烟酸酰胺腺嘌呤二核苷酸磷酸对大鼠缺氧心肌细胞自噬流的影响及机制

Influence of nicotinic acid adenine dinucleotide phosphate on autophagy in hypoxic cardiomyocytes of rats and its mechanism

目的探讨烟酸酰胺腺嘌呤二核苷酸磷酸（NAADP）对大鼠缺氧心肌细胞自噬流的影响及机制。方法取5只SD大鼠乳鼠，处死后取心脏，制备原代心肌细胞用于以下实验。（1）取原代心肌细胞，按随机数字表法分为常氧组、缺氧9h组、缺氧9h＋NAADP组，每组各5孔。常氧组细胞置于37℃恒温培养箱中常规培养9h；缺氧9h组和缺氧9h＋NAADP组细胞置于缺氧培养箱（含体积分数94％氮气、体积分数5％二氧化碳及体积分数1％氧气）培养9h，缺氧9h＋NAADP组细胞缺氧处理前加入终物质的量浓度10μmol／LNAADP。细胞计数试剂盒8检测细胞活力。（2）取原代心肌细胞，按随机数字表法分为常氧组、缺氧9h组、缺氧9h＋NAADP组、缺氧9h＋trans-Ned-19组及缺氧9h＋trans-Ned-19＋NAADP组，每组各2孔。常氧组细胞置于37℃恒温培养箱中常规培养9h；余4组细胞同实验（1）行缺氧处理。缺氧处理前，缺氧9h＋NAADP组细胞加入终物质的量浓度10μmol／LNAADP，缺氧9h＋trans-Ned-19组细胞加入终物质的量浓度1μmol／L trans-Ned-19，缺氧9h＋trans-Ned-19＋NAADP组细胞加入终物质的量浓度10μmol／LNAADP和1μmol／L trans-Ned-19。蛋白质印迹法检测微管相关蛋白1轻链3-Ⅱ型和P62的表达量。（3）取原代心肌细胞，同实验（1）进行分组及处理。免疫荧光法检测溶酶体的酸碱度。对数据行单因素方差分析和LSD检验。结果（1）常氧组细胞活力为1．114±0．024，明显高于缺氧9h组的0．685±0．079，P〈0．01。缺氧9h＋NAADP组细胞活力为0．886±0．06l，明显高于缺氧9h组（P〈0．05）。（2）与常氧组比较，缺氧9h组细胞微管相关蛋白1轻链3-Ⅱ型及P62表达量明显升高（P〈0．01）。与缺氧9h组比较，缺氧9h＋NAADP组细胞P62表达量明显下降（P〈0．01），微管相关蛋白1轻链3-Ⅱ型表达量无明显变化（P〉0．05）；缺氧9h＋trans-Ned-19组细胞微管相关蛋白1轻链3-Ⅱ型及P62表达量均无明显变化（P〉0．05）。与缺氧9h＋NAADP组比较，缺氧9h＋trans-Ned-19＋NAADP组细胞微管相关蛋白1轻链3-Ⅱ型表达量无明显变化（P〉0．05），P62表达量明显升高（P〈0．01）。（3）常氧组细胞绿色荧光强度大，与红色荧光共染好，溶酶体内环境酸性较强。缺氧9h组细胞绿色荧光强度明显低于常氧组，溶酶体内环境酸性较常氧组减弱。缺氧9h＋NAADP组细胞绿色荧光强度较缺氧9h组明显增强，溶酶体内环境酸性较缺氧9h组强，但仍低于常氧组。结论NAADP通过酸化缺氧后心肌细胞溶酶体内环境，促进自噬体降解，减少自噬溶酶体堆积，修复受损自噬流从而改善心肌细胞活力。

