Study On The Mechanism Of Cervical Vascular Stenosis,Astragaloside ? And Exposure To Cigarette Smoke On Cognitive Function In Vascular Dementia

Vascular dementia(VaD)is the second most common dementia in the world after Alzheimer's disease,which imposes a heavy burden on People's daily life and economy.VaD refers to cognitive impairment associated with vascular risk factors and/or vascular causes,including the entire process from mild cognitive impairment to dementia.The etiology includes chronic cerebral hypoperfusion,infarction at key sites,multiple cerebral infarction,microinfarction,microhemorrhage,white matter lesions,CADASIL,and brain amyloid lesions,which further leads to individual cognitive impairment.Chronic cerebral hypoperfusion(CCH)caused by bilateral carotid artery ligation is considered as an ideal model for VaD.Whether this relatively classical model can well simulate the clinical process of VaD patients,this study will combine clinical cases and animal models for further discussion.CCH can cause a series of pathophysiological events,including inflammation,oxidative stress and so on.Neuroinflammation plays an important role in the process of clearing pathogens and phagocytosis of apoptotic cells and debris.However,uncontrolled or chronic neuroinflammation exerts its neurotoxic effects through the production of neurotoxic mediators and pro-inflammatory cytokines.Neuroinflammation is closely related to cognitive function.The release of neuroinflammatory cytokines activates immune system cells and is involved in the regulation of neurogenesis,synaptic plasticity and neuronal survival,thereby influencing the formation of learning and memory.Astrocytes are the most abundant cells in the central nervous system,accounting for 40 percent of the total number of brain cells.GFAP is a marker for astrocytes,the most widely used of which is used to check for nerve damage,deformation and inflammation.When the body is early exposed to stress,the morphology and transients of astrocytes are permanently altered.The NLRP3 inflammasome is the most widely studied inflammasome.Various factors can trigger inflammasomes,such as pathogen-associated molecular patterns(PAMP)and damage-associated molecular patterns(DAMP).Therefore,it is further inferred that if there are drugs that can selectively inhibit the molecules or genes of the above processes,it will be of great therapeutic significance in the treatment of VaD.Or whether anything that causes neuroinflammation can lead to cognitive impairment,which might better mimic the progression of vascular dementia.Therefore,this research is divided into five parts.The first part collects clinical cases to observe the cognitive function of patients with different degrees of cervical vascular stenosis;the second part establishes an animal model of unilateral carotid artery ligation and bilateral carotid artery ligation to verify whether it is consistent with the first part.Part of the clinical results are consistent to further explore the rationality of the vascular dementia model;the third part is the establishment of a vascular dementia model by 2 VO method,and the model animals are given nasogastric astragaloside ?(high and low-dose groups).After observation and application of astragaloside ?,Whether the animal's cognition level has improved;the fourth part detects the SOD and MDA content of the third part of the animal,and analyzes the changes of PGC1?/Nrf2,Sting/NLRP3/caspasel pathway proteins,and explores the possible connection mechanism between them.Part 5 Innovative application of smoking cessation combined with 2VO method for model making,to study whether neuroinflammation still exists after smoking cessation,which affects cognitive function.In this paper,the Morris water maze(MWM)was used for behavioral evaluation,enzyme linked immunosorbent assay(ELISA),Western-blot(WB),immunofluorescence(Immunofluorescence,IF)and other technologies.Observe the effects of neuroinflammation on the cognitive function of VaD rats in terms of function,morphology,and protein expression,and explore its mechanism of action.Part One Clinical study of cognitive function in patients with asymptomatic carotid artery stenosis and vertebral artery stenosisObjective:To observe the cognitive function of patients with severe asymptomatic carotid and vertebral artery stenosis.Methods:This experiment selected asymptomatic unilateral patients who were hospitalized in the Department of Neurology,Hebei Provincial People's Hospital from January 2019 to December 2020 in the Department of Neurology,Hebei Provincial People's Hospital and completed cognitive function teSting.Carotid artery stenosis(stenosis rate 70%-99%,9 cases),bilateral carotid artery stenosis(stenosis rate 70%-99%,9 cases),unilateral carotid artery+Patients with unilateral vertebral artery stenosis(both vascular