健脑解郁方对脑小血管病并发抑郁症模型大鼠不同脑区类淋巴系统功能的保护效应

Protective Effect of Jiannao Jieyu(健脑解郁)Formula on the Glymphatic System Function in Different Brain Regions of the Rat Model of Cerebral Small Vessel Disease Complicated with Depression

目的:探讨健脑解郁方改善脑小血管病并发抑郁症(cerebral small vessel disease related depression,CSVDD)大鼠前额叶皮质和海马类淋巴系统(glymphatic system,GS)功能障碍的潜在作用机制。方法:将6只WKY大鼠分为正常对照组,30只SHR大鼠随机分为:模型对照组、阳性药对照组尼莫地平(21.6 mg/kg)+丁苯酞(54 mg/kg)+氟西汀(5.4 mg/kg)组、健脑解郁方7.02、14.04、28.08 g/kg组,每组6只;大鼠背部皮下注射D-半乳糖400 mg/kg,连续8 w联合双侧颈总动脉狭窄(bilateral common carotid artery stenosis,BCAS)4 w,再用慢性不可预见性温和应激(chronic unpredicted mild stress,CUMS)加孤笼饲养3 w,建立CSVDD大鼠模型。采用Morris水迷宫试验评价大鼠认知记忆能力;激光散斑成像监测大鼠脑血流量;ELISA法检测大鼠血脑屏障(BBB)情况;HE染色观察大鼠脑组织病理情况,小脑延髓池注射观测类淋巴系统流入功能;免疫荧光技术检测脑组织中留存的淀粉样前体蛋白(APP)含量,评估类淋巴系统流出功能;免疫荧光技术检测脑组织中水通道蛋白-4(AQP4)表达水平。结果:与正常对照组比较,模型对照组大鼠逃避潜伏期明显延长,目标象限游动时间显著缩短,目标象限潜伏期显著升高(P<0.01),大鼠脑血流量显著降低,脑脊液/血清白蛋白商值(CALB/SALB)显著升高,大鼠出现明显的血管周围间隙扩大(P<0.01),第三脑室背侧出血及轻微脑出血,大鼠前额叶皮质及海马冠状面全脑荧光面积分数降低(P<0.05或P<0.01),大鼠前额叶皮质、海马CA1、CA2、CA3、DG中APP的荧光强度显著升高(P<0.01),大鼠前额叶皮质、海马CA1、CA2、CA3、DG中AQP4蛋白水平显著降低(P<0.01);与模型对照组比较,健脑解郁方组大鼠逃避潜伏期和目标象限潜伏期均明显缩短(P<0.05或P<0.01),目标象限游动时间显著延长,脑血流量显著升高,CALB/SALB比值显著降低,脑区血管周围间隙扩大情况明显减少,第三脑室背侧出血降低,前额叶皮质和海马的全脑荧光面积分数显著升高(P<0.01),APP荧光强度明显降低(P<0.05或P<0.01),AQP4蛋白表达显著升高(P<0.01)。结论:健脑解郁方可能通过降低血脑屏障损伤和出血情况,促进脑血流量,加强脑区代谢物流入功能和脑内代谢废物的排除,以降低脑组织淋巴系统免疫炎症反应,改善CSVDD模型大鼠的认知功能和类淋巴系统功能障碍。

Objective:To explore the potential mechanism of Jiannao Jieyu(健脑解郁)Formula(JNJYF)in ameliorating the dysfunction of prefrontal cortex and hippocampal glymphatic system in the rat model of cerebral small vessel disease complicated with depression(CSVDD).Methods:Six WKY rats were assigned into a control group,and 30 SHR rats were randomized into model control,positive control[nimodipine(21.6 mg/kg)+butylphthalide(54 mg/kg)+fluoxetine(5.4 mg/kg)],and JNJYF(7.02,14.04,and 28.08 g/kg)groups(n=6).The rat model of CSVDD was established by subcutaneous injection of D-galactose(400 mg/kg)at the back combined with bilateral common carotid artery stenosis for 4 weeks,which was followed by chronic unpredicted mild stress and solitary caging for 3 weeks.The Morris water maze test was carried out to evaluate the cognitive ability and memory of rats.Laser speckle imaging was employed to monitor the cerebral blood flow in rats.The blood brain barrier(BBB)of rats was examined by enzyme-linked immunosorbent assay.Hematoxylin-eosin staining was employed to observe the pathological changes of the brain tissue in rats,and the inflow function of the glymphatic system was observed by cerebellomedullary cistern injection.The immunofluorescence assay was used to measure the content of amyloid precursor protein(APP)in the brain tissue to evaluate the outflow function of the glymphatic system.The level of aquaporin 4(AQP4)in the brain tissue was detected by the immunofluorescence assay.Results:Compared with the control group,the model control group showed prolonged escape latency and target quadrant latency,shortened swimming time in the target quadrant,reduced cerebral blood flow,increased cerebrospinal fluid albumin(CALB)/serum albumin(SALB)ratio,and enlargement of perivascular space in the brain region(P<0.01).In addition,the model control group showed hemorrhage in the back of the third ventricle,reduced fluorescence area percentages of prefrontal cortex and hippocampus(P<0.05 or P<0.01),and enhanced fluorescence intensity of APP and lowered level of AQP in the prefrontal cortex and the CA1,CA2,CA3,and DG regions of the hippocampus(P<0.01).Compared with the model control group,the JNJYF groups showed shortened escape latency and target quadrant latency(P<0.05 or P<0.01),prolonged swimming time in the target quadrant(P<0.01),increased cerebral blood flow(P<0.01),decreased CALB/SALB ratio(P<0.01),and reduced enlargement of perivascular space in the brain region(P<0.01).In addition,JNJYF reduced the hemorrhage in the back of the third ventricle,increased the fluorescence area percentages of prefrontal cortex and hippocampus(P<0.01),decreased the fluorescence intensity of APP(P<0.05 or P<0.01),and elevated the level of AQP4(P<0.01).Conclusion:JNJYF may alleviate the damage and bleeding of BBB,promote cerebral blood flow,and strengthen the inflow function of metabolites and outflow of metabolic waste to reduce the immune inflammation of the glymphatic system in the brain tissue,thereby improving the cognitive function and ameliorating the glymphatic system dysfunction in the rat model of CSVDD.