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

Abstract

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.

Keywords

• ] glucosamine
• myocardial ischemia/reperfusion injury
• apoptosis
• necroptosis