Protective Effects Of P38MAPK,Estradiol And TPX2 In The Hippocampal Apoptosis And Their Effect On Cognition In Mouse Models Of Dementia

Dementia is a syndrome characterized by progressive deterioration of cognitive decline.most commonly of memory,but other domains such as language,behavior,and the most significant executive function are often impaired.It is a progressive,degenerative brain disease causing a loss of cognitive and emotional functioning.Estimates suggest that there are about 24 million people living with dementia worldwide and that this number will be three or four times higher by 2040.dementia of Alzheimer's type(DAT/AD)occurs most frequently(60%of all cases),just followed by vascular dementia(VaD,10%to 20%of all cases).With unknown pathogenesis,cerebral structure and physiological function are damaged in all dementia victims.Pathological features of AD include extracellular senile plaques originating from ?-amyloid protein deposits in hippocampus,and neurofibrillary tangles caused by tau overphosphorylation.VaD brings about cognitive damage triggered by factors like ischemia,hemorrhage,hypoperfusion,embolism and small vessel diseases.AD patients can be X-rayed with diffuse atrophy in hippocampus,temporal lobe and parietal lobe,and VaD patients with vascular disorders(like infarction,hemorrhage,leukoencephalopathy,lacunar infarction)in regions responsible for cognition,like frontal lobe,temporal lobe,hippocampus,parietal lobe,angular gyrus or thalamus.In addition to family history and some types of physical diseases,other risk factors for these two types of dementia include age,gender,educational background or income.In spite of their own clinical features,it is still hard to differentiate them in clinical practice.That AD sometimes comes on with pathologic changes in cardiovascular system is a manifestation of senescence or an overlap of AD and VaD signs.Histopathology examination on brains of VaD patients can point out the anomaly that generally appears in the case of AD,like senile plaques.This phenomenon reflects the resemblance of both types of dementia.What their relation is and how to differentiate them have evoked series researches attempting to reveal the pathogenesis of dementia and promote the clinical prevention.Human hippocampus,an organ responsible for learning and memory,is primarily targeted and invaded in both AD and VaD.Enough evidence has proved that the neuronal apoptosis in hippocampus is susceptible to ischemic hypoferfusion injury and actively involved in the development of both diseases.Alzheimer's disease(AD)is a progressive neurodegenerative disorder that eventually leads to the irreversible loss of neurons as well as deficits in various intellectual abilities,including cognition and memory.AD has become important public health concern,because its symptoms primarily include deficiencies in physical,cognitive,and emotional function,and long-term disabling changes in cognitive and sensorimotor abilities that significantly influence the quality of life of patients.Despite great research efforts and significant advances in terms of the occurrence and development of AD,procedures for making effective diagnoses and employing beneficial techniques remain lacking.As a result,compelling demands for such procedures have fueled interest in the development of therapeutic approaches in the neuroscience community as well as medical industry.According to the classic amyloid hypothesis,A?1-42 plays a pivotal role in the pathogenesis of AD relative to various other amyloid derivatives.A?1-42 is generated from the cleavage of amyloid precursor protein(APP)by beta-secretase(BACE-1)and gamma-secretase.The overproduction of A? protein leads to the formation of amyloid plaques and the initiation of various neuronal dysfunctions including the activation of pro-inflammatory cascades,the promotion of tau phosphorylation,increases in oxidative stress,the deregulation of intracellular signaling pathways,impairments in synaptic plasticity,and the induction of neuronal apoptosis and cell death.Compelling evidence indicates that post-menopausal women experience a sudden drop in estradiol levels in the circulatory system,and that this drop creates higher risks of oxidative stress and accelerated neurodegenerative processes,especially those associated with a vulnerability to AD.On the other hand,experiment studies have shown that estradiol treatment in ovariectomized(OVX)female animals