【作者】 徐征；

【导师】 孙健；

【作者基本信息】 吉林大学 ， 内科学， 2017， 博士

【摘要】 背景:全球糖尿病患者持续不断增加,无论1型还是2型糖尿病,其引起的并发症已经成为正在威胁着人类健康的首要问题,尤其是糖尿病心脏损害。既往研究提示不同刺激下的心脏损害均与组蛋白去乙酰化酶(histone deacetylases,HDAC)激活有关,且HDAC抑制剂减轻了链脲佐菌素(streptozotocin,STZ)诱导糖尿病小鼠的心脏损害。HDAC至少有4种不同的类别,究竟是哪一种HDAC促进了1型糖尿病心肌损伤的发生发展,尚未有报道。目的:研究HDAC3抑制剂RGFP966处理自发性1型糖尿病模型OVE26小鼠后,是否可以预防和/或延缓糖尿病所致导致的心功能降低及病理改变,以及HDAC3抑制后表观遗传学修饰所产生的“心脏保护记忆”是否存在。方法:随机选取雄性OVE26小鼠和年龄匹配的野生型(wild-type,WT)FVB小鼠,首先测定其基线心功能、血糖,血糖达到糖尿病诊断标准的OVE26小鼠每隔一天给予HDAC3特异性抑制剂RGFP966(10 mg/kg,s.c.)、非特异性HDAC抑制剂丙戊酸钠(Valproic acid,VPA,200 mg/kg,s.c.)或溶剂至3个月。小鼠被分为六个实验组,分别为:WT+vehicle,WT+RGFP966,WT+VPA,OVE+vehicle,OVE+RGFP966和OVE+VPA。给药3个月后(此为第一个时间点,3M)超声测定心功能,麻醉处死小鼠并收集心脏。余下的小鼠不给药继续饲养3个月(此时为第二个时间点,6M),同样行超声测定心功能后麻醉处死小鼠并收集心脏用以病理学及分子生物学检测,包括:测量小鼠心脏重量及胫骨长度计算二者比值(HW/TL);免疫荧光法测定HDAC和HDAC3活性;免疫印迹或实时定量聚合酶链反应(q RT-PCR)检测心肌肥厚(ANP),纤维化(CTGF,collagen I,FN-1),氧化应激(3-NT,4-HNE),炎症反应(PAI-1,TNF-α)等指标;天狼星红染色和WGA染色检测纤维化及心肌肥厚;DHE染色测定氧化应激反应产物,硫代巴比妥酸法测定膜脂质过氧化物丙二醛(MDA);免疫印迹检测乙酰化组蛋白H3,组蛋白H3赖氨酸9/14乙酰化水平,胰岛素受体底物1(IRS1),蛋白激酶B(Akt),细胞外信号调节激酶1/2(ERK1/2),葡萄糖转运体4、1(GLUT4、GLUT1),AMP依赖蛋白激酶α亚基(AMPKα),c-Jun氨基末端激酶(JNK),p38丝裂原活化蛋白激酶(p38MAPK),双向特异性磷酸酶5(DUSP5);染色质免疫共沉淀(Ch IP)检测DUSP5启动子区域组蛋白H3乙酰化水平。结果:HDAC3抑制剂RGFP966处理3个月显著抑制了1型糖尿病OVE26小鼠心脏HDAC3和总HDAC活性,并且增加了总体组蛋白H3和组蛋白H3赖氨酸9/14乙酰化水平,但未降低小鼠外周血糖。HDAC3抑制剂明显抑制了糖尿病心肌病的发生,表现为RGFP966有效改善了糖尿病所导致的心功能不全、心肌肥大、纤维化、以及氧化应激损伤、心肌炎症和胰岛素抵抗。这些心脏保护效果在3M和6M时间点均存在。心肌肥厚因子ERK1/2的磷酸化水平(p-ERK1/2)显著增加(尤其是细胞核内的p-ERK1/2水平),同时ERK1/2特异性磷酸化酶DUSP5明显下调,上述改变均被HDAC3抑制剂RGFP966所逆转。HDAC3抑制剂在3M和6M时间点均增加了DUSP5启动子区域组蛋白H3乙酰化水平。非特异性HDAC抑制剂VPA作用与RFGP966并无明显差别。结论:在1型糖尿病模型OVE26小鼠心脏中,抑制HDAC3能够明显预防糖尿病导致的心功能异常、心肌纤维化、肥厚、炎症、氧化应激损伤和胰岛素抵抗,其作用机制可能是通过增加DUSP5启动子区域组蛋白H3乙酰化水平使DUSP5表达增加,从而下调细胞核内p-ERK1/2水平来实现的,而且上述作用在停止给药3个月后仍持续存在,提示HDAC3抑制后的表观遗传学修饰产生了“心脏保护记忆”。本研究结果为HDAC3抑制剂防治糖尿病心肌病提供了一种潜在的可能性。更多还原

