| Objective:With the intensification of the aging process of our country’s population,stroke has become the world’s second largest cause of death in my country,and it is also the primary cause of disability in our country’s population.The harm of stroke is increasing.Ischemic stroke will not only cause continuous damage to the nervous system,but also cause a progressive increase in cognitive impairment.Not only that,the damage of remote organs after ischemic stroke has also been regarded as the key to aggravating stroke damage and affecting its prognosis in recent years.In particular,other systems involving digestion,breathing,and circulation.Complications of these distant organs can lead to brain visceral syndromes and affect the clinical prognosis and treatment of stroke patients.Chronic cerebral hypoperfusion(CCH)is one of the important pathological mechanisms of ischemic cerebrovascular disease and one of the important pathophysiological basis of vascular cognitive impairment(VCI).VCI and its related mechanisms are hot topics in the field of neuroscience research.In recent years,related studies have revealed that the functional changes of the intestinal tissue and the intestinal mucosal barrier have an impact on the changes in cognitive function.The previous research results of our research group showed that CCH can cause cognitive dysfunction in experimental animals for up to 24 weeks,and is accompanied by damage to the intestinal mucosal barrier.This study intends to establish a CCH experimental rat model with permanent bilateral common carotid artery occlusion(BCCAO)method based on the results of the research group’s previous studies,and observe the behavioral indicators of experimental rats dynamically for a long time.Changes,changes in the expression levels of claudin-1,the body reactive protein osteopontin(OPN)and inflammatory response-related nuclear factor kappa-B(NF-κB)pathway proteins in the intestinal tissue of experimental rats,and Changes in the expression of serum OPN and related inflammatory factors to further clarify:(1)After 4 weeks of chronic cerebral hypoperfusion,the changes in cognitive behavior and intestinal mucosal tissue morphology of experimental rats;(2)Quantitative analysis of CCH-induced experimental Changes in cognitive behavior of rats and the expression of claudin-1 and OPN in the intestinal mucosa;(3)To explore the possible mechanism of CCH-induced intestinal mucosal barrier damage in experimental rats,and observe the protective effect of simvastatin on the intestinal mucosal barrier.Part Ⅰ: Study on cognitive impairment and intestinal mucosal barrier permeability changes in rats induced by chronic cerebral hypoperfusion Objective:To explore the correlation between cognitive impairment and intestinal mucosal barrier damage in rats after CCH,quantitatively analyze the changes in cognitive behavior of rats caused by chronic cerebral hypoperfusion,and the expression changes of intestinal mucosal barrier tight junction protein claudin-1 and OPN.Material and method:Thirty male SD rats were randomly divided into chronic cerebral hypoperfusion group(CCH group,n=15)and sham operation control group(SHAM group,n=15)according to the random number table method.The CCH group used the BCCAO method for bilateral common carotid artery ligation,and the SHAM group only found the common carotid artery without ligation.Four weeks later,behavioral experiments were used to detect the changes in rat’s cognitive behavior;HE staining and immunofluorescence staining were used to detect the damage of rat ileum tissue,Western blotting was used to detect the expression of OPN in ileum tissue,and ELISA was used to detect OPN content in rat serum.Results:1.Rats Behavior Experiment: in the open field experiment,the number of standing times and the total distance of exercise of rats in the CCH group were less than those in the SHAM group(CCH group 16.70±7.13 times vs.SHAM group 28.70±10.70 times;CCH group 736.64±136.71 cm vs.SHAM group 1030.45±81.51 cm,P<0.05);in the object discrimination experiment,the discrimination index of rats in the CCH group was significantly lower than that in the SHAM group(CCH group 0.44±0.26 vs.SHAM group0.91±0.07,P<0.05);in the positioning navigation experiment,the escape latency and total swimming distance of the rats in the CCH group were longer than those in the SHAM group,and showed a gradually decreasing trend(P<0.05);in the space exploration experiment,the number of crossings of the rats in the CCH group was less than that in the SHAM group.The latency of crossing was increased compared with the SHAM group(CCH group 3.00±0.82 times vs.SHAM group 7.20±1.81 times;CCH group 29.70±6.28 s vs.SHAM group 9.96±2.95 s,P<0.05);in the working memory experiment,the CCH group was larger The escape latency of the mice was longer than that of the SHAM group,and showed a gradually decreasing trend(P<0.05).2.The histopathological changes of the ileum: the pathological score of the intestinal mucosa of rats in the CCH group was higher than that in the SHAM group(CCH group 4.52±0.27 points vs.SHAM group 1.98±0.34 points,P<0.01);immunofluorescence staining of the ileum tissue,the claudin-1 fluorescence IOD value of the intestinal tissue of rats in the CCH group was lower than that in the SHAM group(CCH group 47154.50±7507.29 vs.SHAM group 