facilitates learning and memory processes and improves performance in the radial arm maze and object recognition task.In clinical situations,women within 5 years of menopause who have maintained higher levels of estradiol exhibit superior performance in measures of executive function compared to women who have been menopausal for 5 years and maintain steady low levels of estradiol.Furthermore,higher endogenous levels of estradiol in postmenopausal women enhance performance on the Stroop task and tests of spatial working memory,delayed recall,and digit ordering.Likewise,women who are taking hormone replacement therapies have better responses on non-spatial working memory and spatial working memory tasks.However,it has also reported that estradiol exerts negative effects on cognition in post-menopausal women.Studies designed to understand these discrepant findings more fully have revealed that neurons exposed to A? proteins exhibit markedly less impairment when pretreated with estradiol whereas there is no estrogenic protection or greater damage in cells that receive estradiol treatment either simultaneously or 5 days after A? protein exposure.Taken together,these findings suggest that estradiol plays a subtle but important role during AD pathology and may exert both positive and negative effects during different stages of this process.Thus,the present study investigated the effects of 17?-estradiol(E2)treatment during different stages of AD pathology(early and late stages)to understand more comprehensively the role that E2 plays in the pathogenesis of AD and to evaluate thoroughly the implications of using E2 as a preventive medicine.The hippocampus is an important functional area in the nervous system which is closely related to human learning and memory.The hippocampus of the brain is not only the most important lesion area of Alzheimer's disease,but also an important damaged area of vascular dementia.Hippocampal neuronal apoptosis plays an important role in the pathogenesis of AD and VaD and is susceptible to ischemia and low perfusion injury.Neuron apoptosis in hippocampus may play an important role in the pathogenesis of vascular dementia.It has been found that decreasing the expression of Bax in Bcl-2,can activate the expression of Caspase-3 and p53,and further induce apoptosis of neurons in hippocampus of rats.Bcl-2 and Caspase-3 are two important molecules involved in the regulation of apoptosis.The overexpression of Bcl-2 can effectively inhibit the activation of Caspase-3.The activation of p38 MAPK(mitogen-activated protein kinase,MAPK)is one of the important signal transduction pathways of neuronal apoptosis induced by ischemia and low perfusion injury.P38 MAPK can affect apoptosis by regulating the expression of Bcl-2,Bax and Caspase-3.SB202190,a specific inhibitor of p38MAPK,has been reported to alleviate cerebral ischemia-reperfusion injury.However,the effects of SB202 190 and SB203580 on the expression of apoptotic factors and learning and memory ability of hippocampal neurons are rarely reported.Especially the effect of SB202190 and SB203580 on apoptosis and Bcl-2,Caspase-3 expression of hippocampal neurons has not been reported at home and abroad.In order to define the effect of this apoptosis on both diseases and human cognition,three experiments are organized.1.Introducing estradiol into AD mouse models to elucidate its protective mechanism in hippocampal apoptosis and cognitive function.In this experiment,AD mouse models are established through bilateral ovariectomy and stereotactic brain injection of A?1-42.After the treatment of estradiol(E2),A?1-42-induced neurotoxicity,its molecular mechanism and the antagonistic function of E2 are analyzed.2.Using p38MAPK as inhibitor to find the protective mechanism in hippocampal apoptosis and cognitive function in VaD mouse models.In this experiment,the effect of SB203580 and SB202190 on rats hippocampal apoptosis,learning and memory is tested.The inhibition of p38MAPK in brain cellular apoptosis is observed.Morris-water maze is employed to test mice's spatial learning and memory.3.TPX2 was used to study the protective mechanism of neuronal apoptosis in Alzheimer's disease model rats.The spatial reference learning and memory ability of rats was detected by hippocampal dependent water maze test.The expression of ERK and p38 MAPK in brain tissue cells was detected by Tune]staining.The expression of ERK and p38 MAPK in brain tissue