Neuroprotective Effects Of Erythropoietin On Hippocampal Neurons Via Suppressing Apoptosis And Autophagy To Improve Cognitive Function In Vascular Dementia Rats

Vascular dementia(VD)is the second most frequent type of dementia after Alzheimer’s disease(AD).According to the World Health Organization,VD affects approximately 60 million people worldwide,and this number is projected to rise to 160 million by 2050.For VD,the pathogenesis,however,is not yet fully understood,and there are no effective pharmacological treatments available.Therefore,it is of tremendous social and economic benefit to deepen research on the pathogenesis of VD and to search for effective drugs for VD prevention and treatment.Erythropoietin(EPO)is a glycoprotein in the type I cytokine superfamily and can bind to receptors on the surface of red blood cells to promote cell proliferation,differentiation,and maturation.In addition to the function of promoting erythropoiesis,EPO also has a wide range of biological activities,among which its role in the nervous system is increasingly being emphasized.Current research has found that EPO and its receptors can be expressed in several parts of various mammals,including central nervous cells,glial cells,and vascular endothelial cells.In response to stressful stimuli such as hypoxia and trauma,endogenous EPO and its receptors are up-regulated in the body,thus conferring neuroprotection through multiple mechanisms.In the central nervous system(CNS),the hippocampus is not only the main region for learning and memory formation but also a vulnerable region to ischemia and hypoxia.Current evidence shows that brain damage induced by ischemia and hypoxia can lead to obvious pathological changes and apoptosis and autophagy overaction in neurons in the hippocampus,eventually leading to the death of these neurons.In the CNS,EPO exerts a neuroprotective effect by binding to the EPO receptor(EPOR)and activating the phosphorylation of EPOR.The phosphorylated EPOR further activates the downstream pathways,including Janus kinase/signal transducer and activator of transcription(JAK/STAT),phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt),mitogen-activated protein kinases(MAPK).Of note,the PI3K/Akt pathway plays an important role in cell survival signal transduction.However,it remains uncertain whether EPO can regulate hippocampal neuron apoptosis and autophagy through the PI3K/Akt pathway to improve the learning and memory abilities of VD rats,which calls for further experimental confirmation.In this study,a VD rat Model was established in male Wistar rats through bilateral common carotid artery occlusion(BCCAO),followed by 4 weeks of continuous intervention with EPO.The learning and memory abilities of rats were assessed with the Morris water maze(MWM)test.The morphological changes of hippocampal neurons were observed with Nissl staining.Cell apoptosis was detected with terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)staining,and the expression and phosphorylation of the PI3K/Akt pathway-related proteins were measured with western blotting to verify whether EPO activated the PI3K/Akt pathway.Given the neuroprotective effects of inhibiting apoptosis and autophagy,this study provides a theoretical basis for the application of EPO to alleviate cognitive impairment.Part I Effect of EPO on cognitive function in VD ratsObjective: To established a rat Model of VD using the BCCAO method before 4-week EPO treatment and evaluated the memory and learning ability of VD rats with the MWM test,thereby analyzing whether EPO improves the cognitive function of VD rats.Methods:1.Forty adult male Wistar rats of clean grade were randomized into sham(n = 13),Model(n = 13),and EPO(n = 14)groups.2.Rats in the sham group were only subjected to bilateral common carotid artery dissection without ligation and then were intragastrically administered the same amount of normal saline for 4 consecutive weeks,serving as a control for the other two groups.3.After bilateral common carotid artery ligation,rats in the Model group were given the same amount of normal saline by gavage for 4consecutive weeks.4.After bilateral common carotid artery ligation,rats in the EPO group were given 5000 UI/kg EPO by gavage for 4 consecutive weeks.5.On the day after the administration,rats were subjected to the MWM test,which consisted of two parts,including the place navigation test and the spatial probe test.In the place navigation test,rats were trained continuously for 5 days to record their escape latency,therefore assessing the spatial learning ability of rats.In the spatial probe test,the hidden platform was removed on the 6th day of the experiment,and the time staying in the original platform quadrant and the frequency of crossing the original platform within120 seconds were recorded to evaluate the spatial memory ability of rats.Results:1.As the number of training days increased,the escape latency of rats was gradually shortened in the three groups.2.On the first day of the place navigation test,there was no significant