摘要
目的:探讨精神分裂症(schizophrenia,SZ)的髓鞘和脑组织间液(interstitial fluid,ISF)相关发病机制,探究熊果酸(ursolic acid,UA)对SZ中髓鞘损伤及其继发的ISF异常引流的治疗效果.方法:使用30只6～8周雌性C57BL/6J小鼠,体质量(20±2)g,随机分为UA治疗组、SZ模型组、对照组3组,每组10只:(1)对照组:腹腔注射(intraperitoneal injection,ip)生理盐水,灌胃(intragastric,ig)给予 1％(质量分数)羧甲基纤维素钠(carboxymethyl-cellulose sodium,CMC-Na);(2)SZ 模型组:ip 给与 2 mg/kg 的地卓西平(dizocilpine maleate,MK-801),ig 给与 1％(质量分数)CMC-Na;(3)UA治疗组:ig给与25 mg/kg的UA,ip给与2 mg/kg的MK-801.治疗组先ig给药UA预治疗一周,然后对3组小鼠进行为期两周的药物干预.在造模完成后依次通过旷场测试和前脉冲抑制实验对小鼠进行行为学评价.行为测试后,通过向脑区注射荧光示踪剂来探究各组ISF分区引流的改变;通过免疫荧光探究各组脑内水通道蛋白4(aquaporin 4,AQP4)极性分布的改变以及蛋白表达的变化;通过激光共聚焦显微镜(laser scanning confocal microscope,LSCM)髓鞘反射光成像研究鼠脑内髓鞘的变化.采用单因素方差分析(one-way ANO-VA)对计量数据进行组间比较,使用TukeyHSD进行组间两两比较.结果:旷场测试发现,模型组在旷场中运动的总路程[(7 949.39±1 140.55)cm vs.(2 831.01±1 212.72)cm,P＜0.001]和中央区域停留时间[(88.43±22.06)svs.(56.85±18.58)s,P=0.011]显著高于对照组,而治疗组在旷场中运动总路程[(2 415.80±646.95)cm vs.(7 949.39±1 140.55)cm,P＜0.001]和中央区域停留时间[(54.78±11.66)s vs.(88.43±22.06)s,P=0.007]较模型组显著降低.前脉冲抑制实验发现,模型组给与预脉冲时对震惊反射的抑制率均显著低于对照组(P均＜0.001);治疗组上述指标较模型组显著增加(P均＜0.001).髓鞘反射光检测发现,模型组小鼠脑内存在显著的脱髓鞘,治疗组脑内脱髓鞘情况得到逆转.荧光示踪发现,模型组示踪剂向头侧皮层区的扩散面积和向尾侧丘脑区的返流面积均显著大于对照组[(13.93±3.35)mm2 vs.(2.79±0.94)mm2,P＜0.001;(2.48±0.38)mm2 vs.(0.05±0.12)mm2,P＜0.001],且脑区间引流分区明显破坏;治疗组示踪剂向头侧皮层区的扩散面积和向尾侧丘脑区的返流面积均较模型组显著减小[(7.93±2.48)mm2 vs.(13.93±3.35)mm2,P＜0.001;(0.50±0.30)mm2 vs.(2.48±0.38)mm2,P＜0.001].免疫荧光染色发现,模型组小鼠脑内AQP4极性分布遭到破坏,且模型组AQP4蛋白表达量较对照组明显下降[(3 663.88±733.77)p.m2 vs.(13 354.92±4 054.05)μm2,P＜0.001];治疗组较模型组 AQP4 极性分布得到改善,AQP4蛋白表达量较模型组显著升高[(11 104.68±3 200.04)μm2vs.(3 663.88±733.77)μm2,P＜0.001].结论:一定剂量的UA干预可以改善SZ小鼠的行为学表现,这种改善表现为运动亢进和焦虑症状得到缓解,感觉运动门控功能得到恢复;其机制可能是通过改善SZ小鼠的脱髓鞘病理改变及ISF分区引流紊乱.
