| Vascular cognitive impairment(VCI)is the second leading cause of cognitive impairment and dementia after alzheimer's disease,including the whole stage from mild cognitive impairment(MCI)to dementia.Especially in the rapidly aging society,the prevalence of vascular cognitive impairment will increase significantly.According to the results of the sixth population census of China in 2010,the number of people over the age of 60 in China has reached 180 million,accounting for 13.3% of the total population.As a result,the whole society will face challenges from diseases that threaten the health of older people,including vascular cognitive impairment and dementia.Chronic cerebral hypoperfusion-induced cognitive impairment(CCH-VCI)is easily neglected in VCI clinical type,mainly due to various reasons lead to a long time continuous ischemia caused by low perfusion of cerebral hemisphere or the whole brain.Essentially its development process is,chronic cerebral hypoperfusion-persistent ischemic and hypoxia-progressing nerve lesion-neural network structure and function damage-cognitive impairment and dementia.In older adults,especially in those with long term suffering for cardiovascular risk factors such as hypertension,diabetes,insignificantly onset of stroke and other cerebrovascular diseases increase,such as intracranial unilateral or bilateral brain arteriosclerosis and progressive stenosis,can cause persistent ischemic hypoperfusion of the cerebral hemisphere or the full brain,nerve damage increase gradually and cause cognitive impairment of memory and executive ability,and can eventually lead to dementia associated with the occurrence of recurrent stroke.It is worth noting that,unlike acute ischemic paroxysmal nerve injury,chronic ischemia-hypoperfusion nerve damage tends to have a relatively insidious and progressive process,so the symptoms of cognitive impairment are often atypical and easy to be ignored even if they are found.For those patients who are unable or temporarily unsuitable for surgical treatment such as angioplasty,the nerve damage caused by continuous ischemia-hypoperfusion will destroy the integrity of neural network structure and function,leading to cognitive impairment and even dementia.Unfortunately,with the exception of few drugs to improve dementia,there are currently no drugs that can improve or reverse nerve damage under the condition of chronic cerebral ischemia and low perfusion.Therefore,it is worth exploring therapeutic targets and intervention strategies that can prevent or improve neural lesion under the condition of chronic ischemia and hypoxia.p75 neurotrophin receptor(p75NTR)is a member of the tumor necrosis factor receptor superfamily and is considered to be the receptor of many neuronutrient precursors.Previous studies have shown that in the serum of patients with ischemic stroke and Alzheimer's disease,the p75 NTR extracellular domain(p75NTR-ECD),which is hydrolyzed by protease,is increased and may be related to the severity of the disease,indirectly suggesting that p75 NTR may play a role in the occurrence or progression of neurological diseases.In view of the sharing of cerebral ischemia and low perfusion in those neurological diseases,it can be speculated that p75 NTR may play a role in CCH-VCI.However,the role of p75 NTR in the process of neural lesion in the context of persistent ischemia and hypoxia induced by chronic cerebral hypoperfusion remains unclear.On the other hand,in these chronic CNS diseases,some studies have reported abnormally elevated inflammatory cytokines,which are believed to be related to nerve damage caused by cerebral ischemia and hypoperfusion.Based on this,this study explored the following experimental steps step by step to find evidence that p75 NTR plays a role in the process of nerve damage caused by chronic ischemia and hypoxia.First,we studied the serum levels of p75NTR-ECD and some inflammatory factors in patients with cognitive