米诺环素对大鼠脑出血后细胞凋亡及Dickkopf-1、B细胞淋巴瘤/白血病-相关X蛋白和B细胞淋巴瘤/白血病表达的影响

Effects of minocycline on apoptosis and expression of Dickkopf -1,B cell lymphoma/leukemia -2 associated X protein and B cell lymphoma/leukemia -2 in rats with intracerebral hemorrhage

目的 观察米诺环素对大鼠脑出血后神经细胞凋亡及Dickkopf-1(DKK-1)、B细胞淋巴瘤/白血病-2相关X蛋白(bax)和B细胞淋巴瘤/白血病-2(bcl-2)表达的影响。方法采用随机数字法将54只成年雄性SD大鼠分成假手术组(n=18)、脑出血组(n=18)及米诺环素组(n=18)。通过颅内注射Ⅶ型胶原酶制作脑出血模型。采用平衡木行走实验、转身实验进行脑出血模型评估,并利用免疫组织化学检测大鼠脑组织中bcl-2、bax及DKK-1蛋白表达水平。利用原位缺口末端标记法(TUNEL)凋亡实验检测大鼠脑出血周围脑组织的细胞凋亡水平。结果脑出血组(4.11±0.75)及米诺环素组(4.28±0.86)大鼠平衡木行走得分及转身实验百分比均高于假手术组(0.50±0.51,P<0.01)。与假手术组比较,各时间点(1、3、7 d)脑出血组的大鼠脑出血部位周围脑组织bcl-2(P<0.05)、bax(均P<0.01)及DKK-1(均P<0.01)表达升高;与假手术组比较,各时间点(1、3、7 d)脑出血组的大鼠脑出血部位周围脑组织bcl-2(36.50±1.54、49.15±2.71、18.76±1.36,P<0.05),bax(41.75±2.46、62.15±1.24、26.54±3.86,P<0.05)及DKK-1(56.48±3.25、70.74±3.23、41.12±2.50,P<0.05)表达也升高。而与脑出血组比较,米诺环素组大鼠出血部位周围脑组织bcl-2表达升高(P<0.05),而bax(P<0.05,P>0.05,P<0.05)及DKK-1(P<0.05)表达下降。与假手术组比较,各时间点(1、3、7 d)脑出血组大鼠出血部位周围脑组织凋亡细胞镜下计数升高[(32.54±5.23)、(45.64±4.84)、(28.26±2.79)个,均P<0.01],而经米诺环素治疗后脑出血部位周围脑组织凋亡细胞显著下降[(15.43±3.04)、(23.15±2.49)、(17.68±4.54)个,P<0.05],同时伴有行为学功能改善:转身运动测验评分(1、3、7 d)优于脑出血组(均P<0.01)。结论 米诺环素通过减少神经细胞凋亡对大鼠脑出血发挥脑保护作用,其分子机制可能是通过减少DKK-1表达来促进bcl-2和抑制bax表达有关。

Objective To observe the effects of minocycline on neuronal apoptosis and expression of Dickkopf-1 (DKK-1), B cell lymphoma/leukemia-2 associated X protein (bax) and B cell lymphoma/leukemia-2 (bcl-2) after intracerebral hemorrhage in rats.Methods Fifty-four adult male SD rats were randomly divided into sham operation group (n=18), cerebral hemorrhage group (n=18) and minocycline group (n=18). Intracerebral injection of type VII collagenase was used to make cerebral hemorrhage model. The model of cerebral hemorrhage was evaluated by balance beam walking test and turn-around test. The expression levels of bcl-2, bax and DKK-1 proteins in rat brain tissues were detected by immunohistochemistry. Apoptosis of brain tissue around intracerebral hemorrhage in rats was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) apoptosis assay.Results The walking score and turning percentage of balance beam in cerebral hemorrhage group (4.11±0.75) and minocycline group (4.28±0.86) were significantly higher than those in sham operation group (0.50±0.51, P<0.01). As compared with sham operation group, the expression of bcl-2 (36.50±1.54, 49.15±2.71, 18.76±1.36, P<0.05), bax (41.75±2.46, 62.15±1.24, 26.54±3.86, P<0.01) and DKK-1 (56.48±3.25, 70.74±3.23, 41.12±2.50, P<0.01) in brain tissue around the site of intracerebral hemorrhage of rats at each time point (1st, 3rd, and 7th day) significantly increased. As compared with sham operation group, the expression of bcl-2 (P<0.05), bax (P<0.01) and DKK-1 (P<0.01) in brain tissue around the site of intracerebral hemorrhage of rats at each time point were also increased. As compared with the cerebral hemorrhage group, the expression of bcl-2 in the brain tissue around the hemorrhage site increased (P<0.05), and that of bax (P<0.05, P>0.05, P<0.05 1st, 3rd, and 7th day respectively) and DKK-1 (P<0.05) decreased in minocycline group. As compared with sham operation group, the number of apoptotic cells in brain tissue around hemorrhagic site was significantly increased at each time point (1st, 3rd, and 7th day) in intracerebral hemorrhage group [(32.54±5.23), (45.64±4.84), (28.26±2.79) cells, all P<0.01], and that in brain tissue around hemorrhagic site decreased significantly in minocycline group [(15.43±3.04), (23.15±2.49), (17.68±4.54) cells, P<0.05]. The scores of turn-around test (1st, 3rd, and 7th day) in minocycline group was better significantly higher or lower than that in cerebral hemorrhage group (P<0.01).Conclusion Minocycline plays a protective role in rats with cerebral hemorrhage by reducing neuronal apoptosis. Its molecular mechanism may be related to the promotion of bcl-2 and the inhibition of bax expression by reducing DKK-1 expression.