甘草素对大鼠肝脏冷缺血损伤的影响及其机制
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摘要
目的探讨甘草素(GL)对大鼠肝脏冷缺血损伤的影响及其机制。方法通过模拟肝移植过程构建大鼠肝脏冷保存模型,观察组大鼠肝脏在含有不同浓度GL的组氨酸-色氨酸-酮戊二酸(HTK)溶液中冷保存24h后,于37℃下再灌注60min,对照组肝脏游离后立即进行再灌注。分别采用qPCR、免疫组化、HE染色、ELISA等方法检测HMGB1、TLR4、NF-κB、TNF-α以及IL-6的表达。分光光度法测定灌流液中乳酸脱氢酶(LDH)水平。TUNEL法检测肝细胞凋亡。结果 HTK溶液中GL的加入可显著降低肝损伤评分,减少LDH渗漏和增加胆汁分泌。而再灌注后,保存在含GL的HTK溶液中的肝脏,HMGB1、TLR4和NF-κB的表达以及肝细胞凋亡均明显降低。灌流液中TNF-α和IL-6的含量也随着GL的加入而显著降低。结论 HTK溶液中GL的加入减弱了冷保存过程中的冷缺血性损伤,其机制可能与HMGB1-TLR4信号通路被抑制有关。
Objective To investigate the protective effect of the glycyrrhizin(GL) on cold ischemic injury of rat liver and its possible mechanism.Methods Construction of rat liver cold storage model by simulating liver transplantation was performed. Livers from rats were reperfused at 37℃ for 60 minutes either immediately or after 24 hours of cold storage in histidine-tryptophan-ketoglutarate(HTK) solution containing different concentrations of GL. The expression of HMGB1, TLR4, NF-κB, TNF-α and IL-6 was observed by real-time PCR, immunohistochemistry, hematoxylin and eosin staining and enzyme-linked immunosorbent assay, respectively. The concentration of LDH in the perfusate was detected by spectrophotometry. Hepatocyte apoptosis was detected by TUNEL. Results Addition of GL to HTK solution significantly attenuated ischemic injury during reperfusion in a dose-dependent way, as evidenced by the lower liver injury scores, lower LDH leakage, and increased bile secretion. After reperfusion, livers stored in HTK solution containing GL had decreased expression of HMGB1, TLR4, and NF-κB, and less apoptosis compared with those stored without GL. The levels of TNF-α and IL-6 in the effluent were also decreased when stored in GL-containing HTK solution. Conclusion The addition of GL to HTK solution attenuates cold ischemic injury during cold storage and the mechanism is at least partially attributable to the inhibition of HMGB1-TLR4 signaling.
Objective To investigate the protective effect of the glycyrrhizin(GL) on cold ischemic injury of rat liver and its possible mechanism.Methods Construction of rat liver cold storage model by simulating liver transplantation was performed. Livers from rats were reperfused at 37℃ for 60 minutes either immediately or after 24 hours of cold storage in histidine-tryptophan-ketoglutarate(HTK) solution containing different concentrations of GL. The expression of HMGB1, TLR4, NF-κB, TNF-α and IL-6 was observed by real-time PCR, immunohistochemistry, hematoxylin and eosin staining and enzyme-linked immunosorbent assay, respectively. The concentration of LDH in the perfusate was detected by spectrophotometry. Hepatocyte apoptosis was detected by TUNEL. Results Addition of GL to HTK solution significantly attenuated ischemic injury during reperfusion in a dose-dependent way, as evidenced by the lower liver injury scores, lower LDH leakage, and increased bile secretion. After reperfusion, livers stored in HTK solution containing GL had decreased expression of HMGB1, TLR4, and NF-κB, and less apoptosis compared with those stored without GL. The levels of TNF-α and IL-6 in the effluent were also decreased when stored in GL-containing HTK solution. Conclusion The addition of GL to HTK solution attenuates cold ischemic injury during cold storage and the mechanism is at least partially attributable to the inhibition of HMGB1-TLR4 signaling.
引文
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13 Tsung A,Sahai R,Tanaka H,et al.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion[J].J Exp Med,2005,201(7):1135-1143
14 刘秀妮,程忠平.氧化/炎性压力诱导HMGB1的出胞及HMGB1与卵巢癌发病机制研究进展[J].医学综述,2017,23(10):1940-1944
15 Zhu H,Li J,Wang S,et al.Hmgb1-TLR4-IL-23-IL-17A axis promote ischemia-reperfusion injury in a cardiac transplantation model[J].Transplantation,2013,95(12):1448-1454
16 Wang J,He GZ,Wang YK,et al.TLR4-HMGB1-,MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury[J].World J Gastroenterol,2015,21(27):8314-8325
2 蒙建源,徐静,樊奇,等.肝移植术后胆道并发症危险因素的Meta分析[J].中华肝胆外科杂志,2012,11:816-822
3 郝建红,席宏杰.Kupffer细胞与肝缺血再灌注损伤[J].胃肠病学和肝病学杂志,2013,9:941-943
4 Lentsch AB,Kato A,Yoshidome H,et al.Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury[J].Hepatology,2000,32(2):169-173
5 Gong G,Xiang L,Yuan L,et al.Protective effect of glycyrrhizin,a direct HMGB1 inhibitor,on focal cerebral ischemia/reperfusion-induced inflammation,oxidative stress,and apoptosis in rats[J].PLoS One,2014,9(3):e89450
6 Ogiku M,Kono H,Hara M,et al.Glycyrrhizin prevents liver injury by inhibition of high-mobility group box 1 production by Kupffer cells after ischemia-reperfusion in rats[J].J Pharmacol Exp Ther,2011,339(1):93-98
7 Wu S,Wohlschlaeger J,de Groot H,et al.Evaluation of a modified HTK solution containing the new iron chelator LK 614 in an isolated rat liver perfusion model[J].J Invest Surg,2009,22(5):340-347
8 Hauet T,Eugene M.A new approach in organ preservation:potential role of new polymers[J].Kidney Int,2008,74(8):998-1003
9 茆玲,冯彩云,刘学燕.甘草酸制剂治疗病毒性肝炎疗效观察[J].湖北中医药大学学报,2016,18(1):67-69
10 Fujisawa Y,Sakamoto M,Matsushita M,et al.Glycyrrhizin inhibits the lytic pathway of complement-possible mechanism of its anti-inflammatory effect on liver cells in viral hepatitis[J].Microbiol Immunol,2000,44(9):799-804
11 Hu X,Zhang K,Xu C,et al.Anti-inflammatory effect of sodium butyrate preconditioning during myocardial ischemia/reperfusion[J].Exp Ther Med,2014,8(1):229-232
12 Lau A,Wang S,Liu W,et al.Glycyrrhizic acid ameliorates HMGB1-mediated cell death and inflammation after renal ischemia reperfusion injury[J].Am J Nephrol,2014,40(1):84-95
13 Tsung A,Sahai R,Tanaka H,et al.The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion[J].J Exp Med,2005,201(7):1135-1143
14 刘秀妮,程忠平.氧化/炎性压力诱导HMGB1的出胞及HMGB1与卵巢癌发病机制研究进展[J].医学综述,2017,23(10):1940-1944
15 Zhu H,Li J,Wang S,et al.Hmgb1-TLR4-IL-23-IL-17A axis promote ischemia-reperfusion injury in a cardiac transplantation model[J].Transplantation,2013,95(12):1448-1454
16 Wang J,He GZ,Wang YK,et al.TLR4-HMGB1-,MyD88- and TRIF-dependent signaling in mouse intestinal ischemia/reperfusion injury[J].World J Gastroenterol,2015,21(27):8314-8325