体外研究己糖激酶Ⅱ在缺血缺氧小鼠心肌细胞自噬流变化中的作用

Role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro

目的探讨己糖激酶Ⅱ在体外培养的缺血缺氧小鼠心肌细胞自噬流变化中的作用。方法取6只1~2 d龄雌雄不限C57BL/6小鼠,分离心脏并培养原代心肌细胞,取原代细胞进行以下实验。(1)按照随机数字表法(分组方法下同)将细胞分为6组,即正常对照3、6、9 h组及缺血缺氧3、6、9 h组,每组4孔。DMEM/F12培养基常规培养(常规培养条件下同)48 h后,正常对照3、6、9 h组更换新鲜DMEM/F12培养基分别培养3、6、9 h,缺血缺氧3、6、9 h组更换无糖无血清培养基后于37℃含体积分数1%氧气、体积分数5%二氧化碳的低氧培养箱(缺氧培养条件下同)内分别培养3、6、9 h。细胞计数试剂盒8(CCK-8)法检测细胞活力。(2)细胞分组及处理同实验(1),每组1孔。蛋白质印迹法检测细胞微管相关蛋白1轻链3 Ⅰ(LC3 Ⅰ)、LC3 Ⅱ、p62和己糖激酶Ⅱ蛋白表达。(3)将细胞分为正常对照组、单纯缺血缺氧9 h组和缺血缺氧9 h+2-脱氧葡萄糖(2-DG)组,每组4孔。常规培养48 h后,正常对照组更换新鲜DMEM/F12培养基培养9 h;单纯缺血缺氧9 h组更换无糖无血清培养基,缺血缺氧9 h+2-DG组更换溶有物质的量浓度为10 mmol/L 2-DG(20 μmol)的无糖无血清培养基,2组均缺氧培养9 h。CCK-8法检测细胞活力。(4)细胞分组及处理同实验(3),每组1孔。蛋白质印迹法检测细胞LC3 Ⅰ、LC3Ⅱ和p62蛋白表达。(5)细胞分组及处理同实验(3),每组2孔。透射电镜下观察心肌细胞中自噬体/自噬溶酶体情况。(6)将细胞分为正常对照组、单纯缺血缺氧9 h组、缺血缺氧9 h+己糖激酶Ⅱ小干扰RNA1(HK-ⅡsiRNA1)组和缺血缺氧9 h+HK-ⅡsiRNA2组,每组4孔。正常对照组和单纯缺血缺氧9 h组常规培养48 h,缺血缺氧9 h+HK-ⅡsiRNA1组和缺血缺氧9 h+HK-ⅡsiRNA2组分别转染200 nmol/L HK-ⅡsiRNA1和HK-ⅡsiRNA2后常规培养48 h。正常对照组更换新鲜DMEM/F12培养基培养9 h,单纯缺血缺氧9 h组、缺血缺氧9 h+HK-Ⅱ siRNA1组和缺血缺氧9 h+HK-ⅡsiRNA2组更换无糖无血清培养基后缺氧培养9 h。CCK-8法检测细胞活力。(7)细胞分组及处理同实验(6),每组1孔。蛋白质印迹法检测细胞LC3 Ⅰ、LC3Ⅱ、p62和己糖激酶Ⅱ蛋白表达。除实验(5)外,各实验均重复3次。对数据行单因素方差分析、LSD-t检验、Bonferroni校正。结果(1)缺血缺氧3、6、9 h组心肌细胞活力分别为0.450±0.022、0.385±0.010、0.335±0.015,分别明显低于对应正常对照3、6、9 h组的0.662±0.026、0.656 ±0.028、0.661±0.021(t=6.21、9.12、12.48,P<0.01)。(2)与对应正常对照3、6、9 h组比较,缺血缺氧3、6、9 h组心肌细胞LC3Ⅱ/Ⅰ比值、p62和己糖激酶Ⅱ蛋白表达均显著增加(t3h=16.15、10.99、5.30,t6 h=6.79、10.42、9.42,t9h=15.76、16.51、7.20,P<0.05或P<0.01)。(3)单纯缺血缺氧9 h组心肌细胞活力为0.353±0.022,较正常对照组的0.673 ±0.027明显降低(t=9.29,P<0.01);缺血缺氧9 h+2-DG组心肌细胞活力为0.472 ±0.025,较单纯缺血缺氧9 h组明显升高(t=3.60,P<0.05)。(4)与正常对照组比较,单纯缺血缺氧9 h组心肌细胞LC3 Ⅱ/Ⅰ比值及p62蛋白表达均明显增加(t=9.45、8.40,P<0.01);与单纯缺血缺氧9 h组比较,缺血缺氧9 h+2-DG组心肌细胞LC3 Ⅱ/Ⅰ比值及p62蛋白表达均明显减少(t=4.39、4.74,P<0.05)。(5)正常对照组心肌细胞中仅可见个别双层膜结构的自噬体/自噬溶酶体;与正常对照组比较,单纯缺血缺氧9 h组心肌细胞中双层膜结构的自噬体/自噬溶酶体明显增多;与单纯缺血缺氧9 h组比较,缺血缺氧9 h+2-DG组心肌细胞中自噬体/自噬溶酶体数量明显减少。(6)单纯缺血缺氧9 h组心肌细胞活力为0.358±0.023,较正常对照组的0.673 ±0.026明显降低(t=9.12,P<0.01);缺血缺氧9 h+HK-Ⅱ siRNA1组和缺血缺氧9 h+HK-Ⅱ siRNA2组心肌细胞活力分别为0.487 ±0.027、0.493±0.022,较单纯缺血缺氧9 h组明显升高(t=3.63、4.28,P<0.05)。(7)与正常对照组比较,单纯缺血缺氧9 h组心肌细胞LC3 Ⅱ/Ⅰ比值、p62和己糖激酶Ⅱ蛋白表达均明显增加(t=6.08、6.31、4.83,P<0.05或P<0.01);与单纯缺血缺氧9 h组比较,缺血缺氧9 h+HK-ⅡsiRNA1组和缺血缺氧9 h+HK-ⅡsiRNA2组心肌细胞中LC3Ⅱ/Ⅰ比值、p62和己糖激酶Ⅱ蛋白表达均明显减少(t=5.10、7.76、15.33,4.17、8.42、12.11,P<0.05或P<0.01)。结论缺血缺氧上调体外培养的小鼠心肌细胞己糖激酶Ⅱ蛋白表达水平,通过损害自噬流导致心肌细胞活力下降;抑制己糖激酶Ⅱ活性或其表达可减轻心肌细胞缺血缺氧损害。

