell death. Whencomposition of neuron is over oxidized, the expression level ofapoptosis-related protein changes promoting apoptosis. Application ofantioxidant can inhibit oxidative stress reducing structure destruction anddysfunction of mitochondria and cell apoptosis. It can improve cognitivefunction of AD patients. SS31is a antioxidant peptide targeting at innermembrane of mitochondria. Studies show SS31can reduce production ofROS in mitochondria, clear of ROS, suppress peroxidation of lipid and otheringredients, suppress change of mitochondria permeability, protectmitochondria function, reduce death of neuron. SS31is effective to all kindsof neurodegenerative diseases that are related to oxidative damage and dysfunction of mitochondria in cells.Objectives: To observe the expression of fusion protein Mfn1(Mitofusin1), Mfn2(Mitofusin2) and the ratio change of Bcl-2(B-celllymphoma-2)/Bax (Bcl-2Assaciated X protein) in the hippocampal neuronsof rapid-aging-model SAMP8(senescence accelerated mouse prone8) miceand SAMR1（senescence accelerated mouse resistant1）mice. To explorewhether mitochondrial-targeting antioxidant peptides SS31has protectiveeffect on the Central nervous system (CNS). And to provide new theoreticalguidance for the prevention and cure of Alzheimer’s disease (AD) and for theaging.Methods:1The experimental animals and groupingwe chose eight-month healthy male SAMP8and SAMR1mice, SAMP8mice were randomly divided into three groups, the first group was notintervened(Model group), the second group was intraperitoneally injected withsaline with a quantity of5mg/kg/d (Vehicle group), the third group wasintraperitoneally injected with SS31with a quantity of5mg/kg/d (SS31).SAMR1mice group was intraperitoneally injected with saline with a quantityof5mg/kg/d(Normal control group), the experimental animals were dividedinto four groups.2HE stainingMice were killed, and the hippocampi were isolated after intervention.Cut the part that was between optic chiasma and superior colliculus in themice into5um hippocampal specimens.we observed the pathological changesunder light microscopy with HE dyeing technology.3ImmunohistochemistryMice were killed, and the hippocampi were isolated after intervention.Cut the part that was between optic chiasma and superior colliculus in themice into5um hippocampal specimens. We observed the expression of Bcl-2and Bax in CA1area and CA3area in hippocampus of the mice from fourgroups with Immunohistochemistry. 4Electron MicroscopeMice were killed, and the hippocampi were isolated after intervention.We cut the hippocampi into cubes of1×1×1mm and dealed the cubes with fourpercent glutaraldehyde, and measured the morphology of tissue with ElectronMicroscope Technology.5Western blotMice were killed, and the hippocampi were isolated after intervention.We extracted total protein from the hippocampi and measured the changes ofMfn1and Mfn2content in the hippocampi with Western Blot technique.Results:1HE stainingIn SAMR1Normal control group, outlines of neurons of CA1area andCA3area in hippocampus were clear. The neurons arranged orderly andclosely. Nucleolus appeared clearly. Karyon had no pyknosis. Compared withSAMR1Normal control group, in SS31treatment group, neurons of CA1areaand CA3area in hippocampus reduced and arranged disorderly. Nucleoluswas not clear. Nuclear atrophied. In Vehicle group and Model group, outlinesof neurons and nucleolus were more unclear. Nuclear atrophying and reducingof neurons were more obvious.2Immunohistochemistry2.1The expression of Bcl-2proteinIn CA1area and CA3area of hippocampus, Bcl-2protein mainlyexpressed in cytoplasm of neurons. Positive cells were pale yellow or tan, andbackground color was light-colored. Positive cells of Vehicle group andModel group mice were much fewer than SAMR1Normal control group.Positive cells in SS31group were more than the former two ones, but werefewer than SAMR1Normal control group. There was no positive cell insection of Negative control group.2.2The expression of Bax proteinIn CA1area and CA3area of hippocampus, Bax protein mainlyexpressed in cytoplasm of neurons. Positive cells were pale yellow or tan, and background color was light-colored. Positive cells of Vehicle group andModel group mice were much more than SAMR1Normal control group.Positive cells in SS31group were fewer than the former two ones, but weremore than SAMR1Normal control group. There was no positive cell insection of Negative control group.3Electron MicroscopeNeurons of CA1area and CA3area in hippocampus in Vehicle group andModel group mice reduced and arranged disorderly. Neurons atrophied,Swelled and degenerated.Mitochondria swelled and vacuolated. There was nopositive cell in neurons of CA1area and CA3area in hippocampus in SAMR1Normal control group mice. Neurons of hippocampus in SS31group weremore than Vehicle group and Model group. Neurons and mitochondriachanged less, but did not recover to the level of SAMR1Normal controlgroup.4Western blot4.1Expression of Mfn1proteinCompared with Vehicle group, the expression of Mfn1protein decreasedin Model group with no significant statistical importance(P＞0.05). Comparedwith SAMR1Normal control group, expression of Mfn1protein decreased inVehicle group and Model group(P＜0.05). Compared with Vehicle group andModel group, the expression of Mfn1protein increased in SS31group(P＜0.05), but decreased compared with SAMR1Normal control group (P＜0.05).4.2Expression of Mfn2proteinCompared with Vehicle group, the expression of Mfn2protein decreasedin Model group with no significant statistical importance(P＞0.05). Comparedwith SAMR1Normal control group, expression of Mfn2protein decreased inVehicle group and Model group(P＜0.05). Compared with Vehicle group andModel group, the expression of Mfn2protein increased in SS31group(P＜0.05), but decreased compared with SAMR1Normal control group (P＜0.05).Conclusion:1In SAMP8mice, neurons in hippocampus decreased. Morphology of neurons and mitochondria was abnormal. The expression of Bax proteinincreased, while the expression of Mfn1, Mfn2and Bcl-2protein decreased. Itrevealed that mitochondria dynamics imbalances is related to neurons injury.2After intervention of SS31, number and morphology abnormity ofneurons and mitochondria in hippocampus recovered in a certain extent. Theexpression of Bax protein in neurons of hippocampus decreased, while theexpression of Mfn1, Mfn2and Bcl-2protein increased. It revealed that SS31could improve the function of mitochondria and reduce apoptosis of neurons.The protection mechanism of the nerves may be related to improving ofbalance disorders of mitochondrial dynamics.