stenosis rate was 70%-99%,8 cases)and completed the cognitive function assessment.All selected patients have signed an informed consent form.The clinical data collection form for patients after enrollment mainly includes gender,age.And collect its Montreal cognitive assessment(Montreal cognitive assessment,MoCA)assessment results.Results:1.There was a positive correlation between the number of cervical vascular stenosis and cognitive impairment.2.There was no statistically significant difference in age among the groups.3.The cognitive comparison between patients with unilateral carotid artery stenosis and patients with bilateral carotid artery stenosis was not statistically significant,but the cognitive comparison between patients with unilateral carotid artery stenosis and patients with unilateral carotid artery stenosis was statistically significant(P<0.05).Conclusions:The degree of cognitive dysfunction is related to the degree and number of cervical artery stenosis.The patients with unilateral carotid stenosis+unilateral vertebral stenosis had the worst cognitive function.Part Two Comparison of cognitive function between unilateral carotid artery stenosis and bilateral carotid artery stenosis in ratsObjective:To observe the cognitive function of rats with unilateral carotid artery stenosis and bilateral carotid artery stenosisMethods:A total of 18 clean-grade healthy male SD(Sprague-Dawley)rats(3 months old),weighing 200-250g,and adaptively reared for 1 week,were randomly divided into 3 groups,each with 6 only.Sham operation group(sham group),unilateral carotid artery occlusion group(100 group),bilateral carotid artery occlusion group(2VO group).After 4 weeks of normal feeding,Morris water maze automatic recorder was used for a 6-day behavioral test.The first 5 days will be a positioning and navigation experiment,and the last day will be a space exploration experiment.Results:1 Place navigation test1.1 Compared with the sham group,there was no significant difference in the escape latency in the 1 VO group.1.2 Compared with the sham group,the escape latency of the 2VO group was prolonged(Day1:P<0.05;Day2-5:P<0.01).1.3 Compared with the 2VO group,the escape latency of 1VO group was shortened(Day2-3:P<0.01;Day4-5:P<0.05).2 Spatial probe test2.1 Compared with the sham group and the 1VO group,the time in the target quadrant of 2VO group was significantly shortened(P<0.01).2.2 Compared with the sham group,the time in the target quadrant of the 1 VO group was not significantly different.Conclusion:The 2VO method is an ideal model for preparing VaD.Part Three Astragaloside ? can alleviate cognitive impairment of the rats with vascular dementiaObjective:The cognitive protective effect of astragaloside ? on rats with vascular dementia.Methods:A total of 40 clean-grade healthy male SD(Sprague-Dawley)rats(3 months old)with a weight of 200-250 g were selected and reared adaptively for 1 week.The rats were randomly divided into 4 groups,each with 10 rats.Sham group(sham group),model group(2VO group),low-dose Astragaloside ?(As-?20 group),and high-dose Astragaloside ?(As-?50 group).The 2VO method was used to prepare the VaD model.The sham group and 2VO group were given double distilled water 1-2ml/time daily for 4 weeks.As-?20 group was given 20mg/kg of As-? per day by gavage,and Ast-?50 group was given As-? 50mg/kg per day by gavage for 4 weeks.Later,the Morris water maze automatic recorder was used for a 6-day behavioral test.The first 5 days are the positioning and navigation experiments,and the last day is the space exploration experiment.Results:1 Place navigation test1.1 Compared with sham group,the 2VO group had a longer escape latency from day 1 to day 5(P<0.01).1.2 Compared with sham group,the As-IV50 group had a shortened escape latency on the first day(P<0.05).There was no significant difference from day 2 to day 5.1.3 There was no statistically significant difference in escape latency between sham group and As-?20.1.4 Compared with 2VO group,the escape incubation period of As-?50 group had a shortened escape latency within 5 days(P<0.01);As-?20 group also had a shortened escape latency(Dayl:P<0.01;Day 2:P<0.05;Day 3:P<0.01;Day 4:P<0.05).1.5 Compared with As-?50 group,the escape latency of As-?20 group was different on the first,third and fourth days,and As-? 