Objective To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro.Methods The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments.(1) The cells were divided into 6 groups according to the random number table (the same grouping method below),i.e.,normal control 3,6,and 9 h groups and ischemia-hypoxia 3,6,and 9 h groups,with 4 wells in each group.After being regularly cultured for 48 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below),the cells in normal control 3,6,and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3,6,and 9 h,respectively,and the cells in ischemia-hypoxia 3,6,and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3,6,and 9 h,respectively.Cell viability was measured by the cell counting kit 8 (CCK-8) method.(2) The cells were grouped and treated the same as those in experiment (1),with 1 well in each group.Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ),LC3Ⅱ,p62,and hexokinase Ⅱ.(3) The cells were divided into normal control group,simple ischemia-hypoxia 9 h group,and ischemia-hypoxia 9 h + 2-deoxyglucose (2-DG) group,with 4 wells in each group.After a regular culture for 48 h,the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h;the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium,and the cells in ischemia-hypoxia 9 h + 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol),and then they were cultured with hypoxia for 9 h.Cell viability was measured by CCK-8 method.(4) The cells were grouped and treated the same as those in experiment (3),with 1 well in each group.Western blotting was used to detect the protein expressions of LC3Ⅰ,LC3 Ⅱ,and p62.(5) The cells were grouped and treated the same as those in experiment (3),with 2 wells in each group.Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes.(6) The cells were divided into normal control group,simple ischemia-hypoxia 9 h group,ischemia-hypoxia 9 h + hexosinase Ⅱ small interfering RNA1 (HK-Ⅱ siRNA1)group,and ischemia-hypoxia 9 h + HK-Ⅱ siRNA2 group,with 4 wells in each group.The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h,and the cells in ischemia-hypoxia 9 h + HK-Ⅱ siRNA1 group and ischemia-hypoxia 9 h + HK-Ⅱ siRNA2 group were respectively transfected with 200 nmol/L HK-Ⅱ siRNA1 and HK-Ⅱ siRNA2 and then also cultured for 48 h.The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h,and the cells in simple ischemia-hypoxia 9 h group,ischemia-hypoxia 9 h + HK-Ⅱ siRNA1 group,and ischemia-hypoxia 9 h + HK-Ⅱ siRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h.Cell viability was measured by CCK-8 method.(7) The cells were grouped and treated the same as those in experiment (6),with 1 well in each group.Western blotting was used to detect the protein expressions of LC3 Ⅰ,LC3 Ⅱ,p62,and hexokinase Ⅱ.Except for experiment (5),each experiment was repeated 3 times.Data were processed with one-way analysis of variance and lest significant difference t test,and Bonferroni correction.Results (1) The viabilities of cardiomyocytes in ischemia-hypoxia 3,6,and 9 h groups were 0.450 ± 0.022,0.385 ± 0.010,and 0.335 ± 0.015,respectively,which were significantly lower than 0.662 ± 0.026,0.656 ± 0.028,and 0.661 ± 0.021 of the corresponding normal control 3,6,and 9 h groups,respectively (t =6.21,9.12,12.48,P <0.01).