Qiu Jia, Wang An, Xu Yingna, Qiao Shigang, An Jianzhong, Li Hua, Wang Chen
Department of Anesthesiology, Second Affiliated Hospital, Soochow University, Suzhou 215004, Jiangsu, China (Qiu J, Wang A); Soochow University, Suzhou 215006, Jiangsu, China (Xu YN); Institute of Clinical Medicine Research, Suzhou Science and Technology Town Hospital, Suzhou 215153, Jiangsu, China (Qiao SG, An JZ); Department of Anesthesiology and Perioperative Medicine, Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou 215153, Jiangsu, China (Li H, Wang C). Corresponding author: Wang Chen, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2018 Feb;30(2):145-150. doi: 10.3760/cma.j.issn.2095-4352.2018.02.010.
To investigate the role of microRNA-1 (miR-1) in cardiac fibroblasts induced by high glucose in rats.
The primary fibroblasts were cultured from the apical tissue of 1-3 day-old Sprague-Dawley (SD) rats. The cells which were passaged to generation 3 or 4, were randomly divided into normal glucose+lentivector-vehicle group (CON+Lv-Vehicle group), normal glucose+lentivector-miR-1 group (CON+Lv-miR1 group), high glucose+lentivector-vehicle group (HG+Lv-Vehicle group), high glucose+lentivector-miR-1 group (HG+Lv-miR1 group), high glucose+Lv-Vehicle+inhibitor group (HG+Lv-Vehicle+CC group), and high glucose+lentivector-miR-1+inhibitor group (HG+Lv-miR1+CC group). The myocardial fibroblasts were cultured in the concentration of 5.5 mmol/L glucose (normal glucose) or 25.0 mmol/L (high glucose) DMEM medium. Then lentiviral vector containing miR-1 silent sequence or the same volume of lentiviral vector was inoculated into the cells. The AMP activated protein kinase (AMPK) inhibitor Compound C (20 μmol/L) was added to the medium at 12 hours before sampling in inhibitor groups. The expression of phosphorylation of AMPK (p-AMPK), collagenIandIII, matrix metalloproteinase (MMP-2, MMP-9), and autophagy flux related protein LC3B-II and p62/SQSTM1 were measured by Western Blot.
The purity of rat myocardial fibroblasts in vitro was 97%. Compared with CON+Lv-Vehicle group, there was no significant difference in the expression of p-AMPK in CON+Lv-miR1 group, the expression of p-AMPK in HG+Lv-Vehicle group was significantly decreased (p-AMPK/t-AMPK: 44.72±3.29 vs. 100.00±7.77, P < 0.01). The expression of p-AMPK in HG+Lv-miR1 group was higher than that in HG+Lv-Vehicle group (p-AMPK/t-AMPK: 60.52±5.16 vs. 44.72±3.29, P < 0.05). Compared with HG+Lv-Vehicle group, the expressions of collagen, MMP, LC3B-II and p62/SQSTM1 in HG+Lv-miR1 group were significantly decreased; after the treatment with AMPK inhibitor, the expressions of collagen, MMP, LC3B-II, p62/SQSTM1 were significantly increased (HG+Lv-Vehicle+CC group vs. HG+Lv-Vehicle group: collagen I/β-actin: 158.74±13.21 vs. 100.00±7.64, collagen III/β-actin: 177.38±17.31 vs. 100.00±5.18, MMP-2/β-actin: 130.09±14.31 vs. 100.00±10.47, MMP-9/β-actin: 215.54±20.92 vs. 100.00±11.28, LC3B-II/β-actin: 159.34±13.83 vs. 100.00±6.44, p62/SQSTM1/β-actin: 201.01±24.02 vs. 100.00±8.62; HG+Lv-miR1+CC group vs. HG+Lv-miR1 group: collagen I/β-actin: 108.69±9.93 vs. 80.83±7.24, collagen III/β-actin: 127.68±10.46 vs. 81.56±9.97, MMP-2/β-actin: 106.66±10.21 vs. 74.80±7.43, MMP-9/ β-actin: 145.65±11.56 vs. 74.63±10.55, LC3B-II/β-actin: 150.15±13.28 vs. 22.98±2.87, p62/SQSTM1/β-actin: 130.48±10.74 vs. 49.90±2.27, all P < 0.05).
miR-1 gene silencing inhibits myocardial fibrosis induced by high glucose, its mechanism may be related to the up-regulation of p-AMPK, which can recover autophagy flux.
