Liu Guodong, Zhang Hongmei, Hao Fengyun, Hao Jing, Pan Lixiao, Zhao Qing, Wo Jinshan
Department of Cardiovascular Surgery, Qingdao West Coast Hospital, the Affiliated Hospital of Qingdao University, Qingdao, China.
Department of Thyroid Surgery, Qingdao West Coast Hospital, the Affiliated Hospital of Qingdao University, Qingdao, China.
Cell Physiol Biochem. 2018;45(3):1003-1012. doi: 10.1159/000487295. Epub 2018 Feb 5.
BACKGROUND/AIMS: Ischemia-reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury.
Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control) or UW solution containing recombinant human apolipoprotein-J (hr clusterin) for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1β, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR assays; IL-1β and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses.
Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1β, and TNF-α, and the pro-apoptotic gene Bax, while it enhanced the expression of the anti-apoptotic gene Bcl-xL in vitro and in vivo. Clusterin inhibited cell apoptosis/death and reduced pre-inflammatory.
Clusterin is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects of clusterin are mediated by NF-κB signaling and Bax/Bcl-xL expression.
背景/目的：缺血再灌注（I/R）损伤是心脏移植过程中不可避免的事件，是移植物功能衰竭和受体长期生存率的关键因素。因此，迫切需要开发新的疗法来预防I/R损伤。簇集素是一种具有抗凋亡功能的异二聚体糖蛋白。在本研究中，我们使用体内大鼠模型和体外细胞培养系统，研究簇集素在心脏移植中对I/R损伤是否具有心脏保护作用，并探讨I/R损伤的潜在机制。
将野生型C57BL/6小鼠的心脏移植物保存在UW溶液（对照组）或含有重组人载脂蛋白-J（hr簇集素）的UW溶液中24小时。将保存的心脏移植到与供体同品系的受体小鼠体内72小时，然后取出心脏移植物进行组织病理学和基因表达分析。构建使用H9C2细胞或H9C2/簇集素cDNA细胞的体外缺血再灌注模型。通过蛋白质印迹法、逆转录-聚合酶链反应（RT-PCR）和定量RT-PCR检测心脏组织和H9C2细胞中簇集素、p65、Bax、Bcl-xL、IL-1β和TNF-α蛋白及mRNA的表达；通过酶联免疫吸附测定法检测IL-1β和TNF-α蛋白；通过电泳迁移率变动分析检测NF-κB活性；通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记和流式细胞术分析检测细胞凋亡。
冷I/R对野生型C57BL/6小鼠的心脏移植物造成严重的形态学心肌损伤，而hr簇集素保存的移植物损伤较小，表现为细胞凋亡/死亡减少、中性粒细胞浸润减少以及心脏正常结构的保留。簇集素在体外和体内均降低了p65、促炎因子IL-1β和TNF-α以及促凋亡基因Bax的表达，同时增强了抗凋亡基因Bcl-xL的表达。簇集素抑制细胞凋亡/死亡并减少炎症前反应。
簇集素是预防心脏移植中冷I/R损伤的一个有前景的靶点。本研究还表明，簇集素产生的保护作用是由NF-κB信号通路和Bax/Bcl-xL表达介导的。
