二氮嗪预处理对大鼠小体积肝移植缺血再灌注损伤的保护作用及对肝再生的影响
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摘要
小体积肝移植在历经缺血再灌注损伤后,还存在复流后的门静脉高灌注损伤,其移植后的肝再生过程也不同于全肝移植和普通肝切除术后的肝再生。因此要提高临床活体肝移植的疗效,必须进一步研究部分肝移植尤其是小体积肝移植的缺血再灌注损伤、血液动力学、肝再生机制及其影响因素。通过建立与临床肝移植相似的动物模型,来寻找减轻肝移植术后移植物的缺血再灌注损伤、促进部分移植物尤其是小体积供肝的快速再生的途径,这对于提高临床活体肝移植的疗效有着非常重要的意义。
线粒体膜上存在对ATP浓度敏感的钾离子通道——mitoKATP,二氮嗪(DA)可以特异性开放该通道,而5-羟基癸酸盐(5-HD)则能将其特异性关闭。大量研究提示,mitoKATP通道开放在心肌、脑、肺、肾缺血再灌注损伤中均有保护作用,但目前国内外有关mitoKATP对肝脏的缺血再灌注损伤影响的研究很少。
通过二氮嗪(DA)预处理供体,观察mitoKATP开放对大鼠60%比例部分肝移植和30%比例小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。
第一部分大鼠30%比例小体积肝移植模型的建立
目的:建立稳定的大鼠30%比例小体积肝移植模型,为后续的移植肝缺血再灌注损伤(IRI)和小体积供肝移植后肝再生的实验研究打下基础。
方法:本部分行100例大鼠原位肝移植术,其中预实验50例,60%比例部分肝移植25例,30%比例小体积肝移植25例,全部采用“二袖套法”完成。全部手术均采用经腹主动脉插管原位快速灌注获取供肝,冷缺血时间均设定为1小时。另外各取5只大鼠,行40%和70%比例肝叶切除,分别观察存活率。
结果:60%和30%比例移植组,供肝与原肝重比分别为59.70±3.21%和31.92±2.43%,手术成功率(术后存活2天以上)分别为92%(23/25)和72%(18/25),1周存活率分别为72%(18/25)和64%(16/25)。肝切除组中,切除肝重与理论原肝重比分别为42.51±3.62%和66.83±4.37%,1周存活率为100%。
结论:在以往动物模型的基础上,通过改进操作方法,成功建立了大鼠60%比例部分肝移植和30%比例小体积肝移植模型。该模型稳定,手术成功率较高,便于操作,容易复制。大鼠部分肝移植模型尤其是小体积模型,与临床部分肝移植如活体肝移植有着相似的手术操作过程和病理生理变化,是研究改善肝移植缺血再灌注损伤、促进肝再生的理想途径。
第二部分二氮嗪预处理对大鼠小体积肝移植缺血再灌注损伤的保护机制研究及对肝再生的影响
目的:研究线粒体ATP敏感的钾通道(mitoKATP)开放,对大鼠部分肝移植尤其是小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。为临床部分肝移植如活体肝移植,寻找减轻缺血再灌注损伤、促进术后肝再生的治疗靶点和途径。
方法:大鼠肝移植模型的建立方法同第一部分。SD大鼠300只,体重220～300g,随机配对成150对,随机分成6组,每组25对,供体体重应比受体轻10～20g。分组为全肝移植组(A组),60%比例部分肝移植组(C组),60%比例部分肝移植+二氮嗪组(DAl组),60%比例部分肝移植+5-羟基癸酸盐+二氮嗪组(HD-DA组),30%比例小体积肝移植组(S组),30%比例小体积肝移植+二氮嗪组(DA2组)。DA组供体术前30分钟腹腔注射二氮嗪(5mg/kg),再行手术;HD-DA组供体术前45分钟腹腔注射5-HD(5mg/kg),15分钟后腹腔注射二氮嗪(5mg/kg),再行手术。另取SD大鼠50只,体重220～300g,随机分成2组,每组25只。分组为40%比例肝切除组(B1组)和70%比例肝切除组(B2组)。
