全反式维甲酸在肠道缺血再灌注中的抗氧化作用

目的 探讨全反式维甲酸（ATRA）在肠道缺血再灌注中的抗氧化作用。方法根据随机数字表法将32只雄性SD大鼠分为假手术组、阻断组、DMSO组和ATRA组，每组8只。采用血管钳钳夹肠系膜上动脉60min后，使血液复流120rain的方法制备大鼠肠道缺血再灌注损伤模型。假手术组仅分离肠系膜上动脉不钳夹。ATRA组大鼠术前5d以15μg／gATRA每日灌胃1次，开腹前6h再予ATRA处理1次；DMSO组则在相同时间点给予等量的DMSO液灌胃处理；应用高效液相色谱法检测大鼠术前5h的血浆ATRA浓度。HE染色观察回肠黏膜病理形态学改变，对肠黏膜组织行Chiu氏评分；比色法测定血清二胺氧化酶（DAO）水平、回肠组织丙二醛（MDA）含量以及超氧化物歧化酶（SOD）活性；Westernblot检测回肠组织中锰超氧化物歧化酶（MnSOD）的蛋白表达量。组问比较采用单因素方差分析，两两比较采用LSD—t检验。结果ATRA组大鼠血浆ATRA浓度为（827±276）μg/L，高于假手术组、阻断组和DMSO组的（48±12）μg/L、（55±15）μg／L和（63±20）μg／L（t=11．242，11．138，11．013，P〈0．05）。假手术组大鼠回肠黏膜形态正常；阻断组和DMSO组大鼠回肠黏膜结构破坏严重；ATRA组大鼠回肠黏膜损伤程度明显减轻。阻断组、DMSO组和ATRA组大鼠回肠黏膜Chiu氏评分分别为（3．83±0．77）分、（3．92±0．87）分和（2．42±0．75）分，显著高于假手术组的（O．37±0．28）分（t=9．803，10．040，5．793，P〈0．05），而ATRA组评分明显低于阻断组和DMSO组（t=4．009，4．247，P〈0．05）。阻断组、DMSO组和ATRA组大鼠血清DAO水平分别为（26．3±4．4）U／L、（25．1±4．3）U／L、（20．8±3．8）U／L，均显著高于假手术组的（14．2±1．9）U／L（t=6．493，5．835，3．534，P〈0．05）；但与阻断组及DMSO组比较，ATRA组血清DAO水平明显降低（t=2．959，2．301，P〈0．05）。阻断组、DMSO组和ATRA组大鼠回肠组织中MDA含量分别为（16．9±4．0）μmol／g、（16．0±3．5）μmol／g、（11．3±3．1）μmol／g，明显高于假手术组的（5．4±1．0）μmol／g（t=7．397，6．821，3．821，P〈0．05）；ATRA组显著低于阻断组和DMSO组（t=3．575，3．000，P〈0．05）。阻断组、DMSO组和ATRA组大鼠回肠组织的SOD活性分别为（108±22）U／mg、（98±19）U／rag和（181±38）U／mg，明显低于假手术组的（243±37）U／mg（t=8．939，9．647，4．106，P〈0．05）；ATRA组明显高于阻断组及DMSO组（t=4．833，5．541，P〈0．05）。阻断组和DMSO组大鼠回肠组织中MnSOD的蛋白相对表达量分别为0．36±0．08、0．28±0．07，显著低于假手术组的0．93±0．13（t=8．972，10．101，P〈0．05）；ATRA组为0．80±0．19，明显高于阻断组和DMSO组（t=6．948，8．077，P〈0．05），但与假手术组比较，差异无统计学意义（t=2．024，P〉0．05）。结论ATRA预处理可以促进氧自由基清除剂MnSOD的表达，增加组织的抗氧化能力，对大鼠肠道缺血再灌注损伤具有保护作用。

Antioxidative effects of all-trans retinotic acid on the intestinal ischemia reperfusion

