Objective: To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro. Methods: The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased (t=12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P<0.05 or P<0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased (t=6.88, 10.56, 5.76, 9.91, P<0.05 or P<0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased (t=6.01, P<0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups (F=1.09, P>0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups (t=43.05, 11.07, 5.39, P<0.05 or P<0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups (t=11.18, 12.71, P<0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased (t=4.82, 4.44, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased (t=4.39, 6.27, P<0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group (t=10.13, P<0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=3.28, P<0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group (t=4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P<0.05 or P<0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased (t=5.51, P<0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased (t=5.97, P<0.05). Conclusions: After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.
目的： 探讨人抗原R在体外培养的小鼠心肌细胞自噬过程中对溶酶体酸化的作用。 方法： 取1～2 d龄C57BL/6小鼠(雌雄不限)20只，分离心脏培养原代心肌细胞，进行以下实验。(1)采用随机数字表法(分组方法下同)将细胞分为5组，即正常对照组和无糖无血清0.5、1.0、3.0、6.0 h组。正常对照组用DMEM/F12培养基常规培养(常规培养条件下同)54.0 h，无糖无血清0.5、1.0、3.0、6.0 h组常规培养53.5、53.0、51.0、48.0 h后更换无糖无血清培养基分别处理0.5、1.0、3.0、6.0 h，蛋白质印迹法检测细胞总蛋白中微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、自噬相关蛋白5、腺苷三磷酸酶V1区E1亚基(ATP6V1E1)蛋白表达。(2)将细胞分为正常对照组和无糖无血清3.0 h组，相应各组细胞处理同实验(1)，激光扫描共聚焦显微镜下观测细胞溶酶体酸化水平。(3)取2个批次细胞，均同实验(1)分组处理，蛋白质印迹法检测一个批次细胞总蛋白及另一个批次细胞胞质、胞核蛋白中人抗原R蛋白表达。(4)将细胞分为正常对照组、单纯对照小干扰RNA(siRNA)组、单纯人抗原R-siRNA1(HuR-siRNA1)组、单纯HuR-siRNA2组、无糖无血清3.0 h组、无糖无血清＋对照siRNA组、无糖无血清＋HuR-siRNA1组、无糖无血清＋HuR-siRNA2组。常规培养48 h后，单纯对照siRNA组及无糖无血清＋对照siRNA组加入阴性对照siRNA、单纯HuR-siRNA1组及无糖无血清＋HuR-siRNA1组加入HuR-siRNA1、单纯HuR-siRNA2组及无糖无血清＋HuR-siRNA2组加入HuR-siRNA2转染6 h，8组均继续常规培养48 h后，正常对照组及各单纯siRNA处理组更换DMEM/F12培养基、其余各组更换无糖无血清培养基培养3 h，蛋白质印迹法检测细胞总蛋白中人抗原R蛋白表达。(5)取2个批次细胞，均分为无糖无血清＋对照siRNA组、无糖无血清＋HuR-siRNA1组，相应各组细胞处理同实验(4)，免疫荧光法观测一个批次细胞人抗原R分布及表达，激光扫描共聚焦显微镜下观测另一批次细胞溶酶体酸化水平。(6)取3个批次细胞，均分为无糖无血清3.0 h组、无糖无血清＋对照siRNA组、无糖无血清＋HuR-siRNA1组、无糖无血清＋HuR-siRNA2组，相应各组细胞处理同实验(4)，蛋白质印迹法检测一个批次细胞总蛋白中组织蛋白酶D、一个批次细胞胞质蛋白中人抗原R及另一个批次细胞总蛋白中ATP6V1E1蛋白表达。(7)将细胞分为正常对照组、无糖无血清3.0 h组、无糖无血清＋对照siRNA组、无糖无血清＋HuR-siRNA1组，相应各组细胞处理同实验(4)，实时荧光定量反转录PCR法检测细胞ATP6V1E1 mRNA表达。各实验样本数均为3。对数据进行独立样本t检验、单因素方差分析、LSD-t检验、Bonferroni校正。 结果： (1)与正常对照组比较，无糖无血清1.0、3.0、6.0 h组细胞总蛋白中LC3Ⅱ、ATP6V1E1蛋白表达显著增加(t＝12.16、4.05、4.82，11.64、3.29、8.37，P<0.05或P<0.01)，无糖无血清0.5、1.0、3.0、6.0 h组细胞总蛋白中自噬相关蛋白5蛋白表达显著增加(t＝6.88、10.56、5.76、9.91，P<0.05或P<0.01)。(2)与正常对照组的1.03±0.08比较，无糖无血清3.0 h组细胞溶酶体酸化水平(2.92±0.30)明显升高(t＝6.01，P<0.01)。(3)5组细胞总蛋白中人抗原R蛋白表达组间总体比较，差异无统计学意义(F＝1.09，P>0.05)。与正常对照组比较，无糖无血清1.0、3.0、6.0 h组细胞胞质蛋白中人抗原R蛋白表达显著增加(t＝43.05、11.07、5.39，P<0.05或P<0.01)，无糖无血清3.0、6.0 h组细胞胞核蛋白中人抗原R蛋白表达显著减少(t＝11.18、12.71，P<0.01)。(4)与单纯对照siRNA组比较，单纯HuR-siRNA1组、单纯HuR-siRNA2组细胞总蛋白中人抗原R蛋白表达显著减少(t＝4.82、4.44，P<0.05)。与无糖无血清＋对照siRNA组比较，无糖无血清＋HuR-siRNA1组、无糖无血清＋HuR-siRNA2组细胞总蛋白中人抗原R蛋白表达显著减少(t＝4.39、6.27，P<0.05)。(5)与无糖无血清＋对照siRNA组比较，无糖无血清＋HuR-siRNA1组细胞胞质中人抗原R分布减少、表达水平显著降低(t＝10.13，P<0.01)。与无糖无血清＋对照siRNA组的1.00±0.06比较，无糖无血清＋HuR-siRNA1组细胞溶酶体酸化水平(0.73±0.06)显著下降(t＝3.28，P<0.01)。(6)与无糖无血清＋对照siRNA组比较，无糖无血清＋HuR-siRNA1组、无糖无血清＋HuR-siRNA2组细胞总蛋白中组织蛋白酶D、胞质蛋白中人抗原R、总蛋白中ATP6V1E1蛋白表达均显著减少(t＝4.16、3.99，4.81、5.07，11.68、12.97，P<0.05或P<0.01)。(7)与正常对照组比较，无糖无血清3.0 h组细胞中ATP6V1E1 mRNA表达显著增加(t＝5.51，P<0.05)。与无糖无血清＋对照siRNA组比较，无糖无血清＋HuR-siRNA1组细胞中ATP6V1E1 mRNA表达显著减少(t＝5.97，P<0.05)。 结论： 小鼠原代心肌细胞在体外无糖无血清处理后自噬激活、溶酶体酸化增强、胞质中人抗原R表达增加，人抗原R功能激活并参与维持小鼠心肌细胞自噬过程中的溶酶体酸化。.
Keywords: Autophagy; Human antigen R; Lysosomal acidification; Myocytes, cardiac.