50 group had good results(P<0.05).2 Spatial probe test2.1 2VO group spent less time in the target quadrant than those in Sham group(P<0.01).2.2 As-?50 group and As-?20 group spent longer time in the target quadrant than the 2VO group(P<0.01).Conclusion:According to the above results,As-? has the ability to significantly improve the cognitive impairment caused by CCH,and the high-dose group can restore the cognitive ability of CCH to the same level as the sham operation group.Part Four Astragaloside ? can reduce CCH-induced neuroinflamma-tory response through the Sting/NLRP3/caspasel pathwayObjective:To observe and explore the role of neuroinflammation in the pathogenesis of VaD.Methods:The preparation and grouping of the experimental models in this part are the same as those in the third part.The western blot method was used to detect the protein expression of Sting,NLRP3,caspase1,GFAP,PGCla,and Nrf2 in rat hippocampus.Double immunofluorescence labeling technology applied to caspasel and GFAP Neun.Results:The expression of these inflammatory factors in 2VO group was significantly higher than that in sham group.1.2VO group was significantly higher than that in the sham group(Sting:P<0.01;NLRP3:P<0.01;caspasel:P<0.01;GFAP protein level:P<0.01).As-? can significantly alleviate this inflammatory response.(Sting:P<0.01,As-?20 group vs 2VO group,P<0.01,As-?50 group vs 2VO group;NLRP3:P<0.05,As-?20 group vs 2VO group,P<0.01,As-?50 group vs 2VO group;caspasel:P<0.01,As-?20 group vs 2VO group,P<0.01,As-?50 group vs 2VO group;GFAP:P<0.01,As-?2O0 vs 2VO group,P<0.01,As-?50 vs 2VO group).2.Compared with the sham group,the levels of PGCla and Nrf2 in the 2VO group decreased(PGC1?:P<0.01;Nrf2:P<0.01;),but after treatment with As-?,the levels of these two factors can be restored(PGC1?:P<0.01,As-?20 group vs.2VO group,P<0.01,As-? 50 group vs.2VO group;Nrf2:P<0.01,As-?20 group vs.2VO group,P<0.01,As-?50 group vs.2VO group).Conclusion:This section found that As-? could play an anti-inflammatory effect by up-regulating the PGC1?/Nrf2 pathway and inhibiting the Sting/NLRP3/caspasel pathway,thereby improving the damage of rat hippocampus and protecting cognitive function.Part Five Past exposure to cigarette smoke aggravates cognitive impairment in a rat model of vascular dementia via neuroinflammationObjective:Past smoking experience can further impair the cognitive function of rats with vascular dementia.Methods:A total of 24 clean-grade healthy male SD(Sprague-Dawley)rats(3 months old),weighing 200-250g,were adaptively reared for 1 week,and the rats were randomly divided into 4 groups.Control group(CT,n=8),early smoke exposure group(ECS,n=6),vascular dementia group(2VO,n=7),early smoke exposure+vascular dementia group(ECS+2VO,n=6))group.We applied passive smoking to the ESC,ESC+2VO group,and carried out passive smoking for 3 months.Then,after routine feeding for 1 month,we used the 2VO method to prepare the vascular dementia model for the 2VO and ESC+2VO group.Morris water maze was used for behavioral assessment after 4 weeks.Western-blot method to detect NLRP3 and caspasel in the hippocampus of rats,and the levels of IL1? in each group were detected by ELISA.Results:1 Place navigation test1.1 Compared with the CT group,the average time of rats in the 2VO and ESC+2VO groups significantly prolonged the escape latency from day 1 to day 5(P<0.05).1.2 Compared with the CT group,there was no significant delay in the escape latency of the ESC group from day 1 to day,but on day 5,the ESC group spent more time(P<0.05).1.3 Compared with the ESC group,the escape latency of the ESC+2VO group was significantly prolonged from day 1 to day 5(P<0.05);the 2VO group prolonged from day 1 to day 4(P<0.05),and there was no significant difference in the escape latency between 2VO and ESC on day 5.1.4 Starting from the fourth day,the difference between the 2VO and ESC+2VO groups became more and more obvious(Day 1-3:P>0.05;Day 4:P<0.05;Day 5:P<0.01).2 Spatial probe testRats in each group had significant differences in the percentage of staying time in the target quadrant.The CT group spent more time in the target quadrant than the other three groups(P<0.05).However,There is no difference between the 2VO group and ESC group in staying time of the target quadrant.3 Western-blot results3.1 The ESC+2VO group showed higher expression of GFAP compared with the other three groups(CT:P<0.001;ESC:P<0.001;2VO:P<0.01).The expression of CT group was lower than that of ESC group and 2VO group(ESC:P<0.01;2VO:P<0.001).There was no difference between the ESC group and the 2VO group.3.2 The ESC+2VO group showed higher expression of NLRP3 than the other three groups(CT:P<0.001;ESC:P<0.01;2VO:P<0.05).The expression of CT group was lower than that of ESC and 2VO group(ESC:P<0.01;2VO:P<0.001).There was no statistical significance between the ESC and 2VO groups.3.3 The expression of caspasel in the ESC+2VO group was higher than the other three groups(CT:P<0.001;ESC:P<0.001;2VO:P<0.01).The expression of 2VO group was higher than that of CT and ESC group(CT:P<0.01;ESC:P<0.05).There is no difference between CT and ESC groups.4 ELISA resultsWe found that the IL1? of the ESC+2VO group was significantly higher than the other three groups(P<0.001).IL1? in CT group was lower than that in ESC and 2VO groups(P<0.01).However,there is no difference between the ESC and 2VO groups.