慢性脑低灌注对大鼠认知功能和肠黏膜屏障的影响

Effects of chronic cerebral hypoperfusion on cognitive function and intestinal mucosal barrier in rats

目的:探讨慢性脑低灌注(chronic cerebral hypoperfusion,CCH)后大鼠认知障碍及肠黏膜屏障损伤之间的相关性,量化分析慢性脑低灌注所致实验大鼠认知行为改变,以及肠黏膜屏障紧密连接蛋白claudin-1与骨桥蛋白(osteopontin,OPN)的表达变化。方法:雄性SD大鼠30只,按照随机数字表法分为慢性脑低灌注组(CCH组)和假手术组(SHAM组),每组15只大鼠。通过双侧颈总动脉永久性结扎法建立CCH模型,SHAM组大鼠只分离颈总动脉不结扎。4周后采用旷场实验、物体辨别实验和Morris水迷宫实验检测大鼠的情绪唤醒能力,对新事物的探索能力及空间学习记忆能力。HE染色及免疫荧光染色实验检测大鼠回肠组织损伤情况,免疫蛋白印迹实验检测回肠组织OPN表达水平,ELISA实验检测大鼠血清OPN水平。采用SPSS 23.0及GraphPad 8.0统计软件处理数据,采用t检验及重复测量方差分析等方法进行数据分析。结果:旷场实验:与SHAM组[(28.70±10.70)次,(1030.45±81.51)cm]比较,CCH组大鼠站立次数和运动总路程[(16.70±7.13)次,(736.64±136.71)cm]减少,差异有统计学意义(t=1.59,4.16,均P<0.05);物体辨别实验:CCH组大鼠的辨别指数(0.44±0.26)低于SHAM组(0.91±0.07),差异有统计学意义(t=-7.76,P<0.05);Morris水迷宫定位航行实验显示,组别和时间主效应均显著(F=383.36,153.87,P<0.05)。简单效应分析显示,与SHAM组比较,CCH组大鼠逃避潜伏期与游泳总路程增加(均P<0.05);空间探索实验显示,与SHAM组[(7.20±1.81)次,(9.96±2.95)s]比较,CCH组大鼠穿越次数[(3.00±0.82)次]减少,穿越潜伏期[(29.70±6.28)s]延长,差异有统计学意义(t=4.65,7.04,均P<0.05)。肠道黏膜病理评分,与SHAM组[(1.98±0.34)分]比较,CCH组评分[(4.52±0.27)分]更高,肠道黏膜损伤更重(t=18.53,P<0.01);免疫荧光实验显示,与SHAM组(125028.58±33077.39)比较,CCH组肠道上皮细胞间的claudin-1累计光密度(47154.50±7507.29)降低(t=-16.10,P<0.01);免疫蛋白印迹实验,与SHAM组(0.38±0.11)比较,CCH组肠道OPN表达含量(1.20±0.95)增加(P<0.05);ELISA实验,与SHAM组[(3.42±0.66)μg/L]比较,CCH组血清OPN含量[(14.92±1.45)μg/L]明显增加(P<0.05)。认知受损程度与肠黏膜上皮claudin-1表达量、血清OPN含量间均呈负相关(均P<0.01);肠黏膜上皮claudin-1表达量与血清OPN含量呈负相关(r=-0.952,P<0.01)。结论:CCH可引发明显的大鼠认知功能受损及肠道黏膜屏障的破坏,血清OPN或可作为CCH致大鼠认知损伤及肠道黏膜屏障破坏的潜在血清学标志物。

Objective To explore the correlation between cognitive impairment and intestinal mucosal barrier injury in rats after chronic cerebral hypoperfusion(CCH),and to quantitatively analyze the changes in cognitive behavior of experimental rats caused by chronic cerebral hypoperfusion,as well as the expression changes of the intestinal mucosal barrier claudin-1 and osteopontin.Methods Thirty male SD rats were randomly divided into CCH group(n=15)and sham operation(SHAM)control group(n=15).The CCH model was established by permanent ligation of bilateral common carotid arteries.Rats in the SHAM group only separated the common carotid artery without ligation.Four weeks later,open field experiment,object discrimination experiment,and Morris water maze experiment were used to detect the emotional arousal ability,the ability to explore new things,and the ability of spatial learning and memory in rats.HE staining and immunofluorescence experiments were conducted to detect the damage of rat ileum tissue.Western blot was used to detect OPN expression,and ELISA was used to detect serum OPN.SPSS 23.0 and GraphPad 8.0 statistical softwares were used to process the data,and the t-test and repeated measures one-way analysis of variance were used for data analysis.Results In the open field test,compared with the SHAM group((28.70±10.70)times,(1030.45±81.51)cm),the number of standing and total exercise distance of rats in the CCH group((16.70±7.13)times,(736.64±136.71)cm)were decreased(t=1.59,4.16,both P<0.05).In the object discrimination experiment,the discrimination index of rats in the CCH group(0.44±0.26)was lower than that of the SHAM group(0.91±0.07,t=-7.76,P<0.05).Morris water maze positioning navigation experiment showed that the group main effect and time main effect were both significant(F=383.36,153.87,P<0.05).Simple effect analysis showed that,compared with the SHAM group,the escape latency and total swimming distance of rats in CCH group increased(P<0.05).Space exploration experiment showed that,compared with SHAM group((7.20±1.81)times,(9.96±2.95)s),the number of crossings of rats in CCH group((3.00±0.82)times)decreased,and the incubation period((29.70±6.28)s)was prolonged(t=4.65,7.04,both P<0.05).The intestinal mucosal pathology score of SHAM group((1.98±0.34)points)was lower than that of the CCH group((4.52±0.27)points),and the difference was significant(t=18.53,P<0.01).Immunofluorescence experiment showed that,compared with SHAM group(125028.58±33077.39),the cumulative optical density of claudin-1 between the intestinal epithelial cells of the CCH group(47154.50±7507.29)decreased(t=16.10,P<0.01).Western blot experiment showed that,compared with the SHAM group(0.38±0.11),the expression of OPN in the intestines of the CCH group(1.20±0.95)increased(P<0.05).ELISA experiment showed that,compared with the SHAM group((3.42±0.66)μg/L),the serum OPN content of the CCH group((14.92±1.45)μg/L)significantly increased(P<0.05).The degree of cognitive impairment was negatively correlated with intestinal mucosal epithelial claudin-1 expression and serum OPN content(P<0.01).Intestinal mucosal epithelial claudin-1 expression was negatively correlated with serum OPN content(r=-0.952,P<0.01).Conclusion CCH may cause obvious cognitive impairment in rats and the destruction of the intestinal mucosal barrier.Serum OPN may be a potential serological marker of CCH-induced cognitive impairment and intestinal mucosal barrier destruction in rats.