二氯甲烷对肝脏缺血再灌注损伤的影响

Effect of methylene chloride upon hepatic ischemic reperfusion injury

目的探讨二氯甲烷对减轻肝脏缺血再灌注损伤的机制。方法将80只雄性SD大鼠随机分为4组（20只／组）：对照组，无任何处理；CoPP（钴-原卟啉）组，供肝收获前24h供体腹腔注射CoPP5mg／kg；ZnPP（锌-原卟啉）组，供肝收获前24h供体腹腔注射ZnPP20mg／kg；MC（二氯甲烷）组，供肝收获前7d，供体口服MC500mg／（kg·d）。受体不作处理，采用双袖套法施行大鼠同基因原位肝移植（每组供体和受体各10只），移植后3d每组各处死5只受体鼠，取出供肝并检测：移植后受体肝功能；Western印迹法和免疫组化法检测供肝血红素氧合酶-1（HO-1）的表达；TUNEL法检测供肝细胞的凋亡；Suzuki标准评分评估供肝的缺血再灌注损伤程度。每组留下5只受体鼠观察移植后的生存时间。结果MC组、CoPP组血清的丙氨酸转氨酶（ALT）[（75±16）、（65±28）U／L]、天冬氨酸转氨酶（AST）[（185±42）、（187±43）U／L]明显较对照组[ALT（346±45）U／L、AST（474±90）U／L]和ZnPP组[ALT（578±75）U／L、AST（1084±128）U／L]低（P〈0.01）。CoPP组肝脏移植物的HO-1表达量中位数为3.05，明显较对照组高（P〈0.05），MC组与对照组间差异无统计学意义（P〉0.05）。MC组和CoPP组的移植物细胞凋亡指数分别为4.1％±0.6％、3.2％±0.8％，明显较对照组（12.5％±2.4％）和ZnPP组（25.8％±3.1％）低（P〈0.05）。MC组、CoPP组比对照组、ZnPP组的肝细胞损伤轻、炎症细胞浸润少，MC组、CoPP组的Suzuki评分分别为0.76±0.52、0.76±0.59，明显较对照组（1.32±0.74）和ZnPP组（1.80±0.70）低（P〈0.05）。MC组、CoPP组的中位生存时间分别为100、93d，较对照组（85d）、ZnPP组（12d）延长（P〈0.05）。结论HO-1上调和二氯甲烷对肝脏移植物缺血再灌注损伤均有保护作用。

Objective To investigate the effects and mechanisms of methylene chloride （MC） in hepatic isehemic reperfusion injury. Methods Eighty SD-rats were divided equally into 4 groups： control group （n =20）, donors without any treatment; CoPP group （n =20）, donors injected with CoPP （5 mg/kg, ip） at 24 h; ZnPP group （n =20）, donors injected with ZnPP （20 mg/kg, ip） at 24 h; MC group （n = 20）, donors fed with MC （500 mg/kg ） per day for 7 days before graft procurement. Syngeneic orthotopic liver transplantation was performed in rats with modified Kamada＇s two-cuff technique. And SD rats were used as donors （n = 10）and recipients respectively. 5 recipients in each group were sacrificed and the grafts were procured at day 3 after transplantation, the post-operative survival time was observed in the remnant. The tests were determined as following： the level of serum ALT, AST in recipients; heme oxygenase-1 （HO- 1 ） expression of graft was tested by immunohistochemistry and Western blot; the index of graft apoptosis examined by TUNEL method; the pathology of graft assessed by Suzuki＇s criteria. Results The level of serum ALT [（65±28） U/L], AST [（187±43） U/L] in CoPP and ALT [ （75±16） U/L], AST [ （185±42） U/L] in MC group was significantly lower than that ALT [ （346±45） U/L], AST [ （474±90） U/L] in control group and ALT [（578±75）U/L], AST [（1084±128） U/L] in ZnPP group（ P 〈 0.01 ）. The median expression of HO-1 in MC group was no significantly different with that in control group（ P 〉 0.05 ）. While the median expression of HO-1 in CoPP group was higher than that in control group（ P 〈 0.05 ）. The index of graft apoptosis in MC group and CoPP group, 4.1%±0.6% and 3.2%±0.8% respectively, was significantly lower than that （ 12.5%±2.4% ） in control group and （25.8%±3.1% ） in ZnPP group（P 〈 0.05）. Compared with the other two groups, MC and CoPP groups had lesser neutrophil infiltration and a lower grade of hepatocytic injury in grafts. Suzuki＇s scores in grafts of MC and CoPP groups were lower than that in control and ZnPP groups （ P 〈 0.05）. The median post-transplantation survival time of the recipients in MC and CoPP groups was 100 and 93 days respectively while that in control and ZnPP groups was 85 and 12 days（P 〈 0.05）. Conclusion Over-expression of HO-1 and MC both have protective effects in hepatic ischemie reperfusion injury.