impairment of chronic cerebral hypoperfusion,and looked for the correlation between the two.Through the study of the extracellular segment of the receptor,we indirectly proved that p75 NTR in the brain of patients with cognitive impairment of chronic cerebral hypoperfusion was likely to have corresponding changes.In order to further confirm the role of p75 NTR in the lesion of neural cells under the condition of ischemia and hypoxia,we constructed a human neuroblastoma cell(SH-SY5Y)oxygen-glucose deprivation(OGD)model in vitro by using tri-gas incubator and sugar-free culture to simulate the changes of p75 NTR expression and its role in the cell injury under the condition of ischemia and hypoxia in vivo.Finally,we made a mouse model of chronic hypoperfusion-induced cognitive impairment by bilateral incomplete carotid artery ligation(residual inner diameter 0.2mm),to study the changes in p75 NTR expression in the brain under continuous ischemia and hypoxia,and its role in inflammatory response,apoptosis and destruction of synaptic plasticity.The research contents of each part are summarized as follows:Part I Serum p75NTR-ECD level and its relationship with inflammatory factors in patients with vascular cognitive impairment induced by chronic cerebral hypoperfusionOBJECTIVE: To examine serum p75NTR-ECD level in patients with vascular cognitive impairment induced by chronic cerebral hypoperfusion(CCH-VCI)and investigate its relationship with tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?)and interleukin-6(IL-6).SUBJECTS AND METHODS: 34 patients with CCH-VCI admitted to Changhai Hospital of the Naval Military University from August to December 2018,as well as 36 healthy middle-aged and elderly patients and 34 patients with ischemic stroke of the same age period were included.Serum levels of p75NTR-ECD,TNF-?,IL-1? and IL-6 in the three groups were measured by ELISA and compared.Correlation between serum p75NTR-ECD and TNF-?,IL-1? and IL-6 levels in patients with CCH-VCI was studied by Spearman correlation analysis.RESULTS: The serum p75NTR-ECD level in the CCH-VCI group was higher than that in the healthy control group and the ischemic stroke group [(544.36(440.88,628.50)pg/m L vs 276.49(262.59,313.87)pg/m L and 366.87(337.09,450.43)pg/m L],and the differences were statistically significant(U=87.500,335.500,P < 0.05).The serum levels of TNF-??IL-1??IL-6 were 196.02(141.20,280.35)pg/m L,68.23(60.79,91.94)pg/m L,51.04(40.24,65.26)pg/m L in the CCH-VCI group,and 218.67(143.76,281.28)pg/m L,76.87(59.10,99.91)pg/m L and 64.45(43.13,86.76)pg/m L in the ischemic stroke group,which were higher than the healthy control group(73.71(56.94,79.81)pg/m L,42.98(34.52,51.34)pg/m L,14.97(11.76,21.19)pg/m L,respectively),with significant differences(U=4.000,31.000,132.000,106.000,13.000,48.000,P < 0.05).Serum p75NTR-ECD level in CCH-VCI patients was correlated with TNF-? level(r = 0.391,P = 0.022),but not IL-1? and IL-6 levels(r = 0.032,0.164,P = 0.855,0.355).CONCLUSION: Serum p75 NTR level may be related to inflammatory factors(TNF-?)after chronic cerebral hypoperfusion,and they may jointly participate in the pathogenesis of CCH-VCI.Part II Expression and effect of p75 neurotrophin receptor in human neuroblastoma cells in the condition of oxygen-glucose deprivationOBJECTIVE: To investigate the expression and effect of p75 neurotrophin receptor(p75NTR)in human neuroblastoma(SH-SY5Y)cells in the condition of oxygen-glucose deprivation(OGD).METHODS: The OGD model of SH-SY5 Y cell was established by an approach of glucose-free and serum-free culturing using tri-gas incubator,and then three groups were being setting up,including serum-free regular culturing group(control),OGD group and OGD+LM11A-31(a competitive blocker of p75NTR)group.After 12 h culturing,the cell viability was measured by MTT assay,LDH release activity by commercial assay kit,cell apoptosis proportion by flow cytometry,and p75 NTR expression