| Objective:Cerebrovascular disease is a high-risk disease in the elderly population.The high disability rate,high mortality rate and complications seriously affect the clinical treatment and prognosis of cerebral ischemia patients.Besides,the brain parenchymal lesions and neurological deficits which caused by cerebrovascular disease,the effects of other systemic changes such as digestive,circulatory,endocrine and immune after cerebral ischemia on the clinical diagnosis and prognosis of patients with cerebrovascular disease have also received much attention in recent years.Patients with stroke or neurodegenerative diseases have symptoms of intestinal microflora disorder,intestinal mucosal damage,intestinal neurogenic reaction and intestinal inflammation in the early stage of the disease.Intestinal ecological imbalance and intestinal dysfunction have been confirmed to be closely related to the occurrence and development of cognitive dysfunction diseases such as dementia.And intestinal mucosal barrier and intestinal function changes play important roles in neurological regulation such as cognitive behavior,regulation of inflammatory response,maintenance of body function.Chronic cerebral hypoperfusion（CCH）,which is an important pathophysiological basis for ischemic brain damage,and has been identified as a notable risk factor and pathophysiological mechanism for vascular cognitive impairment（VCI）and neurovascular dysfunction.However,the effect of CCH on peripheral distal organs has not been clarified.Therefore,based on the previous studies,the present study is designed to observe the dynamic changes and regularity of intestinal mucosal epithelial morphology and intestinal barrier permeability in rat model of CCH.This study also designed to investigate the correlation between the expression level of intestinal tight junction protein claudin-3 and intestinal barrier permeability,and to reveal the effect of CCH on the morphology and function of intestinal mucosal barrier in rats.Material and method:Eighty adult male Sprague-Dawley rats were randomly divided into a sham-operated control group（n=8×4）and CCH model group（n=12×4）,each group.And then divided into 4subgroups,that is 1 week,4 weeks,12 weeks,and 24 weeks group.The rats were subjected to permanent bilateral common carotid artery occlusion（BCCAO）to reproduce CCH.At 1,4,12 and 24 weeks after BCCAO,the morphological changes of intestinal mucosa were observed by HE staining,plasma D-lactic acid content was measured to reflect the dynamic changes of intestinal barrier permeability,immunofluorescence staining and western blot were used to observe the changes in claudin-3 expression and analysis of the correlation between plasma D-lactic acid content and expression of claudin-3 in intestinal tissue.Results:1.CCH induced intestinal mucosal epithelial sustained damage in rats:HE staining results showed that the ileal mucosa structure of Sham group was relatively complete,and the villus morphology was finger-like and arranged neatly.In the CCH group,the damage was obvious,the ileum villi became thicker and dull,the apical part of the villi was detached,and the mucosa was separated from the submucosa.The lamina propria and its blood vessels were exposed.The results of Chiu’s intestinal mucosal injury scoring system showed that the ileal mucosa of CCH group had higher scores from Sham group from 1 week to 24 weeks（2.62±0.52 vs.0.88±0.64,P<0.001;3.88±0.52 vs.1.12±0.64,P<0.001;3.75±0.71vs.1.25±0.64,P<0.001;3.62±0.52 vs.1.50±0.53,P<0.001 at time points of 1,4,12,and24 weeks,respectively）,the differences between the subgroups were statistically significant.There were significant differences in CCH group at different time points(F（3,28）=6.038,P=0.003),and the difference between the 4 weeks group and the 1 week group was significant（P=0.001）.The difference was not statistically significant between 4 weeks,12weeks,and 24 weeks.（12 weeks group vs.4 weeks group P=0.707;24 week group vs.12week group P=0.707）.2.CCH induced plasma D-lactic acid content（ng/μl）sustained and progressively increased in rats:Plasma D-lactate levels were significantly higher in the CCH group from 1week to 24 weeks after modeling（10.25±1.25 vs.5.60±1.32,P<0.001;12.47±1.35 vs.6.04±1.39,P<0.001;12.27.±0.95 vs.6.42±1.10,P<0.001;16.10±0.52 vs.4.96±0.40,P<0.001 at time points of 1,4,12,and 24 weeks,respectively）,the difference between the subgroups was statistically significant.There were significant differences in CCH group at different time points(F（3,16）=22.214,P<0.001).There was no significant difference between12 weeks and 4 weeks（P=0.78）,but 1 week to 24 weeks after model construction.Plasma D-lactic acid content was continuously increased overall（4 weeks group compared with 1week group,P=0.008,24 weeks group compared with 12 weeks group,P<0.001）.3.CCH induced down-regulation of claudin-3 expression in rat ileal mucosal epithelial cells:immunofluorescence results showed that the average fluorescence density of claudin-3in ileal mucosal epithelial cells of CCH group was significantly lower than that of Sham group from 1 week to 24 weeks after model establishment.（0.035±0.0018 vs.0.058±0.0061,P<0.001;0.038±0.0027 vs.0.054±0.0077,P<0.001;0.040±0.0019 vs.0.053±0.0049,P=0.001;0.047±0.0035 vs.0.055±0.0056,P=0.004 at time points of 1,4,12,and 24weeks,respectively）,the difference between the subgroups was statistically significant.There were significant differences in CCH group at different time points(F（3,28）=30.627,P<0.001).The comparison between groups showed that the claudin-3 fluorescence density increased continuously from 1 week to 24 weeks（4 weeks group compared with 1 week group,P=0.014;12 weeks group compared with 4 weeks group,P=0.079,no statistical difference;24 weeks group compared with 12 weeks group,P<0.001）.4.The expression level of claudin-3 was down-regulated in CCH-induced intestinal tissue:the results of western blot showed that the expression level of claudin-3 in ileal mucosal epithelial cells of CCH group was significantly lower than that of Sham group from1 week to 24 weeks after model establishment（0.53±0.073 vs.1.02±0.059,P<0.001;0.56±0.11 vs.1.02±0.072,P<0.001;0.35±0.077 vs.1.04±0.10 P<0.001;0.22±0.054 vs.1.08±0.094,P<0.001 at time points of 1,4,12,and 24 weeks,respectively）,the difference between the two was statistically significant.There were significant differences in CCH group at different time points(F（3,20）=22.552,P<0.001).The comparison between groups showed that the expression of claudin-3 decreased continuously from 1 week to 24 weeks（4weeks group compared with 1 week group P=0.455,no statistical difference;12 weeks group compared with 4 weeks group P=0.001;24 weeks group compared with 12 weeks group P<0.001）.5.Pearson correlation analysis between claudin-3 protein expression and plasma D-lactic acid content showed a high negative correlation（r=-0.909,P<0.001）.Conclusion:This study suggests CCH can cause long-term persistent intestinal mucosal barrier injury in rats,down-regulation of tight junction protein claudin-3 in intestinal mucosal epithelial cells and increase in intestinal barrier permeability.There is a high negative relationship between claudin-3 expression level and intestinal barrier permeability.The result suggests that elevated plasma D-lactic acid content and down-regulation of claudin-3expression may be an early warning index and an important pathological marker for increased intestinal permeability and intestinal mucosal damage caused by CCH.It provides new targets and new directions for the treatment and prognosis of CCH-related neurodegenerative diseases and vascular cognitive disorders. |
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