cells was detected by Tunel staining,and the expression of mRNA in MAPK,ERK and p38 was detected by RT-PCR method.The protective effect of TPX2 on apoptosis of brain tissue treated with A?1-42 was observed,and its mechanism was further discussed by inhibiting the expression of MAPK,ERK and p38 proteins.In conclusion,these three experiments are accomplished to clarify the protective mechanism of p38MAPK and E2 in mice's hippocampal apoptosis and cognitive function.Part one Chronic estradiol administration during the early stage ofAlzheimer's disease pathology rescues adult hippocampal neurogenesis and ameliorates cognitive deficits in A?1-42 miceObjective:AD mouse models are established through bilateral ovariectomy and stereotactic brain injection of A?1-42.After the treatment with estradiol(E2),A?1-42-induced neurotoxicity,its molecular mechanism and the antagonistic function of E2 are analyzed.Methods:1.Bilateral ovariectomy:All mice are bilaterally ovariectomized and immediately implanted with a subcutaneous pellet that has a 60-day continuous release and 0.18 mg of E2 inside.2.Establishment of mouse models:mice in AD group are treated with slow injection of A?1-42(4 ?g/mouse,0.5 ?l/min);mice in placebo group are treated with injection of saline(4 ?g/mouse).3.Injection of BrdU:intraperitoneal injection of BrdU for 3 successive times,with an interval of 6 hours and a dosage of 50 mg/kg.4.Administration of E2:i.administration of E2 in the early stage:injection of A?1-42 into bilateral hippocampus at day 7 after BrdU injection,subcutaneous injection of E2 in the following successive 7 days(3?g/day).ii.Administration of E2 in the later stage:injection of A?1-42 into bilateral hippocampus at day 7 after BrdU injection,subcutaneous injection of E2 at day 7 after A?1-42 injection(3?g/day).5.Morris water-maze experiment:calculation of mice's average swimming speed,latency period;at day 8 of the experiment,the hidden platform is removed;the swimming speed and latency period of mice are evaluated.6.Immunofluorescence staining:NeuN and Brdu staining is performed to demonstrate the genesis of hippocampal neurons.7.ELISA test:Testing serum level of luteinizing hormone,follicle-stimulating hormone,estrogen and progesterone.8.RT-PCR:Hippocampal tissues are removed from brain samples and minced with eye scissors on ice.The total mRNA is extracted by TRIzol Reagent.Reverse transcription of RNA is accomplished with a PrimeScriptTM RT reagent kit.9.Western blotting analysis:Hippocampal tissues are removed from brain samples and minced with eye scissors on ice.Protein extraction is accomplished with radioimmunoprecipitation assay.10.CAMP test:Hippocampal tissues are minced with eye scissors on ice.The pellets are pyrolyzed in lysis buffer for 10 min and centrifugated at 14000 rpm for 10 minutes at 4?.LANCETM cAMP 384 kit is used to test the concentration of cAMP in supernate.11.Transmission electron microscopy:After intraperitoneal anesthesia and cardiac perfusion with 2.5%glutaraldehyde,hippocampal tissues are removed,fixed in 2.5%glutaraldehyde overnight,then in osmium tetroxide at 4? for 2h,cut into ultrathin sections(60 nm),dehydrated with acetone and stained with uranyl acetate.Results:1.Impaired cognitive function and hippocampal neurogenesis in A?1-42 miceTo evaluate cognitive function,particularly spatial learning and memory abilities,after the formation of fibrils due to the injection of A?1-42,the mice performed the Morris water-maze test(Fig.1 A).The "aged" mice that received the A?1-42 injection did not differ from the control group in terms of swim speed or latency to find the visible platform on Days 1-2 of training(visible platform test),which indicates that spatial learning was not immediately affected by the A?1-42 injection In contrast,the latency to find the hidden platform on Days 3-7 of training(visible platform test)was longer in the A?1-42 mice than in the control mice and these differences were significant on Days 5-7,which suggests that memory function was remarkably impaired beginning on Day 5 after the A?1-42 injection.Additional analyses were conducted to determine the percentage of time spent in each quadrant after the platform was removed from the maze(Fig.1B).The percentage of time spent in the quadrant over the position of the virtual platform was significantly reduced in A?1-42 mice;compelling evidence points to the critical role that hippocampal neurogenesis plays in cognitive