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Title: [In vitro study of effects of transient receptor potential vanilloid 1 on autophagy in early hypoxic mouse cardiomyocytes and the mechanism].
Author: Wei JY, Cui L, Lin JZ, Zhang Q, Yuan HP, Xiang F, Song HP, Jia JZ, Lyu YL, Zhang DX, Huang YS.
Journal: Zhonghua Shao Shang Za Zhi; 2019 Mar 20; 35(3):186-192. PubMed ID: 30897864.
Abstract:
Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco' s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups (t(3 h)=4.891, 5.890, 4.928; t(6 h)=9.790, 6.750, 10.590; t(9 h)=6.948, 6.764, 5.049, P<0.05 or P<0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased (t=10.488, P<0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences (t=4.372, 3.026, P>0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (t=15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P<0.05 or P<0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t=4.365, P<0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t=6.216, P<0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased (t=3.489, P<0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t=0.545, P>0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t=12.550, 7.442, P<0.01). Conclusions: TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury. 目的： 初步探讨瞬时受体电位香草酸亚型1(TRPV1)对体外培养的缺氧早期小鼠心肌细胞自噬的影响及机制。 方法： 取120只1～2 d龄雌雄不拘C57BL/6小鼠，分离心脏培养原代心肌细胞，并进行如下实验，分组方法均按照随机数字表法。(1)将细胞分为常氧组及缺氧3、6、9 h组，每组1孔。常氧组细胞行常规培养(下同)，缺氧3、6、9 h组细胞采用不含胎牛血清的DMEM无糖培养基于体积分数1%氧气、5%二氧化碳、94%氮气的低氧条件下分别培养3、6、9 h。蛋白质印迹法检测微管相关蛋白1轻链3(LC3)、Beclin-1及TRPV1的蛋白表达。(2)将细胞分为常氧组和缺氧组，每组2张盖玻片，其中缺氧组细胞同前缺氧处理6 h。免疫荧光法检测TRPV1的阳性表达。(3)将细胞分为4组，每组1孔。单纯缺氧组细胞同前行缺氧处理6 h；缺氧＋0.1 μmol/L辣椒素组、缺氧＋1.0 μmol/L辣椒素组、缺氧＋10.0 μmol/L辣椒素组细胞分别加入0.1、1.0、10.0 μmol/L辣椒素处理30 min后，行缺氧处理6 h。蛋白质印迹法检测LC3、Beclin-1及TRPV1的蛋白表达。(4)将细胞分为5组，每组5孔。缺氧组细胞同前缺氧处理6 h；缺氧＋氯喹组、缺氧＋辣椒素组、缺氧＋辣椒素＋氯喹组细胞分别加入50 μmol/L氯喹、10.0 μmol/L辣椒素、50 μmol/L氯喹＋10.0 μmol/L辣椒素处理30 min后缺氧处理6 h。细胞计数试剂盒8法检测细胞活力。(5)将细胞分为单纯缺氧组及缺氧＋10.0 μmol/L辣椒素组，每组1孔，前者细胞同前缺氧处理6 h；后者细胞加入10.0 μmol/L辣椒素处理30 min后，行缺氧处理6 h。蛋白质印迹法检测溶酶体相关膜蛋白1(LAMP-1)、LAMP-2的蛋白表达。各实验重复3次或5次。对数据行单因素方差分析、LSD-t检验、Bonferroni校正。 结果： (1)与常氧组比较，缺氧3、6、9 h组心肌细胞中LC3、Beclin-1、TRPV1蛋白表达明显增高(t(3 h)＝4.891、5.890、4.928，t(6 h)＝9.790、6.750、10.590，t(9 h)＝6.948、6.764、5.049，P<0.05或P<0.01)；缺氧6 h组3种蛋白表达最高，分别为1.08±0.05、1.12±0.10、0.953±0.071。(2)缺氧组TRPV1在心肌细胞膜和胞内密度明显增加，呈团块状分布，表达明显强于常氧组。(3)与单纯缺氧组比较，缺氧＋0.1 μmol/L辣椒素组心肌细胞中Beclin-1蛋白表达明显增高(t＝10.488，P<0.01)，LC3和TRPV1蛋白表达虽增高但差异无统计学意义(t＝4.372、3.026，P>0.05)；缺氧＋1.0 μmol/L辣椒素组、缺氧＋10.0 μmol/L辣椒素组心肌细胞中LC3和TRPV1、Beclin-1蛋白表达均明显增高(t＝15.505、5.773、13.430，20.915、8.054、16.384，P<0.05或P<0.01)，其中缺氧＋10.0 μmol/L辣椒素组3种蛋白表达均达峰值，分别为2.33±0.09、1.34±0.07、1.246±0.053。(4)与常氧组(0.585±0.045)比较，缺氧组心肌细胞活力明显下降(0.471±0.037，t＝4.365，P<0.05)；与缺氧组比较，缺氧＋氯喹组心肌细胞活力进一步明显下降(0.350±0.023，t＝6.216，P<0.01)，缺氧＋辣椒素组心肌细胞活力则明显升高(0.564±0.047，t＝3.489，P<0.05)；与缺氧＋氯喹组比较，缺氧＋辣椒素＋氯喹组心肌细胞活力无明显变化(0.364±0.050，t＝0.545，P>0.05)。(5)与单纯缺氧组(0.99±0.04、0.54±0.04)比较，缺氧＋10.0 μmol/L辣椒素组心肌细胞中LAMP-1和LAMP-2蛋白表达明显增加(1.49±0.06、0.81±0.05，t＝12.550、7.442，P<0.01)。 结论： TRPV1通过自噬-溶酶体途径进一步促进缺氧早期小鼠心肌细胞自噬相关蛋白的表达，增强自噬活性，改善自噬流，减轻早期缺氧心肌细胞的损伤。.

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