慢性间断性酒精暴露大鼠海马组织MMP-9的表达及神经元突触超微结构改变

Effects of chronic intermittent ethanol exposure on expression of MMP-9 and synaptic ultrastructure in rat hippocampus

目的探讨慢性间断性酒精暴露(CIE)大鼠海马组织中MMP-9的表达水平及海马区突触超微结构的改变。方法将42只6周龄雄性SD大鼠随机分为染毒组和对照组,每组21只。染毒组大鼠间断性灌胃给予酒精4 g/(kg·d),灌胃2 d,戒断2 d,直至第28天。对照组大鼠给予等热量麦芽糖7 g/(kg·d)。实验期间测量大鼠体重、顶空气相色谱法测定血清酒精浓度。第0、26和28天时,每组随机处死7只大鼠,取海马组织,实时荧光定量PCR、免疫蛋白印迹法检测海马组织MMP-9 mRNA及蛋白的表达水平,透射电镜观察海马区突触超微结构并定量分析突触结构参数。结果染毒组大鼠血清酒精浓度达到810 mg/L,对照组血清未检出。暴露第26和28天时,与对照组相比,染毒组海马中MMP-9 m RNA的表达水平明显升高(P<0.05)。染毒组海马中MMP-9蛋白的表达量显著高于对照组(P<0.05),active MMP-9的表达量在第26和第28天时分别升高2.46倍和2.16倍,pro MMP-9蛋白表达量第26和第28天时分别升高1.65倍和1.49倍,总体(total)-MMP-9蛋白表达量在第26和第28天时较对照组分别升高2.07倍和1.82倍。透射电镜结果显示,与对照组相比,染毒组海马突触后膜致密物厚度、突触界面曲率明显增加(P<0.05),突触间隙略增大,但差异无统计学意义。结论慢性间断性酒精暴露可能通过改变突触超微结构从而导致大鼠海马区MMP-9表达升高。本研究为探索MMP-9在酒精依赖过程中的作用提供了新的实验依据。

Objective To understand the effect of chronic intermittent ethanol exposure(CIE) on the expression of matrix metalloproteinase-9(MMP-9) and synaptic ultrastructure in rat hippocampus. Methods A total of 42 male Sprague-Dawley rats were randomly divided into ethanol-treated group and control group,21 in each. The rats in ethanol-treated group were treated with ethanol [ 4 g/(kg·d)] through gavage,two days with a 2-day interval,continuously for 28 days,and the control group rats were treated with maltose [7 g/(kg·d) ]. The weight and serum ethanol concentration of rats were analyzed during the experiment. Following that the samples of hippocampus tissue of seven rats in each group were collected on days 0,26 and28 respectively. The mRNA and protein levels of MMP-9 were detected by real-time PCR and Western blot,and possible synaptic changes in hippocampus were analyzed by transmission electron microscopy. Results The serum ethanol concentration of rats reached 810 mg/L in the ethanol-treated group. Compare with control group,MMP-9 mRNA level in hippocampus were significantly higher on days 26 and 28(P<0.05) in ethanol-treated group. Compared with control group,the protein expression of MMP-9 increased in ethanol-treated group(P<0.05),the protein expression of active MMP-9 increased by 2.46 and 2.16 times on days 26 and 28,the protein expression of pro MMP-9 increased by 1.65 and 1.49 times on days 26 and 28,the protein expression of total MMP-9 increased by 2.07 and 1.82 times on days 26 and 28 respectively. In the CIE model,increased thickness of postsynaptic density(PSD) and synaptic curvature were observed in hippocampus by transmission electron microscope(P<0.05). Otherwise,the synaptic cleft width increased slightly with no statistical significance. Conclusion Chronic intermittent ethanol exposure can increase the expression of MMP-9 and cause synaptic ultrastructure changes in rat hippocampus. The finding suggests that MMP-9 participates in chronic ethanol exposure and ethanol dependence process through synaptic ultrastructures changes.