deficits during the progression of neurodegeneration.In the present study,hippocampal neurogenesis was assessed using NeuN-and BrdU-staining(Fig.1C),and these analyses revealed a significant decrease in the number of NeuN-and BrdU-positive cells in the hippocampi of A?1-42 mice 21 and 28 days after the injection(Fig.1D).These results suggest that there was a serious impairment of hippocampal neurogenesis in mice that received an injection of A?1-42.2.E2 during the early stage of AD pathology increased hippocampal neurogenesis and improved the recovery of AD cognitive function in A?1-42 miceEstradiol plays an important role in hippocampal neurogcncsis,but the undcrlying these effect remain unclear.Thus,the present study aimed to investigate the effects of E2 on the pathology of AD and the process of hippocampal neurogenesis at different stages.To evaluate the effects of E2 during the early stage of AD pathology inA?1-42 mice,E2 was administered immediately after the A?1-42 injection(Fig.2A).E2 treatment in healthy mice did not alter hippocampal neurogenesis relative to control mice,but E2 treatment in A?1-42 mice notably increased the number of NeuN-and BrdU-positive cells compared to A?1-42 mice that did not receive treatment(Fig.2B).Furthermore,the latency to find the hidden platform was significantly reduced and the percentage of time spent in the quadrant over the position of the virtual platforms was markedly increased in A?1-42 mice that received E2 treatment(Figs.2C and 2D).These findings suggest that E2 treatment during the early stage of AD pathology inA?1-42 mice increased hippocampal neurogenesis and improved the recovery of cognitive function.3.E2 treatment during the late stage of AD pathology had no effect on hippocampal neurogenesis or cognitive function in A?1-42 miceTo assess the effects of E2 during the late stages of the AD pathology in A?1-42 mice,E2 was administered 14 days after A?1-42 injection(Fig.3A).E2 administration during the late stage of AD pathology did not alter the number of NeuN-and BrdU-positive cells in the hippocampi of mice that received A?1-42 injections(Fig.3B).Moreover,there were no significant differences in the latency to find the hidden platform or the percentage of time spent in the quadrant over the position of the virtual platform between the E2-treated A?1-42 mice and the A?1-42 mice that did not receive E2 treatment(Figs.3C and 3D).These findings indicate that E2 treatment during the late stage of AD pathology in A?1-42 mice did not affect hippocampal neurogenesis or cognitive function.4.E2 treatment during the early stage of AD pathology upregulated the neurogenesis-associated pathway in the hippocampus in A?1-42 miceTo confirm the efficiency of E2 delivery,the serum levels of LH,FSH,estrogen,and progesterone were measured.There were no significant differences in LH,FSH,estrogen,or progesterone levels prior to the E2 treatment but marked increases in LH,FSH,and estrogen levels and a notable decrease in progesterone levels after the E2 treatment(Figs.4A-D),which indicated that the mice were suitable for further study.Because E2 treatment during the early stage of AD pathology increased hippocampal neurogenesis,the neurogenesis-associated pathway in the hippocampus was analyzed further.First,the levels of BDNF,TrkB and p75NTR,which are thought to play critical roles in neurogenesis,were determined.The mRNA and protein levels of BDNF and the activation levels of TrkB in A?1-42 mice treated with E2 during the early stage of AD pathology were significantly upregulated compared to A?1-42 mice that did not receive E2 treatment and A?1-42 mice that were treated with E2 during the late stage of AD pathology while those of p75N R were reduced(Figs.4A-C).These findings suggest that E2 induced neurogenesis,at least in part,through the BDNF/TrkB pathway.Next,the downstream factors of this pathway were assessed(Figs.4A-C)and the results were consistent with the earlier findings in that the activation of the STAT3/CREB/ERK pathway markedly increased,Additionally,cAMP levels were assessed to confirm the activation of the BDNF/TrkB downstream signaling pathway(Fig.4D).5.E2 treatment during the early stage of AD pathology reduced oxidative stress and cell death-related pathway activity in the hippocampus in A?1-42 miceA growing body of evidence has demonstrated that the application of estradiol reduces oxidative stress and,thus,the levels of nitric oxide(NO)and