氨基葡萄糖通过OGT-RIPK3轴抑制缺血/再灌注引起的心肌细胞死亡

Glucosamine inhibits cardiomyocyte death induced by myocardial ischemia/reperfusion via OGT-RIPK3 axis

目的 探讨氨基葡萄糖(glucosamine, GlcN)对心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MI/RI)中细胞凋亡和程序性坏死的调控作用及其机制。方法 将24只体质量220~250 g雄性SD大鼠按随机数字表法分为4组:假手术组、缺血再灌注组、缺血再灌注+氨基葡萄糖组(氨基葡萄糖组)和缺血再灌注+氨基葡萄糖+OGT-IN-2共处理组(氨基葡萄糖+OGT-IN-2组),每组6只。经开胸行左前降支结扎术30 min后再灌注3 h以收集MI/RI心脏组织和血清样本,假手术组只穿线不结扎。HE染色观察各组大鼠心肌组织损伤程度,ELISA检测各组大鼠血清中心肌损伤标志物CK-MB、LDH水平,TUNEL染色观察细胞凋亡情况,MLKL免疫组化评估程序性坏死水平,Western blot检测各组心肌组织中p-RIPK3、t-RIPK3、p-MLKL、cleaved Caspase-9、cleaved Caspase-3、N-乙酰氨基葡萄糖转移酶(N-acetylglucosaminyltransferase, OGT)的蛋白表达变化。结果 GlcN可明显改善MI/RI后心肌组织损伤程度并降低心肌损伤标志物CK-MB、LDH水平(P<0.05),而OGT特异性抑制剂OGT-IN-2则明显削弱GlcN的心肌保护作用(P<0.05)。TUNEL染色和MLKL免疫组化结果显示GlcN可同时减少细胞凋亡和程序性坏死发生,而OGT-IN-2则明显削弱GlcN对凋亡和程序性坏死的抑制作用。Western blot结果显示,GlcN可上调OGT活性后抑制RIPK3介导的p-RIPK3、p-MLKL、cleaved Caspase-9和cleaved Caspase-3表达(P<0.05),而OGT-IN-2则逆转GlcN的作用(P<0.05)。结论 GlcN可以有效减轻MI/RI,其机制可能是通过上调OGT表达从而抑制RIPK3介导的细胞凋亡和程序性坏死。

Objective To investigate the regulatory effect and specific mechanism of glucosamine(GlcN)on cardiomyoctye apoptosis and necroptosis after myocardial ischemia/reperfusion(I/R)injury.Methods Twenty-four male SD rats(220~250 g)were randomly divided into sham operation group,I/R group,I/R+GlcN group(GlcN group)and I/R+GlcN+OGT-IN-2 group(GlcN+OGT-IN-2 group),with 6 animals in each group.For the mice of the I/R model groups,the left anterior descending artery was ligated for 30 min after thoracotomy followed by perfusion for 3 h,and then the injured cardiac tissues and serum samples were collected.While,the mice from the sham group were inflicted with only threaded but no ligation.HE staining was used to observe the severity of myocardial tissue damage,and ELISA was employed to measure the contents of serum myocardial injury markers CK-MB and LDH.TUNEL assay and immunohistochemical assay for mixed lineage kinase-like(MLKL)protein were applied respectively to measure cardiomyocyte apoptosis and necroptosis.Western blotting was performed to detect the protein changes of p-RIPK3,t-RIPK3,p-MLKL,cleaved Caspase-9,cleaved Caspase-3,and N-acetylglucosaminyltransferase(OGT)in myocardial tissues of each group.Results GlcN treatment significantly improved the severity of myocardial injury and reduced the levels of myocardial injury markers CK-MB and LDH(P<0.05),while the addition of OGT-specific inhibitor OGT-IN-2 greatly weakened the cardioprotective effect of GlcN(P<0.05).TUNEL assay and MLKL immunohistochemistry confirmed that GlcN reduced cardiomyocyte apoptosis and necroptosis at the same time,while OGT-IN-2 also significantly attenuated the inhibitory effect of GlcN on cell death.Western blotting suggested that GlcN enhanced the activity of OGT and then inhibited the expression of p-RIPK3,p-MLKL,cleaved Caspase-9 and cleaved Caspase-3 through RIPK3 mediation(P<0.05),while OGT-IN-2 reversed the above effects of GlcN(P<0.05).Conclusion GlcN can effectively alleviate myocardial I/R injury,and its mechanism may be due to its inhibiting RIPK3-mediated apoptosis and necroptosis by up-regulating the expression of OGT.