各手术组均取5个时间点,分别为术后2小时、1天、3天、5天和7天,每个时间点各取5例处死取材(n=5)。检测血清肝功能指标(ALT、AST),肝组织湿重并计算肝再生率,光镜下HE染色病理检查,电镜下超微结构检查,增殖细胞核抗原标记指数(PCNA),TUNEL法肝细胞凋亡指数,血清、肝组织细胞因子TNF-α、IL-6、IL-1β(ELISA法)检测,肝组织NF-κB活性检测(ELISA法)。
结果:
1.光镜下病理检查:DA组与对照组~*比较,肝细胞肿胀变性坏死及空泡样变性、肝小叶结构排列紊乱、汇管区炎症都较轻。DA组在术后7d分裂相多见,而对照组~*则减少。
2.电镜下超微结构检查:DA组中细胞结构完整,线粒体肿胀及细胞间隙扩张程度较轻,肝窦无扩张,肝巨噬细胞(Kupffer)活跃度低,吞噬体不多见。而对照组~*中细胞间隙明显增宽,窦间隙显著扩张,线粒体肿胀明显,线粒体嵴减少或消失,内质网扩张并有包裹线粒体现象,微绒毛丧失,Kupffer细胞侵入肝实质,吞噬活跃,局部见坏死区。
3.肝再生测定:DA组与对照组~*比较,肝湿重、肝再生率明显增加,术后7d有显著性差异(P＜0.05)。
4.血清肝功能检测:DA组与对照组~*比较,谷丙转氨酶(ALT)、谷草转氨酶(AST)值明显降低,术后2h、1d、3d均有显著性差异(P＜0.05)。
5.免疫组化法检测细胞核增值抗原(PCNA):DA组与对照组~*比较,PCNA阳性率明显增多,术后3d和5d有显著性差异(P＜0.05)。
6.细胞凋亡检测(TUNEL法):DA组与对照组~*比较,凋亡指数(AI)术后2h和1d明显减少(P＜0.05)。
7.肝组织白介素-1β(IL-1β)测定:DA组与对照组~*比较,术后2h和1d明显降低(P＜0.05)。
8.血清、肝组织肿瘤坏死因子-α(TNF-α)测定:血清中,TNF-α表达较肝组织明显降低;DA组与对照组~*比较,TNF-α水平有所降低,但多数无显著性差异(P＞0.05)。肝组织中,DA组与对照组~*比较,TNF-α明显下降,术后2h、1d和3d均有显著性差异(P＜0.05)。
9.血清、肝组织白介素-6(IL-6)测定:DA组与对照组~*比较,血清和肝组织中IL-6均明显升高,术后2h、1d和3d有显著性差异(P＜0.05)。
10.肝组织NF-κB活性的变化:DA组与对照组~*比较,术后2h和1d明显降低(P＜0.05)。
结论:
1.应用mitoKATP开放剂二氮嗪,可以减轻大鼠部分肝移植包括小体积肝移植术后缺血再灌注损伤,其机制可能是通过降低IL-1β和TNF-α等细胞因子的表达,抑制了NF-κB的活性来减轻肝组织损伤。
2.二氮嗪在改善移植物缺血再灌注损伤的同时,能够增加大鼠部分肝移植包括小体积肝移植的肝重、肝再生率和细胞核增值抗原(PCNA)的表达,其机制可能是通过升高IL-6的表达、并维持一定的TNF-α和NF-κB的水平,来促进肝再生。
3.应用二氮嗪所产生的改善缺血再灌注损伤、促进肝再生的效应,可以被5-羟基癸酸盐特异性关闭,证明其作用位点在于mitoKATP。提示mitoKATP可能成为改善小体积肝移植疗效的一个治疗靶点。
PartⅠEstablishing the Model of 30%Small-for-Size Liver Transplantation in Rats
Objective:To establish the model of 30%small-for-size liver transplantation in rats, as a foundation for the forthcoming research on ischemia reperfusion injury(IRI) in small-for-size liver transplantation and liver regeneration post-operatively.
