亚低温联合脂肪间充质干细胞对肝缺血再灌注损伤的保护作用

Mild hypothermia combined with adipose-derived mesenchymal stem cells protects hepatic function in hepatic ischemia-reperfusion injury

背景:肝缺血再灌注损伤的机制复杂多样,是制约肝脏外科手术的瓶颈,如何减轻肝缺血再灌注损伤一直是医学研究的热点与难点之一。目的:探讨亚低温联合脂肪间充质干细胞预处理对大鼠肝缺血再灌注损伤的保护作用机制。方法:将60只雄性SD大鼠(购于昆明医科大学实验动物中心)随机分为6组:假手术组、肝缺血再灌注损伤组、亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组、ERK通路阻断剂组,每组10只,均于缺血前30 min给予亚低温、脂肪间充质干细胞、ERK通路阻断剂干预。肝缺血再灌注12 h后收集大鼠血清及肝组织标本,进行相关指标检测。肝缺血再灌注12 h后收集大鼠血清、肝组织,术后7 d收集大鼠肝组织,进行相关指标检测。结果与结论:①全自动生化分析仪检测肝功能指标:亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组血清中天冬氨酸转氨酶、丙氨酸转氨酶和乳酸脱氢酶活性均低于肝缺血再灌注损伤组(P <0.05),亚低温+脂肪间充质干细胞组血清中3种酶活性低于亚低温组和脂肪间充质干细胞组(P <0.05);②ELISA检测氧化应激指标:亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组肝组织中超氧化物歧化酶、谷胱甘肽过氧化物酶活性显著高于肝缺血再灌注损伤组(P <0.05),丙二醛水平均显著低于肝缺血再灌注损伤组(P <0.05);亚低温+脂肪间充质干细胞组肝组织中超氧化物歧化酶和谷胱甘肽过氧化物酶水平显著高于亚低温组和脂肪间充质干细胞组(P <0.05),而丙二醛水平低于亚低温组和脂肪间充质干细胞组(P <0.05);③Western blotting检测相关蛋白表达:与肝缺血再灌注损伤组相比,亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组肝组织中p-ERK1/2相对表达水平显著升高(P <0.05),各组中ERK1/2的表达差异无显著性意义;④TUNEL检测肝细胞凋亡数量:肝缺血再灌注损伤组和ERK通路阻断剂组肝组织中细胞凋亡率明显高于亚低温组、脂肪间充质干细胞组、亚低温+脂肪间充质干细胞组。同时,Western blotting检测相关凋亡蛋白结果与其一致;⑤结果表明,亚低温联合脂肪间充质干细胞对肝缺血再灌注损伤具有保护作用,其机制与激活ERK通路进而下调肝细胞凋亡有关。

BACKGROUND: Hepatic ischemia-reperfusion injury is the bottleneck restricting liver surgeries as its complex and diverse mechanisms. How to reduce liver ischemia-reperfusion injury has always been one of the hotspots and difficulties in medical research.OBJECTIVE: To explore the protective mechanism of mild hypothermia pretreatment combined with adipose-derived mesenchymal stem cells transplantation for hepatic ischemia reperfusion injury in rats.METHODS: Sixty male Sprague-Dawley rats(provided by the Experimental Animal Center of Kunming Medical University in China) were randomly divided into six groups with 10 rats in each group: sham operation group(Sham), hepatic ischemia-reperfusion injury group(HIRI),mild hypothermia group(MH), adipose-derived mesenchymal stem cells group(ADMSCs), mild hypothermia+ADMSCs group(MH+ADMSCs)and mild hypothermia+ADMSCs+ ERK inhibitor group(MH+ADMSCs+PD98059). The rats were given mild hypothermia, adipose-derived mesenchymal stem cells, or ERK inhibitor intervention at 30 minutes before ischemia. The serum and liver tissue samples were collected 12 hours after hepatic ischemia-reperfusion.RESULTS AND CONCLUSION: The activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase in the serum of groups MH, ADMSCs and MH+ADMSCs were significantly higher than those in the HIRI group(P < 0.05). The activities of aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase in group MH+ADMSCs were significantly lower than those in groups MH and ADMSCs(P < 0.05). Subsequently, compared with group HIRI, the activities of superoxide dismutase and glutathione peroxide were significantly higher in groups MH, ADMSCs and MH+ADMSCs(P < 0.05), whereas the levels of malondialdehyde were significantly lower in the three groups(P < 0.05). The activities of superoxide dismutase and glutathione peroxide in group MH+ADMSCs were significantly higher than those in groups MH and ADMSCs(P < 0.05), whereas the level of malondialdehyde in group MH+ADMSCs was lower than that in groups MH and ADMSCs(P < 0.05). The relative expression level of p-ERK1/2 in the liver tissue was significantly increased in groups MH, ADMSCs and MH+ADMSCs as compared with group HIRI(P < 0.05), but there were no significant differences in the relative expression level of ERK1/2 among the groups. Furthermore, the apoptotic cell proportion of liver tissues in groups HIRI and MH+ADMSCs+PD98059 were higher than that in groups MH, ADMSCs and MH+ADMSCs. These results were in consistent with the findings of western blot detection. To conclude, mild hypothermia combined with adipose-derived mesenchymal stem cells transplantation has a protective effect against hepatic ischemia-reperfusion injury, and the mechanism may be through activating ERK pathway to downregulate the percentage of apoptotic hepatocytes in vivo.