Investigation Of The Effect And Mechanism Of Bcl6 In Cardiomyocyte Hypoxia Injury

Background Coronary heart disease(CHD),one of the leading causes of death worldwide,is mainly caused by myocardial ischemia and hypoxia due to coronary artery stenosis and occlusion.The primary consequence of myocardial ischemia and hypoxia is cardiomyocyte death,which induces contractile dysfunction,conduction dysfunction,and even sudden cardiac death.It's proved that oxidative stress,inflammation,autophagy,and apoptosis all involve the pathological process of cardiomyocyte hypoxia injury.The complex mechanisms of ischemic injury in the heart have not been fully elucidated,although great progress has been made in molecular and cellular mechanisms.Hence,it is of vital importance for scientists to find new therapeutic targets beneficial for human beings in clinical application.Bcl6 is a sequence-specific transcriptional repressor,originally characterized as a regulator of B-lymphocyte development and growth,contributes to the progression of various types of cancer.Research have shown that Bcl6 plays an important role in oxidative stress and cell apoptosis,and participates in many other diseases,but the effects of Bcl6 on cardiomyocyte hypoxia injury are not clear.Thus we hypothesized that Bcl6 participates in the pathological process of cardiac ischemia injury and may become a therapeutic target of cardiac ischemia injury.The aim of this stu dy was to elucidate the functional role and mechanism of Bcl6 in cardiomyocyte hypoxia injury.Further it's beneficial to explore the protective measures of myocardial ischemia and provide the experiment evidences for the drug development and clinical application of coronary heart disease.Objective To investigate the effect and the underlying mechanism of Bcl6 in cardiomyocyte hypoxia injury.Methods Rat cardiac H9c2 cells were pretreated with Bcl6-si RNA or the negative control si RNA,then maintained at 37?C,under a hypoxic atmosphere of 95% N2 and 5% CO2 for 24 h.The cells were divided into four treatment groups: si RNA-Normoxia,si Bcl6-Normoxia,si RNA-Hypoxia,and si Bcl6-Hypoxia.Western blot analysis was used to test Bcl6 expression in cardiomyocytes after treatment with Bcl6-si RNA or the negative control si RNA.Real time-PCR analysis was used todetect the pro-inflammatory cytokine expression in the four groups.Cell viability was detected by MMT assay and ROS level was analyzed by flow cytometry in the indicated groups.To evaluate the underlying mechanism through which Bcl6 affects cardiomyocyte hypoxia injury,the related signaling pathways were screened.Cells were pretreated with P38 inhibitor,JNK inhibitor,and ERK inhibitor to further find which could reverse the Bcl6 knockdown-induced deteriorating phenotype.Results We found that,under physiological conditions,Bcl6 knockdown decreased cell viability and increased pro-inflammatory cytokine(TNF?,IL-1,IL-6)expression in cardiomyocytes.When cells were exposed to hypoxia for 24 h,cell viability decreased markedly,pro-inflammatory cytokine expression increased,and oxidative stress increased,which was further augmented by Bcl6 knockdown.Under baseline conditions,Tunel staining revealed that Bcl6 knockdown increased the proportion of apoptotic cells.Bcl6 silencing resulted in a decrease in the level of the anti-apoptosis protein Bcl2 in both normoxic and hypoxic conditions.Moreover,pro-apoptosis protein Bax was up-regulated by Bcl6 deficiency.Bcl6 silencing also increased cytochrome C expression in both normoxic and hypoxic conditions.We found that Bcl6 silencing increased P38 activation without affecting JNK and ERK phosphorylation in the normoxic state.The phosphorylation levels of P38,JNK,and ERK increased after cells were expose to hypoxia.Interestingly,in the hypoxic state,Bcl6 silencing only increased P38 activation.To further confirm that P38 is the target of Bcl6,cells were pretreated with P38 inhibitor,JNK inhibitor,and ERK inhibitor,then exposed to hypoxia for 24 h.Western blot analysis showed that treatment with a P38 inhibitor reversed the Bcl6 silencing-induced deteriorating phenotype,as evidenced by reduced inflammatory response,improved oxidative stress response,and increased cell viability.Conclusion The results indicate that Bcl6 knockdown causes inflammation,oxidative stress,and apoptosis in cardiomyocytes and aggregates in hypoxia-induced cardiomyocyte injury via activating the P38 pathway.Thus,Bcl6 participates in the physiological and pathological process of cardiomyocytes and may become a new therapeutic target for the treatment of cardiovascular disease.