Methods:100 pairs of Sprague-Dawley(SD) rats were performed orthotopic liver transplantation(OLT) by using Kamada's 2-cuff method,among which 50 pairs were performed 100%OLT before formal experiment,25 pairs were performed 60%OLT, and 25 pairs were performed 30%OLT.The graft liver was procured by situ rapid perfusion through abdominal aorta.The time for cold ischemia was set as 1 hour. Besides,we randomly selected 10 rats to perform hepatectomy,5 rats by 40%and 5 rats by 70%,and observed the survival rate.
Results:For the groups of 60%and 30%partial liver transplantation,the weight ratios of donated liver to receipt liver were 59.70±3.21%and 31.92±2.43%. 92%(23/25) and 72%(18/25) survived for 2 days or above after transplantation. 72%(18/25) and 64%(16/25) were still alive 1 week after transplantation.For the group of hepatectomy,the weight ratios of the excised liver to the theoretical original liver were 42.51±3.62%and 66.83±4.37%,and 100%were alive 1 week after transplantation.
Conclusion:Based on past animal experiments,we successfully established the model of 60%and 30%partial liver transplantation in rats with improvement in methods.This model is stable,workable and replicable with high probability of success.The rat model of partial liver transplantation has similar operation procedures and pathological and physiological change as compared with clinical partial liver transplantation,such as living donor liver transplantation;and is an ideal way for research on improving IRI in liver transplantation and promoting liver regeneration.
PartⅡExperiment Study on the Role of Diazoxide Preconditioning in Alleviation Ischemia Reperfusion Injury after Rat Small-for-Size Liver Transplantation and the Impact on Liver Regeneration
Objective:To investigate the protection mechanism of mitochondrial ATP-sensitive potassium channels(mitoKATP) opening on IRI after rat partial liver transplantation, especially small-for-size liver transplantation,and the impacts on liver regeneration; and offer a better practice to relieve IRI and promote liver regeneration for clinical partial liver transplantation,such as living donor liver transplantation.
Methods:The animal model was built as the same way of PartⅠ.300 SD rats with the average weight of 220-300g were randomized into 150 pairs,and divided to 6 groups with 25 pairs in each group.Receipts should be heavier than donators by 10-20g.The 6 groups were:100%orthotopic liver transplantation(Group A),60%partial liver transplantation(Group C),60%partial liver transplantation with diazoxide(Group DA1),60%partial liver transplantation with 5-hydroxidecaprate and diazoxide (Group HD-DA),30%small-for-size liver transplantation(Group S),and 30% small-for-size liver transplantation with diazoxide(Group DA2).For Group DA, receipts received diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.For Group HD-DA,receipts received 5-HD(5mg/kg) 45 min preoperatively,and diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.Besides,50 SD rats with the average weight of 220-300g were randomized into 2 groups with 25 rats in each group.The 2 groups were respectively performed 40%hepatectomy(Group B1) and 70%hepatectomy(Group B2).
For each experimental group,5 rats were killed(n=5) respectively at 5 points of time,which were 2 hours,1 day,3 days,5 days,and 7 days postoperatively. Observation and detection includes liver function index(ALT,AST),wet weight of liver tissue postoperatively and ratio of liver regeneration,pathological examination by light microscope,examination of ultra-micro structure of cell by electro-microscope,proliferating cell nuclear antigen(PCNA) index,apoptosis of liver cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL),cytokines such as tumor necrosis fator-alpha(TNF-α),interleukin-6(IL-6), and interleukin-1β(IL-1β) in liver tissue and serum by enzyme-linked immunosorbent assay(ELISA),and the activity of NF-κB in liver tissue by ELISA.
Results:
1.Pathological examination by light microscope:as compared with the control group ~*,the liver cells of Group DA showed less swelling,degeneration and necrosis;the vacuolus showed less regeneration;the structure of hepatic lobule was less disorderly; inflammation was less serious.More cell division was found in Group DA than the control group.
2.Ultra-micro structure of cell examined by electro-microscope:the cell structure of Group DA was complete,swelling of mitochondria was less,intercellular space and sinus hepaticus was less distending,Kupffer cell was less active,and phagosome was less observed.However,mitochondrial crista and microvilli of the control group was less observed,endocytoplasmic reticulum was more swelling,Kupffer cell was more active,and some parts were necrosis.