reactive oxygen species(ROS)were evaluated to determine the degrees of oxidative stress and activity in the cell death-related pathway in the hippocampus.The production levels of NO and ROS were significantly reduced in A?1-42 mice that received E2 during the early stage of AD pathology compared to those that did not receive treatment or received E2 treatment during the late stage of AD pathology(Fig.5A).Accordingly,there was a downregulation of activation in the cell death-related Cytochrome-c/Bax/Bcl-2/acspase-3 signaling pathway(Fig.5B-C).These findings indicate that E2 treatment reduced oxidative stress and activity in the cell death-related pathway in the hippocampus during the early stage of AD pathology and probably contributed to the recovery of cognition.6.E2 treatment during the early stage of AD pathology in the hippocampus in A?1-42 miceAn increasing amount of evidence suggests that gliosis-induced proinflammatory responses occur in conjunction with the progression of AD pathology play pivotal roles in cognitive impairment.Thus,the present study investigated the levels of gliosis and gliosis induced pro-inflammatory response in hippocampus.There was a notable reduction of gliosis in the hippocampus in A?1-42 mice treated with E2 during the early stage of AD pathology compared to A?1-42 mice that did not receive treatment or were treated during the late stage of AD pathology(Figure 6A and 6B).The proinflammatory response was also mediated by E2 such that there was an upregulation of activation in anti-inflammatory signaling pathways(CD206,FIZZ1,Ym1,and TGF?)and a downregulation of activation in proinflammatory signaling pathways(IL-1?,TNF?,and IL-6;Figs.6C-E).These findings indicate that microglia switched from an M2-to an M1-skewed activation phenotype following E2 treatment in the early stage of AD pathology,and that this was associated with an amelioration of the cognitive deficits.7.E2 treatment during early stage of AD pathology improved receptor traffic in the hippocampal synapses in A?1-42 miceReceptor traffic in the hippocampal synapses is closely related to cognition and is vulnerable to damage.In the present study,an ultrastructural investigation of synapses in the hippocampus was conducted using TEM.The number of synapses was increased in A?1-42 mice that received E2 treatment during the early stage of AD pathology compared to A?1-42 mice that did not receive treatment or were treated with E2 during the late stage of AD pathology(Fig.7A).To further assess the effects of E2 on different types of synapses,the levels of NMDAR and AMPAR receptors in the hippocampus were determined by Western blot analyses.Although NMDAR receptors(GluN1 and GluN2)levels were not affected,the levels of AMPAR receptors(GluAl and GluA2)significantly increased(Figure 7B and 7C).Additionally,there were elevations in important post-synaptic proteins;the levels of two proteins,hevin and SPARC,that play key roles in synapse formation and function were assessed and there were significant increases in the expressions of both proteins(Figure 7B and 7C).Conc usions:1.Cognitive impairment in A?1-42 mice was associated with hippocampal neurogenesis.The hippocampal nerve of mice was severely damaged after injection of A?1-42 and the damage of hippocampal nerve played an important role in cognitive impairment.2.E2 can facilitate the genesis of hippocampal neurons and the recovery of cognitive function in the early stage of AD,but not in the later stage.3.In the early stage of AD,E2 up-regulates hippocampal neurogenesis-related signaling pathways in the hippocampus of A?1-42 mice.4.In the early stage of AD,E2 reduces oxidative stress and the activity of apoptosis-related signaling pathways.5.In the early stage of AD,E2 inhibits the hyperplasia of microglial cells in the hippocampus of A?1-42 mice.6.In the early stage of AD,E2 increases the number of hippocampal synapses.Part 2:The Effect of P38MAPK Inhibitors on Hippocampus Apoptosis and Learning Memory Ability in Vascular Dementia RatObjectiveIn this study,we used bilateral common carotid artery ligation(2-VO)method to produce vascular dementia(VaD)rat model.The learning and memory disorders of VaD rats were studied by the method of injecting SB202190 and SB203580 into the lateral ventricles of the brain.We observed the effect of p38MAPK inhibitor on the cognitive function of VaD rat hippocampal with Morris water maze.The hippocampus cell apoptosis was detected