125028.58±33077.39,P<0.01).3.Western blot experiment: The relative protein expression of OPN in the ileum tissue of the CCH group was higher than that of the SHAM group(CCH group 1.20±0.95 vs.SHAM group 0.38±0.11,P<0.05).4.ELISA experiment: The serum OPN content of rats in the CCH group was increased compared with that in the SHAM group(CCH group 14.92±1.45 ng/ml vs.SHAM group3.42±0.66 ng/ml,P<0.05).5.Pearson correlation analysis,cognitive impairment is negatively correlated with intestinal mucosal epithelial claudin-1 expression and serum OPN content(all P<0.01),intestinal mucosal epithelial claudin-1 expression is negatively correlated with serum OPN content(r=-0.952,P<0.01).Part Ⅱ: Chronic cerebral hypoperfusion induces rat intestinal mucosal barrier damage through NF-κB pathway and the protective effect of simvastatin Objective:To explore the possible mechanism of CCH-induced ileal mucosal barrier damage in rats and the protective effect of simvastatin on the intestinal mucosal barrier.Material and method:48 male SD rats were randomly divided into chronic cerebral hypoperfusion group(CCH group,n=12),sham operation group(SHAM group,n=12),and solvent group(SC group,n=12)according to the random number table method.)And Simvastatin group(SV group,n=12).The modeling method was the same as the previous chapter.The SC group was given 0.5% sodium carboxymethyl cellulose by gavage,and the SV group was given simvastatin suspension prepared by sodium carboxymethyl cellulose by gavage.After 4weeks,specimens were collected,and the tissue damage was detected by HE staining and immunofluorescence staining of the ileum.Western blotting was used to detect the expression levels of claudin-1,OPN,NF-κB p65 and p-NF-κB p65 in the ileum tissue,and the ELISA test was used to detect the expression levels of claudin-1,OPN,NF-κB p65 and p-NF-κB p65.Serum levels of TNF-α and IL-6 in rats.Results:1.The histopathological changes of the ileum: the pathological score of the intestinal mucosa of rats in the CCH group was higher than that in the SHAM group(CCH group 4.44±0.36 points vs.SHAM group 1.93±0.33 points,P<0.05),rats in the SV group The pathological score of intestinal mucosa was lower than that of SC group(SV group 2.03±0.44 points vs.SC group 4.38±0.34 points,P<0.05);ileum tissue immunofluorescence staining,CCH group rat intestinal tissue epithelial claudin-1 fluorescence IOD value was higher SHAM group decreased(CCH group 41494.15 ± 13296.08 vs.SHAM group120796.32 ± 32563.27,P < 0.05),SV group rat intestinal tissue epithelial claudin-1fluorescence IOD value increased compared with SC group(SV group 109149.71 ±59375.56 vs.SC group 45850.14±12925.51,P<0.05).2.Western blot experiment,the relative protein expression of claudin-1 in the ileum tissue of the CCH group was lower than that of the SHAM group(CCH group 0.35±0.21 vs.SHAM group 0.96±0.30,P<0.05),SV group rat claudin-1 The relative protein expression was higher than that in the SC group(SV group 0.85±0.18 vs.0.36±0.06,P<0.05);the relative protein expression of OPN in the CCH group was higher than that in the SHAM group(CCH group 1.24±0.26 vs.SHAM group 0.40±0.15,P<0.05),the relative protein expression of OPN in SV group was lower than that in SC group(SV group 0.58±0.11 vs.SC group 1.03±0.09,P<0.05);rat p-p65/p65 ratio in CCH group was higher than that in the SHAM group(CCH group 0.57±0.12 vs.SHAM group 0.13±0.07,P<0.05),and the ratio of p-p65/p65 in the SV group was lower than that in the SC group(SV group 0.26±0.06 vs.SC group 0.52±0.10,P<0.05).3.In the ELISA experiment,the serum TNF-α and IL-6 levels in the CCH group increased compared with the SHAM group(CCH group 166.30±25.52 pg/ml,128.48±6.53pg/ml vs.SHAM group 45.91±10.30 pg/ml,40.56±7.86 pg/ml,P<0.05).The serum TNF-αand IL-6 levels in SV group were lower than those in SC group(SV group 64.89±12.96pg/ml,61.67±5.97 pg/ml vs.SC group 162.53±27.64 pg/ml,123.76±5.80 pg/ml,P<0.05).Conclusions:1.The state of chronic cerebral hypoperfusion can cause persistent emotional arousal disorders and impaired spatial learning and memory abilities in experimental animals.2.The state of chronic cerebral hypoperfusion can cause the morphological changes of the intestinal mucosal tissue in rats,leading to the destruction of the intestinal mucosal barrier function.3.There is a correlation between the content of serum osteopontin and the cognitive impairment in experimental rats and the expression of claudin-1 between intestinal mucosal epithelial cells,suggesting that osteopontin may be involved in the intestinal mucosal barrier injury after chronic cerebral hypoperfusion The process may be used as a potential serological marker for the cognitive impairment and the destruction of the intestinal mucosal barrier in rats caused by chronic cerebral hypoperfusion.4.Changes in the expression of proteins related to the NF-κB p65 pathway in the ileum of experimental rats suggest that chronic cerebral hypoperfusion may activate the intestinal NF-κB pathway of experimental animals to increase the expression of inflammatory factors TNF-α and IL-6,thereby destroying the intestine Mucosal barrier.5.Simvastatin has a protective effect on the intestinal mucosal barrier damage while improving the cognitive impairment of experimental rats caused by chronic cerebral hypoperfusion. |
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