3.Index of liver regeneration:as compared with the control group,liver wet weight of Group DA is larger and the liver regeneration ratio increased distinctly.Significant difference was noted 7 days post-operatively(P＜0.05).
4.Index of liver function via serum:as compared with the control group,alanine aminotransferase(ALT) and aspartate aminotransferase(AST) of Group DA decreased distinctly,and significant difference was noted 2 hours,1 day,and 3 days post-operatively(P＜0.05).
5.Proliferating cell nuclear antigen(PCNA) by immunohistochemistry:as compared with the control group,positive ratio of PCNA of Group DA increased distinctly,and significant difference was noted 3 days and 5 days post-operatively(P＜0.05).
6.Apoptosis index(AI) by TUNEL:as compared with the control group,AI of Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).
7.Expression of interleukin-1β(IL-1β) in liver tissue:as compared with the control group,IL-1βof Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).
8.Expression of tumor necrosis fator-alpha(TNF-α) in serum and liver tissue:TNF-αin serum significant decreased as compared with that in liver tissue.As compared with the control group,TNF-αof Group DA decreased but no significant difference (P＞0.05).For liver tissue,TNF-ot of Group DA decreased significantly as compared with the control group and significant difference was noted 2 hours,1 day and 3 days post-operatively(P＜0.05).
9.Expression of interleukin-6(IL-6) in serum and liver tissue:as compared with the control group,IL-6 in both serum and liver tissue of Group DA increased distinctly and significant difference was noted 2 hours,1 day and 3 days post-operatively (P＜0.05).
10.Changes in activity of NF-κB in liver tissue:as compared with the control group, NF-κB of Group DA decreased significantly 2 hours and 1 day post-operatively (P＜0.05).
Conclusion:
1.DA,which opens the mitoKATP,can relieve IRI after partial liver transplantation, including small-for-size graft,by reducing IL-1βand TNF-αand restraining the activity of NF-κB.
2.DA could relieve IRI after transplantation;in the meantime,it could increase the weight,regeneration ratio and PCNA of the transplanted liver,the mechanism of which may be promoting liver regeneration by increasing IL-6 and maintaining essential expression of TNF-αand NF-κB.
3.The effect of improving IRI and promoting liver regeneration by using DA could be specially blocked by 5-HD,and it proves that DA produces the effect through mitoKATP and implies that mitoKATP might be a target for improving the treatment effect of partial liver transplantation,including small-for-size graft liver.
线粒体膜上存在对ATP浓度敏感的钾离子通道——mitoKATP,二氮嗪(DA)可以特异性开放该通道,而5-羟基癸酸盐(5-HD)则能将其特异性关闭。大量研究提示,mitoKATP通道开放在心肌、脑、肺、肾缺血再灌注损伤中均有保护作用,但目前国内外有关mitoKATP对肝脏的缺血再灌注损伤影响的研究很少。
通过二氮嗪(DA)预处理供体,观察mitoKATP开放对大鼠60%比例部分肝移植和30%比例小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。
第一部分大鼠30%比例小体积肝移植模型的建立
目的:建立稳定的大鼠30%比例小体积肝移植模型,为后续的移植肝缺血再灌注损伤(IRI)和小体积供肝移植后肝再生的实验研究打下基础。
