INT-777对颅脑损伤小鼠脑水肿及血脑屏障通透性的影响

Effects of INT-777 on cerebral edema and blood brain barrier permeability in mice with traumatic brain injury

目的探讨G蛋白偶联胆汁酸受体激动剂6R-乙基-23(S)-甲基胆酸(INT-777)对小鼠创伤性颅脑损伤(TBI)后脑水肿及血脑屏障(BBB)通透性的影响。方法52只健康雄性C57BL/6J小鼠按照随机数字表法分为假手术组(Sham组)、颅脑创伤组(TBI组)、DMSO治疗组(TBI+DMSO组)和INT-777治疗组(TBI+INT-777组),每组13只。Sham组仅开骨窗不做处理;TBI组、TBI+DMSO组和TBI+INT-777组开骨窗后采用液压颅脑打击仪建立小鼠TBI模型,TBI组只建模但不干预,TBI+DMSO组和TBI+INT-777组分别经鼻给予10%DMSO(30μL)和INT-777(30μg/kg)后建立模型。建模后48 h,采用干湿质量法测量损伤区以及对侧脑组织含水量;伊文思蓝(EB)外渗法测定BBB通透性;Western blot测定脑组织中基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、闭锁小带蛋白1(ZO-1)、闭合蛋白(Occludin)和紧密连结蛋白-5(Claudin-5)表达水平。结果TBI后48 h,与Sham组比较,其他3组脑组织含水量(损伤侧)和EB含量均显著升高,脑组织中MMP-2和MMP-9蛋白表达上调,ZO-1、Claudin-5和Occludin蛋白表达下降,差异均有统计学意义(P<0.05);与TBI组和TBI+DMSO组比较,TBI+INT-777组脑组织含水量和EB含量显著降低,脑组织中MMP-2和MMP-9蛋白表达下调,ZO-1、Claudin-5和Occludin蛋白表达升高,差异均有统计学意义(P<0.05)。结论INT-777可减轻小鼠TBI后脑水肿,改善BBB的破坏,其机制可能与抑制伤后脑组织MMP-2、MMP-9的表达以及ZO-1、Claudin-5和Occludin的下调有关。

Objective To investigate the effects of G protein coupled bile acid receptor agonist INT-777 on brain edema and blood brain barrier(BBB)permeability after traumatic brain injury(TBI)in mice.Methods A total of 52 healthy male C57BL/6J mice were randomly divided into sham operation group(Sham group),craniocranial trauma group(TBI group),DMSO treatment group(TBI+DMSO group)and INT-777 treatment group(TBI+INT-777 group),with 13 rats in each group.In Sham group,only had the bone window open without treatment.TBI model was established with a brain fluid-percussion instrument in TBI group,TBI+DMSO group and TBI+INT-777 group after the bone window was open.Among them,the TBI group was modeled without intervention,and the TBI+DMSO group and TBI+INT-777 group were modeled after given 10%DMSO(30μL)and INT-777(30μg/kg)by transnasal administration,respectively.48 h after modeling,the water content of the injured area and contralateral brain tissue was measured by dry-wet weight method.The blood-brain barrier permeability was measured by Evans-blue(EB)extravasation method,and the protein expression levels of MMP-2,MMP-9,ZO-1,Claudin-5 and Occludin in brain tissue were measured by Western blot.Results 48 h after TBI,compared with Sham group,the water content(on the injured side)and extravasation of EB in brain tissue in the other three groups were significantly increased,the protein expressions of MMP-2 and MMP-9 in brain tissue were up-regulated,and the protein expressions of ZO-1,Claudin-5 and Occludin were decreased,the difference was statistically significant(P<0.05).Compared with TBI group and TBI+DMSO group,brain tissue water content and EB extravasation in TBI+INT-777 group were significantly decreased,the expression of MMP-2 and MMP-9 in brain tissue was down-regulated,and the expression of ZO-1,Claudin-5 and Occludin in brain tissue was increased,the difference was statistically significant(P<0.05).Conclusion INT-777 can reduce the cerebral edema after TBI in mice and improve the damage of the blood-brain barrier.The mechanism may be related to the inhibition of MMP-2 and MMP-9 expression and the down-regulation of ZO-1,Claudin-5 and Occludin in brain tissue after injury.