【Abstract】 Background: With the high prevalence of diabetics,the concerns regarding its complications caused by both type 1 and type 2 diabetes are growing,especially the diabetic cardiomyopathy(DCM),which is the most challenging health problem today.Recent studies have shown that activation of histone deacetylases(HDAC)is associated with DCM and total HDAC inhibition attenuated diabetic cardiac injury in the streptozotocin(STZ)-induced diabetic mice.However,there are,at least,4 different types of HDAC and its roles in DCM remain under debate.Aims: To investigate whether HDAC3 inhibitor RGFP966 can prevent the development of DCM,and whether this epigenetic modification produces the phenomenon of “cardiac protective memory”.Method: Male type 1 diabetic OVE26 and wild-type mice were randomized the baseline cardiac function and blood glucose data being collected.Specific HDAC3 inhibitor RGFP966(10 mg/kg,s.c.),pan-inhibitor Valproic acid(VPA,200 mg/kg,s.c.)and vehicle were injected into the mice every other day for 3 months.Animals were divided into six experimental groups: WT + vehicle,WT + RGFP966,WT + VPA,OVE + vehicle,OVE + RGFP966,and OVE + VPA.At the end of the 3-month treatment,the cardiac function of all the mice were examined,then five to seven mice from each group were killed as the time point of 3 months(labeled as 3 M)cohort and heart tissues were collected.The remaining animals(n=5 at least)in each group were kept for another 3 months without HDAC inhibitors’ treatment(labeled as 6 M).After determining the cardiac function at the end of 6 months,,heart tissue were collected for the pathological examination,including the ratio of heart weight and tibia length,total HDAC and HDAC3 activities,hypertrophy,fibrosis,oxidative stress,inflammation.Acetylated Histone H3,acetylated Histone H3K9/14,insulin receptor substrate 1(IRS1),phosphorylated protein kinase B(p-Akt),phosphorylated extracellular signal-regulated kinases 1/2(p-ERK1/2),glucose transporter 4(GLUT4),GLUT1,phosphorylated AMP-activated protein kinase α(p-AMPKα),phosphorylated c-Jun N-terminal kinase(p-JNK),phosphorylated p38 mitogen activated protein kinases(p-p38MAPK),dual specificity phosphatase 5(DUSP5)were detected by Western Blot and real-time PCR method.The acetylated level of histone H3 on the DUSP5 gene promoter region was determined by chromatin immunoprecipitation(Ch IP).Results: In the present study,HDAC3 inhibitor RGFP966 significantly inhibited total HDAC and HDAC3 activity in the typa 1 diabetic OVE26 mice and increased the total acetylated Histone H3 and H3K9/14,but didn’t affect the blood glucose.Pathological and histological examination showed that inhibition of HDAC3 blocked the development of diabetic cardiomyopathy,as evidenced by improved diabetes-induced cardiac dysfunction,hypertrophy,and fibrosis,along with diminishing cardiac oxidative stress,inflammation and insulin resistance.All these cardioprotection of RGFP966 were found at 3M and 6M.Furthermore,phosphorylated ERK 1/2,a well-known initiator of cardiac hypertrophy,was significantly increased,while DUSP5,an ERK1/2 nuclear phosphatase,was substantially decreased in diabetic hearts.Both of these changes were prevented by RGFP966.CHIP assay showed that HDAC3 inhibition elevated histone H3 acetylation on the DUSP5 gene promoter at both two time points.Conclusion: In the type 1 diabetic OVE26 mice,inhibiting HDAC3 significantly prevented the diabetes-induced cardiac dysfunction,hypertrophy,fibrosis,oxidative stress,inflammation and insulin resistance.The possible mechanism might be that inhibition of HDAC3 up-regulates DUSP5 expression via acetylating Histone H3 at DUSP5 gene promoter region,leading to the inactivation of nuclear ERK1/2.These cardioprotection persisted for another 3 months after termination of RFGP966 treatment,which indicates the phenomenon of “cardiac protective memory” induced by epigenetic modification and the potential application of HDAC3 inhibitor for the prevention of DCM.更多还原