NBD多肽预处理改善大鼠肝移植缺血再灌注损伤的研究
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摘要
目的采用SD-SD大鼠原位肝移植模型,观察NBD多肽预处理供肝对术后肝脏缺血再灌注损伤(ischemia reperfusion injury,IRI)的影响,并探讨其可能的保护机制。方法 105只SD大鼠采用抽签法随机分为3组,NBD预处理组(24对动物,供体术前2 h经腹腔注射8 mg/kg的NBD多肽并建立原位肝移植模型)、移植组(24对动物,注射同体积的生理盐水作为对照)和假手术组(9只动物)。术后3、6、24 h分别处死动物,检测3组中ALT水平,ELISA检测血清中TNF-α含量。取NBD预处理组和移植组肝脏组织,HE染色观察肝脏病理学改变。采用TUNEL检测肝细胞凋亡,EMSA检量NF-κB转录活性,Western blot检测p-IKK-2和IκBα在肝组织的表达。结果 NBD预处理组和移植组术后各时间点的ALT水平、TNF-α含量均明显高于假手术组。NBD预处理组中ALT水平、NF-κB活性、p-IKK-2表达、TNF-α含量较移植组均明显降低(P<0.05);IκBα表达则较移植组明显增强(P<0.05);术后6 h,NBD预处理组中病理损伤、凋亡细胞较移植组明显减轻。结论 NBD多肽预处理供体可通过抑制供肝中NF-κB活性,有效改善肝移植IRI。
Objective NF-κB essential modulator binding domain peptides( NBD peptides) interfere the formation of IκB kinase complex( IKK) and down-regulate the classical NF-κB signaling pathway.The aim of this study was to determine the effects of NBD peptides pretreatment on ischemia reperfusion injury( IRI)after orthotopic liver transplantation in rats.Methods A total of 105 Sprague Dawley( SD) rats were randomly divided into 3 groups,NBD group( donors were given 8 mg /kg NBD peptides intra-peritoneally in 2 h before surgery and performed orthotropic liver transplantation according to the Kamada technique,totally 24pairs),control group( treated with the same volume of physiological saline,totally 24 pairs) and shamoperation group( n = 9).Animals were sacrificed at 3,6,and 24 h after surgery.The serum levels of alanine aminotransferase( ALT) and tumor necrosis factor( TNF-α) were measured in the 3 groups.The levels of IKK phosphorylation and IκBα degradation,NF-κB transcriptional activity and cell apoptosis were detected by Western blotting,EMSA and TUNEL respectively.Morphological changes of liver tissues were observed after HE staining.Results The levels of ALT and TNF-α in the both NBD group and control group were significantly higher than that of sham-operation group( P < 0.05).Compared with the control group,pretreatment with NBD peptides had significantly lower ALT level,NF-κB transcriptional activity,IKK complex phosphorylation,and TNF-α levels( P < 0.05),while enhanced IκBα expression( P < 0.05).NBD peptides pretreatment also ameliorated the pathological damages and cell apoptosis in 6 h after operation.Conclusion NBD peptides pretreatment attenuates hepatic IRI by preventing NF-κB activation.
Objective NF-κB essential modulator binding domain peptides( NBD peptides) interfere the formation of IκB kinase complex( IKK) and down-regulate the classical NF-κB signaling pathway.The aim of this study was to determine the effects of NBD peptides pretreatment on ischemia reperfusion injury( IRI)after orthotopic liver transplantation in rats.Methods A total of 105 Sprague Dawley( SD) rats were randomly divided into 3 groups,NBD group( donors were given 8 mg /kg NBD peptides intra-peritoneally in 2 h before surgery and performed orthotropic liver transplantation according to the Kamada technique,totally 24pairs),control group( treated with the same volume of physiological saline,totally 24 pairs) and shamoperation group( n = 9).Animals were sacrificed at 3,6,and 24 h after surgery.The serum levels of alanine aminotransferase( ALT) and tumor necrosis factor( TNF-α) were measured in the 3 groups.The levels of IKK phosphorylation and IκBα degradation,NF-κB transcriptional activity and cell apoptosis were detected by Western blotting,EMSA and TUNEL respectively.Morphological changes of liver tissues were observed after HE staining.Results The levels of ALT and TNF-α in the both NBD group and control group were significantly higher than that of sham-operation group( P < 0.05).Compared with the control group,pretreatment with NBD peptides had significantly lower ALT level,NF-κB transcriptional activity,IKK complex phosphorylation,and TNF-α levels( P < 0.05),while enhanced IκBα expression( P < 0.05).NBD peptides pretreatment also ameliorated the pathological damages and cell apoptosis in 6 h after operation.Conclusion NBD peptides pretreatment attenuates hepatic IRI by preventing NF-κB activation.
引文
[1]Montalvo-Jave E E,Escalante-Tattersfield T,Ortega-Salgado J A,et al.Factors in the pathophysiology of the liver ischemia-reperfusion injury[J].J Surg Res,2008,147(1):153-159.
[2]Abu-Amara M,Yang S Y,Tapuria N,et al.Liver ischemia/reperfusion injury:processes in inflammatory networks--a review[J].Liver Transpl,2010,16(9):1016-1032.
[3]Matsui N,Kasajima K,Hada M,et al.Inhibiton of NF-kappaB activation during ischemia reduces hepatic ischemia/reperfusion injury in rats[J].J Toxicol Sci,2005,30(2):103-110.