by TUNEL method after the injection of drugs,western blot method was used to detect p-P38MAPK in the rat hippocampus,and mmunofluorescence and confocal laser scanning microscopy were used into observe Bcl-2 and Caspase-3 expression in hippocampus of rats.We investigated the role of apoptosis of hippocampal neurons in the pathogenesis of vascular dementia.MethodsVascular dementia(VaD)model was established by two vessels method(bilateral common carotid artery occlusion(2VO),100 male Wistar rats were randomly divided into sham-operation group,vascular dementia model group(control group),SB203580 group and SB202190 group,Rats were injected medication into the lateral ventricle,the rats of SB202190 group injected 10?l SB202190(10?lmol/L)solution by a microsyringe into lateral ventricle.The model group and sham operation group rats were injected 10?l%DMSO solution.The changes of learning and memory ability of rats in each group were tested by Morris water maze test after a week of injection.The hippocampus cell apoptosis was detected by TUNEL method after the injection of drugs,western blot method was used to detect P38MAPK in the rat hippocampus,and mmunofluorescence and confocal laser scanning microscopy were used into observe Bcl-2 and Caspase-3 expression in hippocampus of rats.we investigated the role of apoptosis of hippocampal neurons in the pathogenesis of vascular dementia in order to provide more theoretical basis for the prevention and treatment of vascular dementia.Results1.The platforms escape latent period and spatial learning or rats' treatment with SB203580 or SB202190 were studied in this study.The result show that the platform escape latent period in groups of treatment with SB203580 and SB202190 was shortened obviously compared to sham-operated and control groups(**P<0.01),indicating spatial learning improvement.2.We also observed significant differences on phosphoactivation of P38MAPK expression levels in hippocampal among the treatment groups(**P<0.01,SB203580-treated or SB202190-treated vs sham-operated and control groups)after five-day treatment.Our result demonstrated that P38MAPK expression was significantly reduced after treatment with SB203580 or SB202190.There is no significant difference between SB203580 and SB202190 groups.3.SB203580 and SB202190 could protect hippocampal neurons against apoptosis of chronic ischemia-induced.In order to explore whether the SB203580 and SB202190 is effective on hippocampus apoptosis and learning memory ability in vascular dementia rat agent in vitro,we tested the therapeutic activity of t SB203580 and SB202190 on HT22 cells.Our results demonstrated that apoptosis of hippocampal neurons were decreased after treatment with SB203580 or SB202190 compared to control group.We also confirmed the efficient of SB203580 and SB202190 on hippocampus apoptosis and learning memory ability in vascular dementia rat agent in vivo.Five days after treatment with SB203580 and SB202190 by ICV injection,rats were sacrificed and TUNEL-positive(Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling,green fluorescent)cell numbers were significantly decreased in the sham-operated group compared to in control,SB203580 and SB202190 groups(**P<0.01).However,the number of TUNEL-positive cells in SB203580 or SB202190 was significantly lower in the SB203580 and SB202190 groups compared to control group,and the number of TUNEL-positive cells in SB203580 were lower than SB202190 groups.Taken together,SB203580 and SB202190 could protect hippocampal neurons against apoptosis of chronic ischemia-induced.4.This study investigated the anti-apoptosis ability of SB203580 and SB202190 for hippocampus of VaD rat model.Five days after intraventricular injection,rats were sacrificed and hippocampus of VaD rat model.The experiments were performed(n=3)and the number of apoptosis cells in experimental groups were determined and calculated after 48 hours of incubation with SB203580 or SB202190.After incubation with P38MAKA,HT22 cells appeared shrinkage,fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells.As shown in the study,the results indicated that P38MAKA could reduce apoptosis of HT22 cells.However,Apoptotic HT22 cells in groups added SB203580 or SB202190 were significant lower than control group.5.The relationship between P38MAKA and apoptosis-related genes was studied in the hippocampus of VaD rat model.Expressing levels of mRNA related to P38MAKA-induced apoptosis were