(2) Compared with those of corresponding normal control 3,6,and 9 h groups,the LC3 Ⅱ / Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3,6,and 9 h groups were significantly increased (t 3h =16.15,10.99,5.30,t 6h =6.79,10.42,9.42,t9h=15.76,16.51,7.20,P <0.05 or P <0.01).(3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353 ±0.022,which was significantly lower than 0.673 ± 0.027 of normal control group (t =9.29,P < 0.01).The viability of cardiomyocytes in ischemia-hypoxia 9 h + 2-DG group was 0.472 ± 0.025,which was significantly higher than that of simple ischemia-hypoxia 9 h group (t =3.60,P <0.05).(4) Compared with those of normal control group,the LC3 Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t =9.45,8.40,P <0.01).Compared with those of simple ischemia-hypoxia 9 h group,the LC3 Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h + 2-DG group were significantly decreased (t =4.39,4.74,P < 0.05).(5) In cardiomyocytes of normal control group,only single autophagosome/autolysosome with bilayer membrane structure was observed.Compared with that of normal control group,the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly.Compared with that of simple ischemia-hypoxia 9 h group,the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h + 2-DG group was significantly decreased.(6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358 ±0.023,which was significantly lower than 0.673 ±0.026 in normal control group (t =9.12,P < 0.01).The viabilities of cardiomyocytes in ischemia-hypoxia 9 h + HK-Ⅱ siRNA1 group and ischemia-hypoxia 9 h + HK-Ⅱ siRNA2 group were 0.487 ±0.027 and 0.493 ± 0.022,respectively,which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t =3.63,4.28,P <0.05).(7) Compared with those of normal control group,the LC3 Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t =6.08,6.31,4.83,P <0.05 orP <0.01).Compared with those of simple ischemia-hypoxia 9 h group,the LC3 Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h + HK-Ⅱ siRNA1 group and ischemia-hypoxia 9 h + HK-Ⅱ siRNA2 group were significantly decreased (t =5.10,7.76,15.33,4.17,8.42,12.11,P <0.05 or P <0.01).Conclusions Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro,which decreases the viability of cardiomyocytes by impairing autophagic flow.To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.