[4]Wu C,Wang P,Rao J,et al.Triptolide alleviates hepatic ischemia/reperfusion injury by attenuating oxidative stress and inhibiting NF-κB activity in mice[J].J Surg Res,2011,166(2):e205-e213.
[5]Giakoustidis D E,Giakoustidis A E,Iliadis S,et al.Attenuation of liver ischemia/reperfusion induced apoptosis by epigallocatechin-3-gallate via down-regulation of NF-kappaB and c-Jun expression[J].J Surg Res,2010,159(2):720-728.
[6]May M J,D’Acquisto F,Madge L A,et al.Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex[J].Science,2000,289(5484):1550-1554.
[7]Baima E T,Guzova J A,Mathialagan S,et al.Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides[J].J Biol Chem,2010,285(18):13498-13506.
[8]Dave S H,Tilstra J S,Matsuoka K,et al.Amelioration of chronic murine colitis by peptide-mediated transduction of the IkappaB kinase inhibitor NEMO binding domain peptide[J].J Immunol,2007,179(11):7852-7859.
[9]Long Y M,Chen K,Liu X J,et al.Cell-permeable Tat-NBD peptide attenuates rat pancreatitis and acinus cell inflammation response[J].World J Gastroenterol,2009,15(5):561-569.
[10]Desai A,Singh N,Raghubir R.Neuroprotective potential of the NF-κB inhibitor peptide IKK-NBD in cerebral ischemia reperfusion injury[J].Neurochem Int,2010,57(8):876-883.
[11]Kamada N.Animal models of hepatic allografl rejection[J].Semin Liver Dis,1992,12(1):1-15.
[12]Huang H J,Sugimoto S,Lai J,et al.Maintenance of IKKbeta activity is necessary to protect lung grafts from acute injury[J].Transplantation,2011,91(6):624-631.
[13]Schwabe R F,Brenner D A.Mechanisms of Liver Injury.I.TNF-alpha-induced liver injury:role of IKK,JNK,and ROS pathways[J].Am J Physiol Gastrointest Liver Physiol,2006,290(4):G583-G589.
[14]Peralta C,Fernandez L,Panes J,et al.Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat[J].Hepatology,2001,33(1):100-113.
[15]Rajesh M,Pan H,Mukhopadhyay P,et al.Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress,inflammatory response,and apoptosis[J].J Leukoc Biol,2007,82(6):1382-1389.
[2]Abu-Amara M,Yang S Y,Tapuria N,et al.Liver ischemia/reperfusion injury:processes in inflammatory networks--a review[J].Liver Transpl,2010,16(9):1016-1032.
[3]Matsui N,Kasajima K,Hada M,et al.Inhibiton of NF-kappaB activation during ischemia reduces hepatic ischemia/reperfusion injury in rats[J].J Toxicol Sci,2005,30(2):103-110.
[4]Wu C,Wang P,Rao J,et al.Triptolide alleviates hepatic ischemia/reperfusion injury by attenuating oxidative stress and inhibiting NF-κB activity in mice[J].J Surg Res,2011,166(2):e205-e213.
[5]Giakoustidis D E,Giakoustidis A E,Iliadis S,et al.Attenuation of liver ischemia/reperfusion induced apoptosis by epigallocatechin-3-gallate via down-regulation of NF-kappaB and c-Jun expression[J].J Surg Res,2010,159(2):720-728.
[6]May M J,D’Acquisto F,Madge L A,et al.Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex[J].Science,2000,289(5484):1550-1554.
[7]Baima E T,Guzova J A,Mathialagan S,et al.Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides[J].J Biol Chem,2010,285(18):13498-13506.
[8]Dave S H,Tilstra J S,Matsuoka K,et al.Amelioration of chronic murine colitis by peptide-mediated transduction of the IkappaB kinase inhibitor NEMO binding domain peptide[J].J Immunol,2007,179(11):7852-7859.
[9]Long Y M,Chen K,Liu X J,et al.Cell-permeable Tat-NBD peptide attenuates rat pancreatitis and acinus cell inflammation response[J].World J Gastroenterol,2009,15(5):561-569.
[10]Desai A,Singh N,Raghubir R.Neuroprotective potential of the NF-κB inhibitor peptide IKK-NBD in cerebral ischemia reperfusion injury[J].Neurochem Int,2010,57(8):876-883.
[11]Kamada N.Animal models of hepatic allografl rejection[J].Semin Liver Dis,1992,12(1):1-15.
[12]Huang H J,Sugimoto S,Lai J,et al.Maintenance of IKKbeta activity is necessary to protect lung grafts from acute injury[J].Transplantation,2011,91(6):624-631.
[13]Schwabe R F,Brenner D A.Mechanisms of Liver Injury.I.TNF-alpha-induced liver injury:role of IKK,JNK,and ROS pathways[J].Am J Physiol Gastrointest Liver Physiol,2006,290(4):G583-G589.
[14]Peralta C,Fernandez L,Panes J,et al.Preconditioning protects against systemic disorders associated with hepatic ischemia-reperfusion through blockade of tumor necrosis factor-induced P-selectin up-regulation in the rat[J].Hepatology,2001,33(1):100-113.
[15]Rajesh M,Pan H,Mukhopadhyay P,et al.Cannabinoid-2 receptor agonist HU-308 protects against hepatic ischemia/reperfusion injury by attenuating oxidative stress,inflammatory response,and apoptosis[J].J Leukoc Biol,2007,82(6):1382-1389.