Objective To investigate influence of nicotinic acid adenine dinucleotide phosphate （NAADP） on autophagy in hypoxic cardiomyocytes of rats and its mechanism. Methods Five neonatal Sprague-Dawley rats were collected and sacrificed to harvest the hearts, and primary cardiomyocytes were separated for the following experiments. （ 1 ） Primary cardiomyocytes were collected and divided into normox-ia group, hypoxia 9 h group, and hypoxia 9 h ＋ NAADP group according to random number table, with 5 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. Cells in hypoxia 9 h group and hypoxia 9 h ＋ NAADP group were cultured in hypoxic in-cubator with volume fraction 94% nitrogen, 5% carbon dioxide, and 1% oxygen for 9 hours. Before hypoxi-a, cells in hypoxia 9 h ＋ NAADP group were dealt with final amount-of-substance concentration 10 μmol/L NAADP. Cell counting kit 8 was used to measure cell viability. （2） Primary cardiomyocytes were collected and divided into normoxia group, hypoxia 9 h group, hypoxia 9 h ＋ NAADP group, hypoxia 9 h ＋ tran-Ned-19 group, and hypoxia 9 h ＋ trans-Ned-19 ＋ NAADP group according to the random number table, with 2 wells in each group. Cells in normoxia group were cultured routinely in the constant temperature incubator at 37 ℃ for 9 hours. And cells in the other 4 groups were cultured in hypoxic incubator as that in experiment （ 1 ） Before hypoxia, cells in hypoxia 9 h ＋ NAADP group were dealt with amount-of-substance concentration 10 μmol/L NAADP, cells in hypoxia 9 h ＋ tran-Ned-19 group were deah with amount-of-substance concen- tration 1 μmol/L trans-Ned-19, and ceils in hypoxia 9 h ＋ trans-Ned-19 ＋ NAADP group were dealt with a-mount-of-substance concentration 10 μmol/L NAADP and 1 μmol/L trans-Ned-19. Protein expressions of microtubule associated protein 1 light chain 3-Ⅱ and 1762 were detected by Western blotting. （3） Primary cardiomyocytes were collected and grouped as those in experiment （1）. The lysosomal acidity was deter-mined by immunofluoreseence method. Data were processed with one-way analysis of variance and least-sig-nificant difference test. Results （ 1 ） The cell viability in normoxia group was 1.114 ± 0. 024, which was significantly higher than 0. 685 ±0. 079 of ceils in hypoxia 9 h group （ P 〈0.01 ）. The cell viability of hy-poxia 9 h ＋ NAADP group was 0. 886 ± 0. 061 , which was obviously higher than that of ceils in hypoxia 9 h group （ P 〈 0.05 ）. （2） Expressions of microtubule-associated protein 1 light chain 3-Ⅱ and P62 of cells in hypoxia 9 h group were significantly higher than those of cells in normoxia group （ P 〈 0.01 ）. Compared with those in hypoxia 9 h group, expression of P62 in hypoxia 9 h ＋ NAADP group was significantly de-creased （ P 〈 0.01 ） , while expression of microtubule-associated protein 1 light chain 3- Ⅱ did not change significantly （ P 〉 0.05 ）. There were no significantly statistical difference in expressions of microtubule-as-sociated protein 1 light chain 3- Ⅱ and P62 between hypoxia 9 h group and hypoxia 9 h ＋ trans-Ned-19 group （ P 〉 0.05）. Compared with those of cells in hypoxia 9 h ＋ NAADP group, expression of P62 of cells in hy-poxia 9 h ＋ trans-Ned-19 ＋ NAADP group was obviously increased （ P 〈0. 01 ） , while expression of micro-tubule-associated protein 1 light chain 3-Ⅱ did not change significantly （ P 〉 0.05）. （3） The intensity of green fluorescence of cells in normoxia group was strong and co-localized well with red fluorescence, and in-ternal environment of lysosome was with stronger acidity. The intensity of green fluorescence in cells of hy-poxia 9 h group was significantly lower than that of cells in normoxia group, and acidity of internal environ-ment of lysosome was weakened. The intensity of green fluorescence and acidity of internal environment of ly-sosome in hypoxia 9 h ＋ NAADP were significantly stronger than those of cells in hypoxia 9 h group, but sig-nificantly lower than those of cells in normoxia group. Conclusions NAADP can improve myocardial cell viability through acidifying internal environment of lysosome of cardiomyocyte after hypoxia, promoting degra-dation of autophagosomes, reducing autophagic lysosomal accumulation, and repairing damaged autophagic flow.