Objective To investigate the antioxidative effects of all-trans retinoic acid （ATRA） on the intestinal ischemia reperfusion. Methods Thirty-two male SD rats were randomly divided into the sham operationgroup, occlusion group, dimethyl sulfoxide （DMSO） group and the ATRA group according to the random number table. There were 8 rats in each group. Rat models of intestinal ischemia reperfusion were established by clamping the superior mesenteric arteries for 60 minutes, and then restore the blood flow for 120 minutes. Superior mesen- teric arteries were only separated without clamping in the sham operation group. Rats in the ATRA group received ATRA pretreatment through intragastric infusion at the dosage of 15μg/g for 5 days, and then ATRA pretreatment at 6 hours before operation. Rats in the DMSO group received intragastric infusion of DMSO at the same dosage. The concentration of ATRA at 5 hours before operation was detected by high performance liquid chromatography. The pathomorphological changes of the ileal mucosa were detected by hematoxylin-eosin staining, and the Chiu＇s scores on the ileal mucosa were evaluated. The serum content of diamine oxidase （DAO） , tissue level of malonal- dehyde （MDA） and activity of superoxide dismutase （SOD） were detected by colorimetry. Protein expression of manganese superoxide dismutase （MnSOD） in the ileal tissues was detected by Western blot. All data were analyzed using the analysis of variance or LSD-t test. Results The concentrations of ATRA in the ATRA group was （827 ±276） μg/L, which was significantly higher than （48 ± 12） μg/L of the sham operation group, （55 ± 15 ） μg/L of the occlusion group and （63 ± 20） μg/L of the DMSO group （ t = 11. 242, 11.138, 11.013, P 〈 0.05 ）. The morphology of the ileal mucosa was normal in the sham operation group, while the ileal mucosa was severely damaged in the occlusion group and the DMSO group. The injury of the ileal mucosa in the ATRA group was slight. The Chiu＇s scores of the occlusion group, DMSO group and the ATRA group were 3.83± 0.77, 3.92 ± 0.87 and 2.42 ± 0.75, which were significantly higher than 0.37 ± 0.28 of the sham operation group （ t = 9. 803, 10. 040, 5. 793, P 〈 0.05）. The Chiu＇s scores of the ATRA group was significantly lower than that of the occlu- sion group and the DMSO group （t = 4. 009, 4. 247, P 〈 0.05）. The DAO levels of the occlusion group, DMSO group and the ATRA group were （26.3 ± 4.4）U/L, （25.1 ± 4.3 ）U/L and （20.8 ± 3.8 ）U/L, which were sig- nificantly higher than （ 14.2 ± 1.9） U/L of the sham operation group （ t = 6. 493, 5. 835, 3. 534, P 〈 0.05 ）. The level of DAO of the ATRA group was significantly lower than that of the occlusion group and the DMSO group （ t = 2. 959, 2. 301, P 〈0.05）. The levels of MDA of the occlusion group, DMSO group and the ATRA group were （ 16.9 ± 4.0） μmol/g, （ 16.0 ± 3.5 ） μmol/g and （ 11.3 ± 3.1 ） μmol/g, which were significantly higher than （5.4 ± 1.0） μmol/g of the sham operation group （t = 7. 397, 6. 821,3. 821, P 〈 0.05）. The level of MDA of the ATRA group was signifcantly lower than that of the occlusion group and the DMSO group （t = 3. 575, 3. 000, P 〈 O. 05 ）. The SOD activity of the occlusion group, DMSO group and the ATRA group were （108 ± 22）U/mg, （98 ± 19）U/mg and （181 ±38）U/mg, which were significantly lower than （243 ± 37）U/rag of the sham operation group （t = 8. 939, 9. 647, 4. 106, P 〈0.05）. The SOD activity of the ATRA group was significantly higher than that of the occlusion group and the DMSO group （ t = 4. 833, 5. 541, P 〈 0.05 ）. The relative protein expressions of the MnSOD of the occlusion group and the DMSO group were 0.36 ± 0.08 and 0.28 ± 0.07, which were significantly lower than 0.93 ± 0. 13 of the sham operation group （ t = 8. 972, 10. 101, P 〈 0.05 ）. The relative protein expression of the MnSOD of the ATRA group was 0.80 ± 0.19, which was significantly higher than that of the occlusion group and the DMSO group （ t = 6. 948, 8. 077, P 〈 0.05 ） , while it was not significantly different from that of the sham operation group （ t = 2. 024, P 〉 0.05）. Conclusion Through up-regulating the expression of MnSOD and improving the antioxidative capacity of tissue, ATRA pretreatment can attenuate intestinal ischemia and reperfusion injury.