方法:本部分行100例大鼠原位肝移植术,其中预实验50例,60%比例部分肝移植25例,30%比例小体积肝移植25例,全部采用“二袖套法”完成。全部手术均采用经腹主动脉插管原位快速灌注获取供肝,冷缺血时间均设定为1小时。另外各取5只大鼠,行40%和70%比例肝叶切除,分别观察存活率。
结果:60%和30%比例移植组,供肝与原肝重比分别为59.70±3.21%和31.92±2.43%,手术成功率(术后存活2天以上)分别为92%(23/25)和72%(18/25),1周存活率分别为72%(18/25)和64%(16/25)。肝切除组中,切除肝重与理论原肝重比分别为42.51±3.62%和66.83±4.37%,1周存活率为100%。
结论:在以往动物模型的基础上,通过改进操作方法,成功建立了大鼠60%比例部分肝移植和30%比例小体积肝移植模型。该模型稳定,手术成功率较高,便于操作,容易复制。大鼠部分肝移植模型尤其是小体积模型,与临床部分肝移植如活体肝移植有着相似的手术操作过程和病理生理变化,是研究改善肝移植缺血再灌注损伤、促进肝再生的理想途径。
第二部分二氮嗪预处理对大鼠小体积肝移植缺血再灌注损伤的保护机制研究及对肝再生的影响
目的:研究线粒体ATP敏感的钾通道(mitoKATP)开放,对大鼠部分肝移植尤其是小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。为临床部分肝移植如活体肝移植,寻找减轻缺血再灌注损伤、促进术后肝再生的治疗靶点和途径。
方法:大鼠肝移植模型的建立方法同第一部分。SD大鼠300只,体重220～300g,随机配对成150对,随机分成6组,每组25对,供体体重应比受体轻10～20g。分组为全肝移植组(A组),60%比例部分肝移植组(C组),60%比例部分肝移植+二氮嗪组(DAl组),60%比例部分肝移植+5-羟基癸酸盐+二氮嗪组(HD-DA组),30%比例小体积肝移植组(S组),30%比例小体积肝移植+二氮嗪组(DA2组)。DA组供体术前30分钟腹腔注射二氮嗪(5mg/kg),再行手术;HD-DA组供体术前45分钟腹腔注射5-HD(5mg/kg),15分钟后腹腔注射二氮嗪(5mg/kg),再行手术。另取SD大鼠50只,体重220～300g,随机分成2组,每组25只。分组为40%比例肝切除组(B1组)和70%比例肝切除组(B2组)。
各手术组均取5个时间点,分别为术后2小时、1天、3天、5天和7天,每个时间点各取5例处死取材(n=5)。检测血清肝功能指标(ALT、AST),肝组织湿重并计算肝再生率,光镜下HE染色病理检查,电镜下超微结构检查,增殖细胞核抗原标记指数(PCNA),TUNEL法肝细胞凋亡指数,血清、肝组织细胞因子TNF-α、IL-6、IL-1β(ELISA法)检测,肝组织NF-κB活性检测(ELISA法)。
结果:
1.光镜下病理检查:DA组与对照组~*比较,肝细胞肿胀变性坏死及空泡样变性、肝小叶结构排列紊乱、汇管区炎症都较轻。DA组在术后7d分裂相多见,而对照组~*则减少。
2.电镜下超微结构检查:DA组中细胞结构完整,线粒体肿胀及细胞间隙扩张程度较轻,肝窦无扩张,肝巨噬细胞(Kupffer)活跃度低,吞噬体不多见。而对照组~*中细胞间隙明显增宽,窦间隙显著扩张,线粒体肿胀明显,线粒体嵴减少或消失,内质网扩张并有包裹线粒体现象,微绒毛丧失,Kupffer细胞侵入肝实质,吞噬活跃,局部见坏死区。
3.肝再生测定:DA组与对照组~*比较,肝湿重、肝再生率明显增加,术后7d有显著性差异(P＜0.05)。
4.血清肝功能检测:DA组与对照组~*比较,谷丙转氨酶(ALT)、谷草转氨酶(AST)值明显降低,术后2h、1d、3d均有显著性差异(P＜0.05)。
5.免疫组化法检测细胞核增值抗原(PCNA):DA组与对照组~*比较,PCNA阳性率明显增多,术后3d和5d有显著性差异(P＜0.05)。
6.细胞凋亡检测(TUNEL法):DA组与对照组~*比较,凋亡指数(AI)术后2h和1d明显减少(P＜0.05)。
7.肝组织白介素-1β(IL-1β)测定:DA组与对照组~*比较,术后2h和1d明显降低(P＜0.05)。
8.血清、肝组织肿瘤坏死因子-α(TNF-α)测定:血清中,TNF-α表达较肝组织明显降低;DA组与对照组~*比较,TNF-α水平有所降低,但多数无显著性差异(P＞0.05)。肝组织中,DA组与对照组~*比较,TNF-α明显下降,术后2h、1d和3d均有显著性差异(P＜0.05)。
9.血清、肝组织白介素-6(IL-6)测定:DA组与对照组~*比较,血清和肝组织中IL-6均明显升高,术后2h、1d和3d有显著性差异(P＜0.05)。
10.肝组织NF-κB活性的变化:DA组与对照组~*比较,术后2h和1d明显降低(P＜0.05)。
结论:
1.应用mitoKATP开放剂二氮嗪,可以减轻大鼠部分肝移植包括小体积肝移植术后缺血再灌注损伤,其机制可能是通过降低IL-1β和TNF-α等细胞因子的表达,抑制了NF-κB的活性来减轻肝组织损伤。
2.二氮嗪在改善移植物缺血再灌注损伤的同时,能够增加大鼠部分肝移植包括小体积肝移植的肝重、肝再生率和细胞核增值抗原(PCNA)的表达,其机制可能是通过升高IL-6的表达、并维持一定的TNF-α和NF-κB的水平,来促进肝再生。
3.应用二氮嗪所产生的改善缺血再灌注损伤、促进肝再生的效应,可以被5-羟基癸酸盐特异性关闭,证明其作用位点在于mitoKATP。提示mitoKATP可能成为改善小体积肝移植疗效的一个治疗靶点。
PartⅠEstablishing the Model of 30%Small-for-Size Liver Transplantation in Rats
Objective:To establish the model of 30%small-for-size liver transplantation in rats, as a foundation for the forthcoming research on ischemia reperfusion injury(IRI) in small-for-size liver transplantation and liver regeneration post-operatively.