by western blotting.RESULTS: SH-SY5 Y OGD model was successfully established,and then cultured for 12 h for the following group tests.The viability rate(%)of cells in the OGD group was significantly lower than that in control(50.34 ± 5.55 vs.94.80 ± 4.06,P < 0.05).The viability rate in OGD+LM11A-31 group was also lower than that in control(64.68 ± 4.59 vs 94.80 ± 4.06,P < 0.05),but higher than that in OGD group(64.68 ± 4.59 vs 50.34 ± 5.55,P < 0.05).LDH releasing activity(U/L)in OGD group was significantly higher than that in control(353.09 ± 30.67 vs 46.93 ± 5.49,P < 0.05).LDH release activity of OGD+LM11A-31 group was also higher than that in control(282.20 ± 25.60 vs.46.93 ± 5.49,P < 0.05),but lower than that in OGD group(282.20 ± 25.60 vs.353.09 ± 30.67,P < 0.05).The proportion of apoptosis in OGD group(Q2+Q4,%)was significantly higher than that in control group(14.98 ± 2.59 vs.1.82 ± 0.45,P < 0.05).The proportion of apoptotic cells in the OGD + LM11A-31 group was also higher than that in control(7.36 ± 1.98 vs.1.82 ± 0.45,P < 0.05),but lower than that in the OGD group(7.36 ± 1.98 vs.14.98 ± 2.59,P < 0.05).The relative expression of p75 NTR in OGD group was higher than that in control group(0.41 ± 0.02 vs.0.06 ± 0.01,P < 0.05).The expression of p75 NTR in OGD + LM11A-31 group was also higher than that in control group(0.19 ± 0.03 vs.0.06 ± 0.01,P < 0.05),but lower than that in OGD group(0.19 ± 0.03 vs.0.41 ± 0.02,P < 0.05).CONCLUSION: The expression of p75 NTR increased in SH-SY5 Y cells under oxygen-glucose deprivation,which may promote neural lesion and apoptosis.Part III Effects of p75 NTR on neural lesion in mice model of chronic cerebral hypoperfusion-induced cognitive impairmentOBJECTIVE: To investigate the expression of p75 NTR in the brain of mouse model of chronic cerebral hypoperfusion-induced cognitive impairment(CCH-VCI)and the its effect on neural lesion under the condition of chronic cerebral ischemia and hypoxia.METHODS: CCH-VCI mouse model(5-month old female c57BL/6 mice)was established by bilateral incomplete carotid artery ligation.Setting up the normal control,Sham group,the operation model group(CCH-VCI)and drug-administered group(CCH-VCI + LM11A-31),using ELISA to detect peripheral blood levels of inflammatory factors,histochemistry and Western blotting methods were used to detect NF-?B expression and phosphorylation levels,nerve apoptosis and JNKs expression and phosphorylation level,dendric spine density and PSD-95 expression,the expression of p75 NTR in each group.RESULTS: CCH-VCI mouse model was successfully established by incomplete ligation of bilateral carotid artery.Group experiments showed that,serum TNF-?,IL-1? and IL-6 levels in the CCH-VCI group were increased compared with the sham group(TNF-?: 129.17 ± 5.42 vs.29.64 ± 4.27;IL-1?: 40.67 ± 3.69 vs.16.13 ± 2.41;IL-6: 50.59 ± 8.88 vs.14.43 ± 2.93;pg/ml,P<0.05),p75 NTR expression was upregulated(0.400 ± 0.037 vs.0.053 ± 0.005,P<0.05),NF-?B p65 expression and phosphorylation level were increased(p65/?-Actin: 0.349 ± 0.033 vs.0.073 ± 0.003,P<0.05).p-p65/p65: 0.587 ± 0.055 vs.0.310 ± 0.018,P<0.05),increased JNKs expression and phosphorylation(JNKs/?-Actin: 0.348 ± 0.009 vs.0.057 ± 0.001,P<0.05;p-JNKs/JNKs: 0.463 ± 0.015 vs.0.282 ± 0.049,P<0.05),increased apoptosis(TUNEL positive staining area ratio %: 1.587 ± 0.128 vs.0.363 ± 0.021,P<0.05),the expression of PSD-95 decreased(0.144 ± 0.004 vs.0.495 ± 0.019,P<0.05),and the density of dendritic sphine decreased(0.160 ± 0.055 vs.0.480 ± 0.084,P<0.05),the above changes decreased when p75 NTR was blocked,and no statistic differences between the normal control and the sham group.CONCLUSION: p75 NTR plays a mediating and regulating role in neural inflammation,apoptosis and destruction of synaptic plasticity in chronic hypoperfusion-induced cognitive impairment,which is a potential target for effective intervention. |
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