组蛋白去乙酰化酶6抑制剂对糖尿病性心肌细胞缺氧复氧损伤的保护作用及其机制研究

Inhibition of Histone Deacetylase 6 Activity Confers Protective Effects Against Hypoxia-Reoxygenation Injury in Diabetic Myocardiocytes

目的:评价组蛋白去乙酰化酶6（HDAC6）抑制剂Tubacin（TBA）对糖尿病性心肌细胞缺氧复氧损伤的保护作用和机制。方法:正常培养的H9c2细胞,采用随机数字表法分为4组（n=24）:高糖常氧组（N组）、高糖缺氧复氧组（H/R组）、高糖缺氧复氧+Tubacin组（H/R+TBA组）、高糖缺氧复氧+Tubacin+Akt抑制剂MK-2206（H/R+TBA+MK组）。高糖培养基（葡萄糖浓度33mmol/L）培养H9c2细胞24h。H9c2心肌细胞缺氧（95%N2+5%CO2）4h,复氧（90%O2+10%CO2）2h制备H9c2心肌细胞缺氧复氧损伤模型。TBA组H/R前6h给予Tubacin（2μmol/L）。Akt抑制剂组H/R前1h给予MK-2206（300nmol/L）。采用ELISA法检测培养上清液乳酸脱氢酶（LDH）含量,采用CCK-8试剂盒检测细胞存活率,HDAC6活性检测试剂盒检测心肌细胞HDAC6活性。Western Blot法检测磷酸化蛋白激酶B （p-Akt,ser473）、总蛋白激酶B（t-Akt）、磷酸化叉头蛋白O3a（p-Foxo3a,ser253）和总叉头蛋白O3a（t-Foxo3a）的表达水平。结果:与N组比较,H/R组LDH释放量和细胞凋亡水平增加（P<0.05）,p-Akt、t-Akt、p-Foxo3a、t-Foxo3a蛋白表达无显著差异（P>0.05）。与H/R组比较,H/R+TBA组LDH释放量和细胞凋亡水平降低（P<0.05）,同时p-Akt和p-Foxo3a表达升高,t-Akt和t-Foxo3a无显著差异（P>0.05）。与H/R+TBA组相比,H/R+TBA+MK组LDH释放量和细胞凋亡水平升高（P<0.05）,同时p-Akt和p-Foxo3a表达显著降低（P<0.05）,t-Akt和t-Foxo3a无显著差异（P>0.05）。结论:HDAC6抑制剂TBA对糖尿病性心肌细胞缺氧复氧损伤的保护效应是通过激活Akt/Foxo3a通路而产生的。

Objective:To evaluate the effects of histone deacetylase 6（HDAC6）inhibitor Tubacin（TBA）on hypoxia-reoxygenation（H/R）induced injury in diabetic myocardiocytes.Methods:H9c2 cells cultured in high-glucose culture atmosphere were randomly divided into four group（n=24）:（1）high glucose control（N）,（2）cells exposed to high glucose followed by H/R（H/R）,（3）H/R cells pretreated with TBA（H/R +TBA）,and（4）H/R cells pretreated with TBA and Akt inhibitor MK-2206（H/R +TBA+MK）.H9c2 cardiomyocytes were exposed to HG medium for 24 hfollowed by 4 hof hypoxia（94% N2 and 5% CO2）and 2 h（90% O2 and 10% CO2）reoxy-genation.TBA（2μmol/L）or vehicle was administered 6 hbefore H/R.MK-2206（300 nmol/L）or vehicle was given 1 hbefore H/R.At the end of reoxygenation the lactate dehydrogenase（LDH）were measured by enzyme-linked immunosorbent assay,the cell survival rate was detected by using cell counting kit-8 assay.HDAC6 activity was evaluated by commercial HDAC6 activity assay kit.Phosphate-Akt（ser473）,total-Akt,phosphate-Foxo3 a（ser253）,total-Foxo3 a was detected by Western blot.Results:Compared with N group,serum LDH level and cell apoptosis rate were increased in H/R group（P<0.05）.However,there was no significant changes in HDAC6 activity,and expression of p-Akt,t-Akt,p-Foxo3 aand t-Foxo3 a（P>0.05）.Compared with H/R group,serum LDH level and cell apoptosis rate were decreased in TBA group（P<0.05）.TBA administration significantly decreased HDAC6 activity but increased p-Akt and p-Foxo3 aexpression（P<0.05）.There were no significant changes in t-Akt and t-Foxo3 aexpression（P>0.05）.Compared with TBA group,serum LDH level and cell apoptosis rate were increased in Akt inhibition group（P<0.05）.MK-2206 administration decreased p-Akt and p-Foxo3 alevels（P<0.05）.However,there were no significant changes in HDAC6 activity,and t-Akt and t-Foxo3 aexpression（P>0.05）.Conclusion:Inhibition of histone deacetylase 6 activity confers protective effects against hypoxia-reoxygenation injury in diabetic myocardiocytes via modulating Akt/Foxo3 apathway.