HMGB 2加重心肌梗死缺血再灌注损伤及机制研究

Myocardial ischemia-reperfusion (I/R) injury has been implicated in myocardial necrosis and dysfunction after acute myocardial infarction, however the mechanisms remain poorly unknown. In our study, we found that HMGB2, a novel ligand for receptor of advanced glycation end products (RAGE), has pro-inflammatory property. The levels of HMGB2 were significantly increased in rat with myocardial I/R injury and in H9C2 myocytes with hypoxia-reoxygenation. This protein, in turn, aggravated pathology of myocardial I/R injury in rats and strengthened inflammatory reaction in H9C2 with hypoxia-reoxygenation. Moreover, HMGB2 significantly upregulated autophagy-related Beclin-1 and LC3-II levels in these H9C2 cells. Collectively, accumulating evidence including our results suggests that HMGB2 could promote myocardial I/R injury through RAGE/TLR-4-mediated autophagy dysfunction mechanism. Thus, in the next step, we will verify our results of HMGB2-myocardial I/R injury using wide-type, HMGB2+/- and HMGB2-/-mice; and investigate whether RAGE-/- and TLR-4-/- mediates the impact of HMGB2; confirms whether dysfunction of autophagy mediates the pathophysiology of HMGB2 effect by Beclin-1-/- mice. Finally, induction of HMGB2 by HMGB1 will be probed using HMGB1(f)/(f) mice. In vitro as well as in vivo experiments will be performed to clarify the mechanisms regarding the aggravation of HMGB2 on myocardial I/R injury.

急性心肌缺血再灌注（I/R）损伤使心梗后预后变差，但机制了解有限。我们发现终末糖化产物受体（RAGE）新配体HMGB2具促炎症作用，它在大鼠心肌I/R损伤和心肌细胞缺氧复氧后显著升高（远高于HMGB1），添加HMGB2又加重大鼠心肌I/R损伤和心肌细胞缺氧复氧中炎症反应，并显著上调促自噬相关蛋白水平（Beclin-1、LC3-II等）。结合文献提示HMGB2直接和经RAGE/TLR-4受体介导促进自噬异常，结果导致心肌I/R损伤。后续将在体内外实验中：以野生型、HMGB2-KO、RAGE-KO、TLR-4-KO， Beclin-1-KO和HMGB1(f)/(f)等小鼠，通过导入对照和HMGB2，探讨HMGB2与心肌I/R损伤和心肌细胞缺氧复氧损伤的关系和致病途径；明确HMGB2是否经自噬异常导致上述疾病并阐明机制；了解HMGB1对HMGB2的诱导作用。阐明HMGB2加重心肌I/R损伤机制。

【背景】：免疫炎症在心肌梗死后的心脏缺血损伤中发挥重要作用。高迁移率族蛋白B（HMGB）参与了多种炎性疾病的发生发展。高迁移率族蛋白B2（HMGB2）是HMGB家族中的一员，其可被分泌至胞外并且可在血清中被检测到。本研究旨在探讨血清HMGB2水平与ST段抬高型心肌梗死(STEMI)患者心梗损伤严重程度和心梗后一个月内的主要心血管不良事件（MACE）发生率的关系，并为临床诊断、治疗及预后判断提供帮助。.【研究方法】：在临床方面，连续入组432个STEMI患者和312个冠脉造影阴性的患者。收集血清后用ELISA法检测血清中HMGB2水平；心超评估心功能；并进行一个月的随访，评估MACE发生率。在基础实验方面，制作大鼠心肌梗死模型，心肌内注射HMGB2（10ug）或生理盐水，应用心超评估心功能，TTC染色评估梗死面积，ROS染色检测活性氧，TUNEL染色评估凋亡细胞数目等。同时，体外培养H9C2大鼠心肌细胞系，利用siRNA敲低HMGB2，建立缺氧模型，通过荧光定量PCR、免疫印迹等方法检测HMGB2及其他炎症指标变化情况，并检测了缺氧后活性氧产生和凋亡细胞。.【主要结果】：心肌梗死患者血清中HMGB2水平远高于对照组（7.74 (3.01~13.90) vs 1.42 (0.78~2.51)，ng/ml）。HMGB2升高与心梗后MACE发生率正相关，与心梗后射血分数负相关。大鼠心肌梗死后，心肌内注射HMGB2增加心梗面积，心功能恶化，ROS增加，凋亡细胞增加，应用ROS抑制剂APO可以改善缺血损伤。进一步实验证明，HMGB2可能通过激动终末糖基化产物受体（RAGE）发挥上述作用。.【结论】：血清HMGB2水平与心肌梗死后缺血损伤的严重程度和心肌梗死后一月内MACE发生率正相关。其作用机制可能是HMGB2通过激动RGAE，增加ROS产生，促进了心肌梗死后缺血损伤。