INT-777对颅脑损伤小鼠脑水肿及血脑屏障通透性的影响

doi:10.3877/cma.j.issn.2095-9141.2021.06.002

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（30 μL）和INT-777（30 μg/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。

关键词: 颅脑损伤, INT-777, 脑水肿, 血脑屏障

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 (30 μL) and INT-777 (30 μg/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.

Key words: Trauma brain injury, INT-777, Brain edema, Blood brain barrier

表1 各组小鼠脑损伤后48 h脑组织含水量、EB含量比较（±s）

组别	只数	脑组织含水量（%）		EB含量（μg/g）
组别	只数	健侧	伤侧	EB含量（μg/g）
Sham组	5	74.58±0.22	74.62±0.41	2.71±1.44
TBI组	5	74.91±0.31	82.02±0.33^a	10.97±1.04^a
TBI+DMSO组	5	74.87±0.29	81.87±0.28^a	11.20±0.62^a
TBI+INT-777组	5	74.80±0.27	78.09±0.46^abc	7.53±0.61^abc
F值		1.496	436.653	80.705
P值		0.254	<0.001	<0.001

表1 各组小鼠脑损伤后48 h脑组织含水量、EB含量比较（±s）

组别	只数	脑组织含水量（%）		EB含量（μg/g）
组别	只数	健侧	伤侧	EB含量（μg/g）
Sham组	5	74.58±0.22	74.62±0.41	2.71±1.44
TBI组	5	74.91±0.31	82.02±0.33^a	10.97±1.04^a
TBI+DMSO组	5	74.87±0.29	81.87±0.28^a	11.20±0.62^a
TBI+INT-777组	5	74.80±0.27	78.09±0.46^abc	7.53±0.61^abc
F值		1.496	436.653	80.705
P值		0.254	<0.001	<0.001

图1 各组小鼠脑损伤后48 h脑组织血脑屏障通透性检测A：Sham组；B：TBI组；C：TBI+DMSO组；D：TBI+INT-777组

图2 各组小鼠脑损伤后MMP-2、MMP-9蛋白表达变化A：Western blot电泳图；B：各组小鼠MMP-2、MMP-9蛋白表达比较；与Sham组比较，^aP<0.05；与TBI组比较，^bP<0.05；与TBI+DMSO组比较，^cP<0.05；MMP-2：基质金属蛋白酶-2；MMP-9：基质金属蛋白酶-9

图3 各组脑组织中Claudin-5、Occludin和ZO-1蛋白表达变化A：Western blot电泳图；B：4组的Claudin-5、Occludin和ZO-1的蛋白表达比较；与Sham组比较，^aP<0.05；与TBI组比较，^bP<0.05；与TBI+DMSO组比较，^cP<0.05；ZO-1：闭锁小带蛋白1；Occludin：闭合蛋白；Claudin-5：紧密连结蛋白-5

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

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