米诺环素减轻大鼠循环死亡后供肝肝移植缺血再灌注损伤的研究

Effect of minocycline on ischemia-reperfusion injury in liver transplantation after circulatory death in rats

目的研究米诺环素对大鼠循环死亡后肝移植缺血再灌注损伤（IRI）的作用及其分子机制。方法采用＂磁环法＂建立大鼠心脏停跳后肝移植模型，供、受者均为雄性SD大鼠。分为假手术组（SHAM组）、肝移植组、米诺环素组（MIN组）、苍术苷（线粒体通透性转换孔开放剂）＋米诺环素组（ATR＋MIN组），每组供、受鼠共24对。MIN组肝移植后经阴茎背静脉注射米诺环素（10 mg/kg），ATR＋MIN组在MIN组基础上再注射苍术苷（2 mg/kg）。术后2、6、24 h检测线粒体通透性转换孔（mPTP）开放状态；免疫印迹法及免疫组织化学法检测含半胱氨酸天冬氨酸蛋白酶3（caspase3）和细胞色素c（cyt c）蛋白的表达；检测受鼠血清谷氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）的水平。受鼠肝组织行HE染色，观察病理学改变并计算Suzuki评分。计算各组受鼠存活率。结果与肝移植组和ATR＋MIN组比较，术后2、6、24 hMIN组mPTP开放明显减少（P〈0.05），组织中cyt c和caspase3蛋白水平、血清ALT和AST水平、肝组织损伤程度明显降低（P〈0.05），术后30 d存活率升高（P〈0.05）；肝移植组和ATR＋MIN组之间各指标的差异，无统计学意义（P〉0.05）。结论米诺环素可减轻大鼠循环死亡后供肝肝移植的IRI，其机制可能是通过抑制mPTP开放，阻止cyt c释放和caspase3激活以减轻肝细胞的凋亡所致，且这种作用可以被mPTP开放剂阻断。

ObjectiveTo study the protective effect of minocycline against ischemia-reperfusion injury after liver transplantation and its molecular mechanism after circulatory death in rats.MethodsThe rat donation after circulatory death （DCD） liver transplantation model was established by using ＂ magnetic-ring method＂ . The donor and recipient were male SD rats. The rats were divided into sham operation group （SHAM group）, liver transplantation group, minocycline group （MIN group）, atractyloside＋ minocycline group （ATR＋ MIN group）, 24 rats in each group. In the MIN group, 10 mg/kg minocycline was injected through the dorsum vein of the penis after reperfusion. The ATR ＋ MIN group was injected with 2 mg/kg atractyloside. The open status of mitochondrial permeability transition pore （mPTP） was detected at 2, 6, and 24 h after operation. Western blotting and immunohistochemistry were used to detect the expression of caspase3 and cyt c. Serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels were determined. Liver tissues were stained with hematoxylin-eosin （HE） and pathological changes were evaluated by Suzuki＇s standard. The survival of each group was calculated.ResultsAs compared with liver transplantation group and ATR＋ MIN group, the mPTP opening of MIN group decreased （P〈0.05）, the expression of caspase3 and cyt c and the serum ALT and AST levels decreased significantly （P〈0.05）, liver tissues injury was alleviated （P〈0.05）, and the survival rate increased significantly after 30 days （P〈0.05）. There was no significant difference between liver transplantation group and ATR＋ MIN group （P〉0.05）.ConclusionMinocycline reduces ischemia-reperfusion injury in DCD liver transplantation in rats probably by inhibiting the mPTP opening, and preventing cyt c release and caspase3 activation to reduce hepatocyte apoptosis. This effect can be blocked by mPTP opener.