橙皮素减轻胸主动脉瘤/夹层发生发展作用的实验研究

Experimental study on the treatment of thoracic aortic aneurysm/dissection by hesperetin

目的:探究橙皮素对胸主动脉瘤/夹层形成的作用。方法:利用β-氨基丙腈(BAPN)饮水28 d的方法构建小鼠主动脉瘤/夹层模型。选择C57BL/6小鼠20只,分为实验组(n=10)和对照组(n=10),实验组自第一天开始饮用含有BAPN(1 g·kg^-1·day^-1)的ddH2O,对照组饮用普通ddH2O。采用大体图和弹力纤维染色观察每周主动脉变化情况。②选取C57BL/6小鼠40只,分为橙皮素20 mg/kg组(n=10)、橙皮素50 mg/kg组(n=10)、对照组(n=10)。在第14天开始给药, 1次/d,实验组给予相应剂量橙皮素溶液,对照组给予等量0. 9%氯化钠溶液。实验结束后处死存活小鼠,统计TAAD发生率和病死率;大体图观察血管直观变化;采用弹力纤维染色观察胸主动脉弹力纤维板断裂情况。③荧光标记法检测橙皮素处理后Hep G2细胞上清中基质金属蛋白酶(MMPs)活性变化;原位荧光明胶酶染色检测小鼠主动脉组织上明胶酶(MMP-2、MMP-9)活性表达。结果:给予BAPN饮水28 d的小鼠生存曲线较普通饮水组,差异有统计学意义(P<0.001),且从第14 d天后的小鼠胸主动脉开始出现不同程度病理变化,并随着时间加重。橙皮素50 mg/kg实验组与0. 9%氯化钠溶液对照组相比,小鼠病死率和TAAD形成率明显下降,差异有统计学意义(P<0.05),且主动脉扩张程度减轻,弹力纤维紊乱与断裂有明显改善。20 mg/kg实验组也有类似效果,但不如前者明显。此外,橙皮素处理后的Hep G2细胞上清中MMPs活性明显下降(P<0.0001);实验组小鼠主动脉组织上明胶酶活性减弱。结论:橙皮素能够减轻主动脉瘤/夹层的发生发展,其机制可能与抑制基质金属蛋白酶活性有关。

Objective: To study the effect of hesperetin on thoracic aortic aneurysm and dissection in mice. Methods: A mouse aortic aneurysm/dissection model was established by drinking water with-aminopropionitrile(BAPN) for 28 days.Twenty C57 BL/6 mice were selected and divided into the experimental group(n=10) and the control group(n=10). The experimental group was given ddH2 O containing BAPN(1 g·kg-1·day-1) from the first day, and, the control group was given ordinary water. The aorta was observed by gross image and elastic fiber staining.②To replicate the aortic aneurysm/dissection model in mice, 40 C57 BL/6 mice were selected and divided into hesperidin 20 mg/kg group(n=10), hesperidin 50 mg/kg group(n=10) and control group(n=10). The administration was started on the 14 th day, once a day. The experimental group was given the corresponding 20 mg-1·kg-1/50 mg-1·kg-1 dose of hesperidin solution, and the control group was given the same amount of 0.9% sodium chloride solution. The surviving mice were sacrificed at 28 days, and visual changes in blood vessels were observed. Morphological changes were observed by EVG staining, TAAD formation rate was analyzed in each group.③Hep G2 cells were treated with 10 uM hesperetin for 36 h and then the cell supernatants was collected to detect changes in matrix metalloproteinases(MMPs)activity by Generic MMP Activity Kit. Activity of gelatinase on mouse aorta was observed by in situ MMP zymography. Results: Thoracic aortic elastic fiber sheets of mice after 14 days of treatment with BAPN(1 g·kg-1·day-1) began to show different degrees of disorder. Compared with the control group, mortality and TAAD formation rate of the hesperetin 50 mg/kg group decreased significantly, and the survival curve was statistically significant(P<0.05). The degree of aortic dilation was reduced, and the elastic fiber disorder and fracture were significant improvement than the control group. The hesperetin 20 mg/kg group also had a similar effect, but it was not as obvious as the hesperetin 50 mg/kg group. In addition, In addition, the activity of MMPs in the supernatant of Hep G2 cells treated with hesperetin was significantly decreased(P<0.0001);activity of gelatinase in aorta tissue of the hesperetin group was weakened. Conclusions: Hesperetin can ameliorate the occurrence and development of thoracic aortic aneurysm and dissection, and its mechanism may be related to the inhibition of metalloproteinase activity.