analyzed.As shown in the study,apoptosis inhibiting genes of BAX and Bcl-2 were increased in hippocampus of VaD rat model after treatment with SB203580 or SB202190 compared to sham-operated and control groups.And caspase-8,caspase-3,and caspase-9 expression were significantly lower in hippocampus from the SB202190 or SB203580 groups compared to the control groups.But apoptosis suppressing gene of BAX and Bcl-2 in SB202190-treated group was slightly high in the SB203580 group,suggesting that SB202190 may reduce apoptosis by inhibiting activation of P38MAPK and the mitochondria-dependent apoptosis pathway may be activated by chronic ischemia.6.The remaining animals from experimental groups in VaD rat model continued to be followed to determine the long-term efficacy of each treatment.Rats in sham-operated and control groups had sacrificed by day 30.In contrast,about 30%,60%animals in SB202190 and SB203580 need sacrifice(**P<0.01).However,SB202190 treatment further extended the survival time of rats,which represented a more prolongation compared to SB203580 treatment.Therein,superior therapeutic effects of SB202190 compared to SB203580 translated into a significant survival advantage for VaD rats(**P<0.05).Conclusion1.In this study,It was showed that P38MAPK inhibitor SB202190 and SB203580 had exact curative effect in improving the learning and memory ability of vascular dementia rats.2.This study also demonstrated that the expression of p38MAPK decreased significantly after treatment with P38MAPK inhibitor in VaD rats,which effectively reduced the apoptosis of hippocampal neurons,promoted the reconstruction of memory impairment,and had good tolerance.3.SB203580 and SB202190 reduce apoptosis by inhibiting the activity of P38 MAKA,which helps hippocampal neurons resist the apoptosis induced by chronic ischemia.The mitochondrial pathway of apoptosis can be activated by chronic ischemia.4.The survival time of rats treated with SB203580 and SB202190 was prolonged,and the survival time of rats was further prolonged compared with that of SB203580.Part 3:Protective effects and mechanism of TPX2 on neurocytes apoptosis of rats with Alzheimer's disease modelObjective:It was investigated the protective effects and mechanism of TPX2 on neurocytes apoptosis of rats with Alzheimer's disease model.Methods:90 rats were randomly divided into drug group,control group and blank group with 30 rats in each group.The rats in the drug treatment group and the blank group were anesthetized with 10%chloral hydrate(0.5ml/100g dose)and A?1-42 5ul(concentration 1 ?g/ul)was injected into the bilateral hippocampal region.The configured TPX2 inhibitor was made into solution.After the establishment of the model,the rats in each group were given different treatment methods,the rats in the drug group were connected with lOul microsyringe,and the rats in the control group were injected with the same amount of 1%DMSO solution in the lateral ventricle.The blank group was normal health group,and no operation or drug treatment was performed in this group.The brain tissue of rats was divided into two parts,one was fixed,dehydrated and embedded in paraffin,and the thickness of 5um was made.TUNEL staining was used to detect the apoptosis of brain tissue,HE staining was used to observe the expression of ERK,MAPK and p38 in brain cells,and RT-PCR method was used to detect the expression of mRNA in MAPK,ERK and p38.Results:one week later,TUNEL staining showed that the apoptosis rate of brain tissue in the drug group was significantly higher than that in the control group and blank group,and the difference was statistically significant(P<0.05).After using TPX2 inhibitor,the mRNA expression level of TPX2 in the drug group was significantly lower than that in the control group(P<0.05).The mRNA expression levels of MAPK and p38 protein in the drug group were significantly higher than those in the control group and blank group(P<0.05).There was no significant difference in the expression of MAPK and p38 between MAPK and p38,but there was no significant difference between them(P>0.05).However,the expression of MAPK and p38 in the drug group was significantly higher than that in the control group,while the gray value of MAPK in the blank group was significantly lower than that in the control group(P<0.05).Conclusion:TPX2 has protective effect on apoptosis of brain cells treated with A?1-42 by inhibiting the expression of MAPK,ERK and p38 protein.