Calreticulin Protects Microvasular Endothelial Cell Against Hypoxia/Reoxygenation Injury

Backgroud and Objective: Restoration of blood flow (reperfusion) timely to the ischemic myocardium is the preferred strategy to rescue ischemic myocardium after myocardial infarction. However, the efficacy of the therapy is attenuated by ischemia/reperfusion (I/R) injury. No-reflow phenomenon is a major manifestation of I/R injury and a key contributor to final myocardial injury.Recently, autophagy garners the attention associated with endothelial cells injury,Therefore, it is important to explore the pharmacological agents targeting on autophagy of MECs against I/R injury. It is reported that expression of CRT in the surface of cell membrane is associated with autophagy suppression. However, it is unknown whether exogenous CRT ameliorates H/R-induced MECs injury by inhibiting excessive autophagy. A rat model of I/R injury in vivo was used to observe the effects of CRT pretreatment on myocardial I/R injury. A model of H/R-induced MECs injury in vitro was used to observe the effects of CRT pretreatment on autophagy against H/R injury.Methods1. Establishment of a rat I/R-model and MECs model of H/R injury: The left anterior descending artery was ligated for 45min, followed by reperfusion for 24 h.Human MECs were subjected to 8 h-hypoxia followed 16 h-reoxygenation.2. Analysis of cell viability, LDH activity and TnT content: Cell viability were measured by CCK-8 kit. LDH activity and TnT level were measured by using the automatic biochemical analyzer.3. Myocardial structure: The myocardial structure was observed by hematoxylin-eosin staining and transmission electron microscope.4. Autophagosome formation: Autophagosome formation was observed by using immunofluorescent staining of LC3.5. Expression of autophagy-related protein: Western blot was employed to detect protein expression of p-mTOR, Beclinl, and LC3.6. Statistical analysis: The SPSS v13.0 program was used for statistical analysis.Values are presented as mean ± SD. For multiple-group comparisons,one-way analysis of variance followed by Newman-Keuls post hoc analysis were performed. P < 0.05 was considered to be statistically significant.Results1. CRT pretreatment attenuated myocardial I/R injury1.1 CRT pretreatment significantly reduced myocardial infarct size and improved cardiac function after I/R injury1.2 CRT alleviated I/R-induced cardiac injury: CRT at high dose alleviated LDH leakage and TnT level and myocardial structure disorder, myocardial necrosis,hemorrhage, and inflammatory cells infiltration.1.3 CRT alleviated I/R-induced autophagy: The effect of CRT on Beclin 1 expression was detected by western blot.2. CRT attenuated H/R-induced MECs injury2.1 CRT pretreatment significantly attenuated LDH leakage.2.2 CRT pretreatment significantly increased the viability of cells exposed to H/R2.3 CRT pretreatment alleviated the ultrastructural lesions, including cells swelling, endoplamic reticulum dilation, and mitochondria vacuolization.3. CRT attenuated H/R-induced MECs injury through mTOR pathway3.1 Rapamycin aggravated H/R-induced MECs injury.3-MA significantly increased the viability of cells exposed to H/R.3.2 CRT suppressed the autophagy induced by rapamycin with or without H/R.3.3 CRT alleviated autophagosome formation and ultrastructural lesions.3.4 CRT pretreatment attenuated H/R-induced MECs injury by up-regulating mTOR phosphorylation, diminishing Beclinl expression and LC3?/LC3?.Conclusion1.CRT pretreatment attenuated autophagy and myocardial ischemic reperfusion injury2. CRT pretreatment allievated H/R-induced MECs through inhibiting the autophagy via mTOR pathway.