Methods:100 pairs of Sprague-Dawley(SD) rats were performed orthotopic liver transplantation(OLT) by using Kamada's 2-cuff method,among which 50 pairs were performed 100%OLT before formal experiment,25 pairs were performed 60%OLT, and 25 pairs were performed 30%OLT.The graft liver was procured by situ rapid perfusion through abdominal aorta.The time for cold ischemia was set as 1 hour. Besides,we randomly selected 10 rats to perform hepatectomy,5 rats by 40%and 5 rats by 70%,and observed the survival rate.
Results:For the groups of 60%and 30%partial liver transplantation,the weight ratios of donated liver to receipt liver were 59.70±3.21%and 31.92±2.43%. 92%(23/25) and 72%(18/25) survived for 2 days or above after transplantation. 72%(18/25) and 64%(16/25) were still alive 1 week after transplantation.For the group of hepatectomy,the weight ratios of the excised liver to the theoretical original liver were 42.51±3.62%and 66.83±4.37%,and 100%were alive 1 week after transplantation.
Conclusion:Based on past animal experiments,we successfully established the model of 60%and 30%partial liver transplantation in rats with improvement in methods.This model is stable,workable and replicable with high probability of success.The rat model of partial liver transplantation has similar operation procedures and pathological and physiological change as compared with clinical partial liver transplantation,such as living donor liver transplantation;and is an ideal way for research on improving IRI in liver transplantation and promoting liver regeneration.
PartⅡExperiment Study on the Role of Diazoxide Preconditioning in Alleviation Ischemia Reperfusion Injury after Rat Small-for-Size Liver Transplantation and the Impact on Liver Regeneration
Objective:To investigate the protection mechanism of mitochondrial ATP-sensitive potassium channels(mitoKATP) opening on IRI after rat partial liver transplantation, especially small-for-size liver transplantation,and the impacts on liver regeneration; and offer a better practice to relieve IRI and promote liver regeneration for clinical partial liver transplantation,such as living donor liver transplantation.
Methods:The animal model was built as the same way of PartⅠ.300 SD rats with the average weight of 220-300g were randomized into 150 pairs,and divided to 6 groups with 25 pairs in each group.Receipts should be heavier than donators by 10-20g.The 6 groups were:100%orthotopic liver transplantation(Group A),60%partial liver transplantation(Group C),60%partial liver transplantation with diazoxide(Group DA1),60%partial liver transplantation with 5-hydroxidecaprate and diazoxide (Group HD-DA),30%small-for-size liver transplantation(Group S),and 30% small-for-size liver transplantation with diazoxide(Group DA2).For Group DA, receipts received diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.For Group HD-DA,receipts received 5-HD(5mg/kg) 45 min preoperatively,and diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.Besides,50 SD rats with the average weight of 220-300g were randomized into 2 groups with 25 rats in each group.The 2 groups were respectively performed 40%hepatectomy(Group B1) and 70%hepatectomy(Group B2).
