下调硫酸乙酰肝素酶减轻大鼠心肌缺血再灌注损伤的机制

doi:10.3969/j.issn.1006-5725.2023.21.012

摘要/Abstract

摘要：

目的探讨硫酸乙酰肝素酶（heparinase， HPSE）对大鼠心肌缺血再灌注损伤（Myocardial Ischemia-Reperfusion，MI/R）的影响及机制。方法选取60只SD大鼠，随机分为对照组、I/R组，I/R+shRNA-NC组和I/R+Heparanase-shRNA组，每组15只。对照组开胸后仅穿线但不结扎，其他各组大鼠均采用冠脉左前降支结扎手术制备MI/R模型。采用蛋白质印迹实验（WB）检测各组大鼠心脏功能及心肌损伤标记物；采用HE染色和TUNEL染色观察大鼠心肌病理情况；采用WB检测凋亡相关蛋白（Caspsae-3、Bax、Bcl-2）和线粒体未折叠蛋白反应（mitochondrial unfolded protein response，UPRmt）标志蛋白（LonP₁、HSP70）的表达情况；采用试剂盒检测超氧化物歧化酶（SOD）、丙二醛（MDA）的含量；采用酶联免疫吸附法（ELISA）检测外周血中白细胞介素-6（IL-6），肿瘤坏死因子-α（TNF-α）含量水平。结果 HE染色发现，对照组大鼠心肌组织、肌外膜、心肌纤维均完整且正常；I/R组大鼠和I/R+shRNA-NC组大鼠心肌纤维弯曲，肌红蛋白溶解，同时心肌肌细胞核固缩、溶解，肌束膜破裂；I/R+Heparanase-shRNA组大鼠心肌外膜较为完整、心肌纤维弯曲显著改善。相比对照组，I/R组肌酸激酶同工酶（CK-Mb）、心肌肌钙蛋白（cTnI）、Mb、Caspase-3蛋白、Bax蛋白、MDA、IL-6、TNF-α、HSP70水平显著升高，HR、LVEF、LVWT、Bcl-2蛋白、SOD、LonP₁蛋白水平显著降低，差异均有统计学意义；与I/R+shRNA-NC组比较，I/R+Heparanase-shRNA组CK-Mb、cTnI、Mb、Caspase-3蛋白、Bax蛋白、MDA、IL-6、TNF-α水平显著降低，而HR、LVEF、LVWT、Bcl-2蛋白、SOD、HSP70和LonP₁水平显著升高，差异均有统计学意义。结论下调HPSE可能通过抑制UPRmt信号通路减轻细胞氧化应激、炎症反应及细胞凋亡，从而缓解大鼠心肌缺血再灌注损伤。

关键词: 心肌缺血再灌注, 硫酸乙酰肝素酶, 线粒体未折叠蛋白反应

Abstract:

Objective To explore the effects of heparinase （HPSE） on myocardial apoptosis， inflammatory response and oxidative damage in myocardial ischemia reperfusion （MI/R） rats. Methods Sixty SD rats were randomly assigned （1∶1∶1∶1） tocontrol group， I/R group， I/R+shRNA-NC group and I/R+Heparanase-shRNA group. The control group was only threaded without ligation after thoracotomy， and the MI/R model was prepared by ligation of the left anterior descending branch of coronary artery. The western blot assay （WB） was used to detect heart function and markers of heart damage in each group. HE staining and TUNEL staining were used to observe the myocardial pathology. The expression of apoptosis-related proteins （Caspase-3， Bax， Bcl-2） and UPRmt marker proteins （LonP₁， HSP70） were detected by WB. The contents of superoxide dismutase （SOD） and malondialdehyde （MDA） were detected by kit. The levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in peripheral blood were detected by enzyme-linked immunosorbent assay （ELISA）. Results HE staining results showed that the myocardium， epimyma and myocardial fibers were intact and normal in control group. However， the myocardial fibers were bent， myoglobin was dissolved， myocardial muscle nucleus was pyknotic and dissolved， and muscle bundle membrane was ruptured in I/R group and I/R+shRNA-NC group. The epimyocardium was intact in I/R+Heparanase-shRNA group. Moreover， the myocardial fiber bending was significantly improved in I/R+Heparanase-shRNA group. Additionally， compared with the control group， the expression levels of CK-MB， cTnI， Mb， Caspase-3 protein， Bax protein， MDA， IL-6， TNF-α and HSP70 in I/R group were significantly increased. And the difference was statistically significant. Furthermore， the expression levels of HR， LVEF， LVWT， Bcl-2 protein， SOD and LonP₁ protein were significantly decreased. Additionally， compared with the I/R+shRNA-NC group， the expression levels of CK-MB， cTnI， Mb， Caspase-3 protein， Bax protein， MDA， IL-6 and TNF-α in I/R+Heparanase-shRNA group were significantly decreased. And the difference was statistically significant. However， the expression levels of HR， LVEF， LVWT， Bcl-2 protein， SOD， LonP₁ and HSP70 were significantly increased. And the difference was statistically significant. Conclusion Down-regulation of HPSE alleviates myocardial injury in ischemia-reperfusion rats by inhibiting oxidative stress， inflammation， and apoptosis. The mechanism may be related to the inhibition of UPRmt signaling pathway.

Key words: myocardial ischemia reperfusion, heparanase, mitochondrial unfolded protein response

中图分类号:

R542.2

李招兵,刘雨露,黄云辉. 下调硫酸乙酰肝素酶减轻大鼠心肌缺血再灌注损伤的机制[J]. 实用医学杂志, 2023, 39(21): 2761-2767.

Zhaobing LI,Yulu LIU,Yunhui HUANG. The mechanism of down⁃regulation of HPSE alleviating myocardial ischemia⁃reperfusion injury in rats[J]. The Journal of Practical Medicine, 2023, 39(21): 2761-2767.

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