Abstract
Objective:To unveil the pathological changes associated with demyelination in schizophre-nia(SZ)and its consequential impact on interstitial fluid(ISF)drainage,and to investigate the thera-peutic efficacy of ursolic acid(UA)in treating demyelination and the ensuing abnormalities in ISF drainage in SZ.Methods:Female C57BL/6J mice,aged 6-8 weeks and weighing(20±2)g,were randomly divided into three groups:control,SZ model,and UA treatment.The control group received intraperitoneal injection(ip)of physiological saline and intragastric administration(ig)of 1％carboxy-methylcellulose sodium(CMC-Na).The SZ model group was subjected to ip injection of 2 mg/kg dizo-cilpine maleate(MK-801)and ig administration of 1％CMC-Na.The UA treatment group underwent ig administration of 25 mg/kg UA and ip injection of 2 mg/kg MK-801.The treatment group received UA pretreatment via ig administration for one week,followed by a two-week drug intervention for all the three groups.Behavioral assessments,including the open field test and prepulse inhibition experiment,were conducted post-modeling.Subsequently,changes in the ISF partition drainage were investigated through fluorescent tracer injection into specific brain regions.Immunofluorescence analysis was employed to examine alterations in aquaporin 4(AQP4)polarity distribution in the brain and changes in protein expres-sion.Myelin reflex imaging using Laser Scanning Confocal Microscopy(LSCM)was utilized to study modifications in myelin within the mouse brain.Quantitative data underwent one-way ANOVA,followed by TukeyHSD for post hoc pairwise comparisons between the groups.Results:The open field test re-vealed a significantly longer total distance[(7 949.39±1 140.55)cm vs.(2 831.01±1 212.72)cm,P＜0.001]and increased central area duration[(88.43±22.06)s vs.(56.85±18.58)s,P=0.011]for the SZ model group compared with the controls.The UA treatment group exhibited signifi-cantly reduced total distance[(2 415.80±646.95)cm vs.(7 949.39±1 140.55)cm,P＜0.001]and increased central area duration[(54.78±11.66)s vs.(88.43±22.06)s,P=0.007]compared with the model group.Prepulse inhibition test results demonstrated a markedly lower inhibition rate of the star-tle reflex in the model group relative to the controls(P＜0.001 for both),with the treatment group dis-playing significant improvement(P＜0.001 for both).Myelin sheath analysis indicated significant demy-elination in the model group,while UA treatment reversed this effect.Fluorescence tracing exhibited a significantly larger tracer diffusion area towards the rostral cortex and reflux area towards the caudal thala-mus in the model group relative to the controls[(13.93±3.35)mm2 vs.(2.79±0.94)mm2,P＜0.001 for diffusion area;(2.48±0.38)mm2 vs.(0.05±0.12)mm2,P＜0.001 for reflux area],with sig-nificant impairment of drainage in brain regions.The treatment group demonstrated significantly reduced tracer diffusion and reflux areas[(7.93±2.48)mm2 vs.(13.93±3.35)mm2,P＜0.001 for diffusion area;(0.50±0.30)mm2 vs.(2.48±0.38)mm2,P＜0.001 for reflux area].Immunofluorescence staining revealed disrupted AQP4 polarity distribution and reduced AQP4 protein expression in the model group compared with the controls[(3 663.88±733.77)μm2 vs.(13 354.92±4 054.05)μm2,P＜0.001].The treatment group exhibited restored AQP4 polarity distribution and elevated AQP4 protein expression[(11 104.68±3 200.04)μm2 vs.(3 663.88±733.77)μm2,P＜0.001].Conclusion:UA intervention ameliorates behavioral performance in SZ mice,Thus alleviating hyperactivity and anxiety symptoms and restoring sensorimotor gating function.The underlying mechanism may involve the improve-ment of demyelination and ISF drainage dysregulation in SZ mice.
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