difference in the escape latency of rats among the three groups.On the second day of the test,the escape latency of rats was significantly longer in the Model group than in the sham group(P < 0.05).On days 3-5 of the test,the escape latency in the EPO and sham groups was significantly shorter than that in the Model group(P < 0.05).3.The spatial probe test demonstrated that compared with those in the Model group,rats in the sham and EPO groups spent significantly longer time in the original target quadrant,with a substantially elevated frequency of crossing the platform(P < 0.05).Conclusions:1.The rat Model of VD established with the BCCAO method is an ideal animal Model for VD because it mimics the pathogenesis of clinical VD.2.It is indicated that EPO can effectively ameliorate the cognitive impairment caused by chronic hypoperfusion in VD rats.Part II Effect of EPO on the histomorphology of the hippocampus in VD ratsObjective: To evaluate the neuroprotective effect of EPO by analyzing the histomorphological changes of hippocampal neurons in VD rats.Methods:1.After the MWM test,the rats were anesthetized,perfused,fixed,and euthanized.Next,the brain tissues were embedded in paraffin and sectioned.2.After the sections were stained with Nissl,the morphological changes of hippocampal pyramidal cells were observed under a light microscope.Results:1.Under the microscope at 100 x magnification,rats in the sham group were observed to have a high number of hippocampal pyramidal cells,which were tightly arranged.In the Model group,hippocampal pyramidal cells were loosely arranged and the number of neurons was reduced.The number and density of hippocampal pyramidal cells were higher in the EPO group than in the Model group.2.Under the microscope at 400 x magnification,hippocampal pyramidal cells in the sham group were tightly arranged and uniform in size,with intact structures,darker cytoplasm staining,regular and plump shapes,and clear nucleoli.In the Model group,hippocampal pyramidal cells were loosely arranged and disordered in size,and some cell nucleoli disappeared and became vacuolated,accompanied by lighter cytoplasm staining and pyknotic and deeply stained cell nuclei.The morphological changes of hippocampal pyramidal cells in the EPO group were significantly improved as compared to those in the Model group.Conclusions:1.It is concluded that BCCAO Modeling causes significant pathological changes in the hippocampal pyramidal cells of rats.2.After 4 weeks of EPO treatment,hippocampal neuron damage is alleviated,indicating the neuroprotective effect of EPO.Part III Effect of EPO on the expression of apoptosis and autophagy related proteins in hippocampal tissuesObjective: To investigate the effect of EPO on the PI3K/Akt pathway and apoptosis-and autophagy-related proteins in hippocampal neurons by examining the phosphorylation of PI3K/Akt pathway-related proteins,the expression of apoptosis-and autophagy-related proteins,and changes in apoptosis and autophagic flux in hippocampal cells.Methods:1.Western blotting was used to test the phosphorylation levels of EPOR,Akt,PI3 K,and cyclic AMP-response element-binding protein(CREB)and in the hippocampus of VD rats and in vitro cultured neurons,as well as the expression of apoptosis-related proteins B-cell lymphoma-2-Associated X(Bax),Cleaved-Caspase 3,and Cleaved-Caspase 9 and autophagy-related proteins light chain 3B(LC3B),Beclin 1,and autophagy-related 5(ATG5).2.TUNEL staining was used to detect apoptosis at the single-cell level.The nucleus of apoptotic cells was yellow-green,which could be observed under a fluorescence microscope.Since the number of TUNEL-positive cells was generally proportional to the degree of apoptosis,five fields of view were randomly selected under a fluorescence microscope to count the number of TUNEL-positive cells and total cells.Afterward,the apoptosis index was calculated: AI = the number of positive cells in each area/total number of cells.3.Western blotting was utilized to measure the expression of LC3II/LC3 I and P62 in hippocampal cells of VD rats and in vitro cultured neurons.Results:1.The effect of EPO on EPOR,Akt,PI3 K,and CREB phosphorylation in hippocampal neurons of VD rats was analyzed.The results unveiled that compared with the Model group,the sham group had statistically significant lower phosphorylation levels of EPOR,Akt,PI3 K,and CREB(P < 0.05),illustrating that EPOR expression is increased in hippocampal neurons under hypoxic conditions,which improves the adaptability of rats to environmental changes.Additionally,exogenous EPO treatment statistically substantially enhanced the phosphorylation level of EPOR,Akt,PI3 K,and CREB in the hippocampal neurons of VD rats(P < 0.05),meaning that exogenous EPO can compensate for its insufficient endogenous secretion and bind to highly expressed EPOR,further activating EPOR,PI3 K,Akt,and CREB.2.The effect of EPO on the expression of apoptosis and autophagy related proteins was evaluated in the hippocampal neurons of VD rats.The protein expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG was statistically prominently higher in the Model group than in the sham group(P < 0.05),highlighting that obvious apoptosis and autophagy occurs in the hippocampal neurons of VD rats under low perfusion conditions.Exogenous EPO treatment elicited a statistically significant reduction in the protein expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG5 in the hippocampal neurons of VD rats(P < 0.05),implying that EPO can significantly inhibit the excessive activation of autophagy and apoptosis in hippocampal neurons under chronic hypoperfusion conditions.3.In vitro experiments revealed that in normal cells,EPO treatment increased the phosphorylation level of EPOR(P < 0.05)but did not significantly alter the phosphorylation levels of PI3 K and Akt and the expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG5,meaning that exogenous EPO can stimulate EPOR phosphorylation in hippocampal neurons,but its physiological effect may not be related to neuroprotection.Compared with the control group,the control +EPO + inhibitor group presented with unchanged protein expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG5 and elevated phosphorylation of EPOR,similar to the trend in the control +inhibitor group.This result indicates the presence of basic autophagy and apoptosis in the body.Under the condition of oxygen and glucose deprivation,exogenous EPO treatment statistically significantly elevated EPOR,PI3 K,and Akt phosphorylation while statistically markedly reducing the protein expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG5,illustrating that under hypoxia conditions,exogenous EPO can enhance EPOR phosphorylation and further activate the downstream PI3K/Akt pathway to regulate apoptosis and autophagy.Under the condition of oxygen and glucose deprivation plus inhibitors,addition with exogenous EPO increased the phosphorylation of EPOR,accompanied by no significant alteration in the protein expression of Bax,Cleaved-Caspase 3,Cleaved-Caspase 9,LC3 B,Beclin 1,and ATG5 and the phosphorylation of PI3 K and Akt.4.According to TUNEL staining results,apoptosis-positive cells were almost invisible in the sham group.A small number of green apoptosis positive cells were observed in the EPO group.The proportion of stained apoptotic cells in the sham and EPO groups was significantly lower than that in the Model group.5.The autophagic flux was tested.In vivo experiment results showed that compared with the Model group,the LC3II/LC3 I ratio was statistically markedly diminished and P62 protein expression was statistically significantly increased in the sham and EPO groups.In vitro experiment results displayed that compared with the hypoxia group,the EPO group showed a statistically markedly lower ratio of LC3II/LC3 I and statistically significantly higher P62 protein expression.Conversely,the LC3II/LC3 I ratio and P62 protein expression were essentially unchanged after addition of inhibitors or both EPO and inhibitors.Conclusions:1.EPO could increase the survival of hippocampal neurons under chronic hypoperfusion conditions possibly by up-regulating the phosphorylation of EPOR and activating the PI3K/Akt pathway.This pathway represses hypoxia-induced excessive apoptosis and autophagy of hippocampal neurons,thereby facilitating the survival of hippocampal neurons after stress stimulation.2.The detection of apoptosis by TUNEL staining indicates the potential neuroprotective effect of EPO.Part IV Differences of EPO levels between individuals with and without vascular cognitive impairmentObjective: To investigate the differences in EPO levels between individuals with and without vascular cognitive impairment,further providing clinical evidence for the treatment of cognitive impairment with EPO.Methods: A retrospective study was conducted to determine the relationship between EPO levels and age in patients with or without vascular cognitive impairment.This study enrolled 42 participants who were hospitalized in the Department of Neurology at the Hebei Provincial People’s Hospital from January 2022 to January 2023,including 21 participants with vascular cognitive impairment and 21 participants without vascular cognitive impairment.In this study,vascular cognitive impairment was diagnosed based on the 2019 Chinese Guidelines for the Diagnosis and Treatment of Vascular Cognitive Impairment.Results:1.There was no statistical difference in gender,age,blood pressure,blood sugar,cerebrospinal fluid biochemical results,and EPO levels in cerebrospinal fluids and serum between the two groups.2.In patients with vascular cognitive impairment,the concentration of EPO in cerebrospinal fluids was significantly correlated with age,but not with the concentration of EPO in the serum.Conclusions:1.For patients with vascular cognitive impairment,the concentration of EPO in cerebrospinal fluid might be positively correlated with age.2.For patients with vascular cognitive impairment,the concentration of EPO in the cerebrospinal fluid could be not related to its concentration in serum.