For each experimental group,5 rats were killed(n=5) respectively at 5 points of time,which were 2 hours,1 day,3 days,5 days,and 7 days postoperatively. Observation and detection includes liver function index(ALT,AST),wet weight of liver tissue postoperatively and ratio of liver regeneration,pathological examination by light microscope,examination of ultra-micro structure of cell by electro-microscope,proliferating cell nuclear antigen(PCNA) index,apoptosis of liver cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL),cytokines such as tumor necrosis fator-alpha(TNF-α),interleukin-6(IL-6), and interleukin-1β(IL-1β) in liver tissue and serum by enzyme-linked immunosorbent assay(ELISA),and the activity of NF-κB in liver tissue by ELISA.
Results:
1.Pathological examination by light microscope:as compared with the control group ~*,the liver cells of Group DA showed less swelling,degeneration and necrosis;the vacuolus showed less regeneration;the structure of hepatic lobule was less disorderly; inflammation was less serious.More cell division was found in Group DA than the control group.
2.Ultra-micro structure of cell examined by electro-microscope:the cell structure of Group DA was complete,swelling of mitochondria was less,intercellular space and sinus hepaticus was less distending,Kupffer cell was less active,and phagosome was less observed.However,mitochondrial crista and microvilli of the control group was less observed,endocytoplasmic reticulum was more swelling,Kupffer cell was more active,and some parts were necrosis.
3.Index of liver regeneration:as compared with the control group,liver wet weight of Group DA is larger and the liver regeneration ratio increased distinctly.Significant difference was noted 7 days post-operatively(P＜0.05).
4.Index of liver function via serum:as compared with the control group,alanine aminotransferase(ALT) and aspartate aminotransferase(AST) of Group DA decreased distinctly,and significant difference was noted 2 hours,1 day,and 3 days post-operatively(P＜0.05).
5.Proliferating cell nuclear antigen(PCNA) by immunohistochemistry:as compared with the control group,positive ratio of PCNA of Group DA increased distinctly,and significant difference was noted 3 days and 5 days post-operatively(P＜0.05).
6.Apoptosis index(AI) by TUNEL:as compared with the control group,AI of Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).
7.Expression of interleukin-1β(IL-1β) in liver tissue:as compared with the control group,IL-1βof Group DA decreased significantly 2 hours and 1 day post-operatively(P＜0.05).
8.Expression of tumor necrosis fator-alpha(TNF-α) in serum and liver tissue:TNF-αin serum significant decreased as compared with that in liver tissue.As compared with the control group,TNF-αof Group DA decreased but no significant difference (P＞0.05).For liver tissue,TNF-ot of Group DA decreased significantly as compared with the control group and significant difference was noted 2 hours,1 day and 3 days post-operatively(P＜0.05).
9.Expression of interleukin-6(IL-6) in serum and liver tissue:as compared with the control group,IL-6 in both serum and liver tissue of Group DA increased distinctly and significant difference was noted 2 hours,1 day and 3 days post-operatively (P＜0.05).
10.Changes in activity of NF-κB in liver tissue:as compared with the control group, NF-κB of Group DA decreased significantly 2 hours and 1 day post-operatively (P＜0.05).
Conclusion:
1.DA,which opens the mitoKATP,can relieve IRI after partial liver transplantation, including small-for-size graft,by reducing IL-1βand TNF-αand restraining the activity of NF-κB.
2.DA could relieve IRI after transplantation;in the meantime,it could increase the weight,regeneration ratio and PCNA of the transplanted liver,the mechanism of which may be promoting liver regeneration by increasing IL-6 and maintaining essential expression of TNF-αand NF-κB.
3.The effect of improving IRI and promoting liver regeneration by using DA could be specially blocked by 5-HD,and it proves that DA produces the effect through mitoKATP and implies that mitoKATP might be a target for improving the treatment effect of partial liver transplantation,including small-for-size graft liver.
引文
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