纳络酮预处理对脑出血大鼠脑组织水通道蛋白4及基质金属蛋白酶9表达的影响

Naloxone hydrochloride preconditioning suppresses expressions of aquaporin protein-4 and matrix metalloproteinase-9 in rat brain tissue around cerebral hemorrhagic focus

目的探讨纳络酮预处理对脑出血大鼠的保护效应及相关作用机制。方法将30只雄性Sprague-Dawley大鼠(体质量280～350g)按随机数字表法随机分为假手术对照组(Sham组)、脑出血(intracere bralhemorrhage,ICH)组和纳络酮预处理(Naloxone preconditioning,NP)组,每组10只大鼠。复制上述动物模型之前,NP组大鼠预先给予盐酸纳络酮(2.0mg/kg),ICH组给予相同体积的生理盐水,随后制备ICH模型。切取并收集大鼠脑出血灶周围脑组织,分别采用RT-PCR和Westernblot检测大鼠病变脑组织中水通道蛋白(aquaporin protein4,AQP4)在转录水平和蛋白水平上的变化情况;同时采用免疫组织化学检测大鼠病变脑组织中基质金属蛋白酶(matrix metalloproteinase9,MMP-9)的阳性表达情况。结果相对于Sham组,ICH组大鼠脑组织中含水量明显增加(P<0.05),而经过纳络酮预处理后,大鼠脑组织中含水量明显减少(P<0.05);相对于ICH组,NP可有效降低脑出血大鼠脑组织中AQP4mRNA和蛋白水平(P<0.01);同时,NP组大鼠脑组织中MMP-9阳性表达率明显下调(P<0.01)。结论纳络酮作为脑出血的一种有效的保护措施,其具体作用机制可能与其降低脑组织中AQP4的表达,从而减少脑水肿的发生及下调的MMP-9水平密切相关。

Objective To investigate the protective effect and mechanism of naloxone preconditioning （ NP） for rats with cerebral hemorrhage. Methods Totally 30 healthy male Sprague Dawley （ SD） rats were randomly separated into Sham operatopm group,intracerebral hemorrhage （ ICH） and NP treatment groups （ n =10） . Before the establishment of rat cerebral hemorrhage model,rats in the NP group were given an intraperitoneal injection of naloxone hydrochloride （ 2. 0 mg/kg） ,and those of ICH group received an injection of normal saline at same volume. Then,the ICH model was made by injecting 0. 4 IU collagen Ⅶ into caudate nucleus while. The brain tissue samples around cerebral hemorrhagic focus after ICH were collected. RT-PCR and Western blotting were used to detect the transcriptional level and protein level of aquaporin protein 4 （ AQP4） in the brain tissue,respectively. Meanwhile,immunohistochemistry was employed to determine the expression of matrix metalloproteinase 9 （ MMP-9） in the rat brain tissue. Results Brain water content in ICH group was larger than that in Sham group （ P〈0. 05） . However,NP preconditioning effectively decreased the brain water content （ P〈0. 05） compared with the other 2 groups. Compared with the ICH group,NP group had significantly decreased mRNA and protein levels of AQP4 in brain tissue （ P〈0. 0,P〈0. 01） . Meanwhile,the positive expression percentage of MMP-9 in the brain tissue was also significantly down-regulated after naloxone hydrochloride preconditioning. Conclusion Naloxone hydrochloride preconditioning exerts its effective protective effect by decreasing AQP4 expression at mRNA and protein levels and thus leads to attenuate and down-regulate MMP-9 in the ICH brain tissue.