探讨微小RNA-1（miR-1）在高糖诱导的大鼠心脏成纤维细胞中的作用。
从1-3日龄的Sprague-Dawley（SD）大鼠的顶端组织中培养原代成纤维细胞。将传代至第3或第4代的细胞随机分为正常葡萄糖+慢病毒载体空载组（CON+Lv-Vehicle组）、正常葡萄糖+慢病毒载体-miR-1组（CON+Lv-miR1组）、高糖+慢病毒载体空载组（HG+Lv-Vehicle组）、高糖+慢病毒载体-miR-1组（HG+Lv-miR1组）、高糖+Lv-Vehicle+抑制剂组（HG+Lv-Vehicle+CC组）和高糖+慢病毒载体-miR-1+抑制剂组（HG+Lv-miR1+CC组）。心肌成纤维细胞在含5.5 mmol/L葡萄糖（正常葡萄糖）或25.0 mmol/L（高糖）的DMEM培养基中培养。然后将含有miR-1沉默序列的慢病毒载体或相同体积的慢病毒载体接种到细胞中。在抑制剂组取样前12小时，向培养基中加入AMP激活的蛋白激酶（AMPK）抑制剂Compound C（20 μmol/L）。通过蛋白质免疫印迹法检测AMPK磷酸化（p-AMPK）、I型和III型胶原蛋白、基质金属蛋白酶（MMP-2、MMP-9）以及自噬通量相关蛋白LC3B-II和p62/SQSTM1的表达。
体外培养的大鼠心肌成纤维细胞纯度为97%。与CON+Lv-Vehicle组相比，CON+Lv-miR1组p-AMPK的表达无显著差异，HG+Lv-Vehicle组p-AMPK的表达显著降低（p-AMPK/t-AMPK：44.72±3.29 vs. 100.00±7.77，P<0.01）。HG+Lv-miR1组p-AMPK的表达高于HG+Lv-Vehicle组（p-AMPK/t-AMPK：60.52±5.16 vs. 44.72±3.29，P<0.05）。与HG+Lv-Vehicle组相比，HG+Lv-miR1组胶原蛋白、MMP、LC3B-II和p62/SQSTM1的表达显著降低；用AMPK抑制剂处理后，胶原蛋白、MMP、LC3B-II、p62/SQSTM1均显著增加（HG+Lv-Vehicle+CC组与HG+Lv-Vehicle组比较：I型胶原蛋白/β-肌动蛋白：158.74±13.21 vs. 100.00±7.64，III型胶原蛋白/β-肌动蛋白：177.38±17.31 vs. 100.00±5.18，MMP-2/β-肌动蛋白：130.09±14.31 vs. 100.00±10.47，MMP-9/β-肌动蛋白：215.54±20.92 vs. 100.00±11.28，LC3B-II/β-肌动蛋白：159.34±13.83 vs. 100.00±6.44，p62/SQSTM1/β-肌动蛋白：201.01±24.02 vs. 100.00±8.62；HG+Lv-miR1+CC组与HG+Lv-miR1组比较：I型胶原蛋白/β-肌动蛋白：108.69±9.93 vs. 80.83±7.24，III型胶原蛋白/β-肌动蛋白：127.68±10.46 vs. 81.56±9.97，MMP-2/β-肌动蛋白：106.66±10.21 vs. 74.80±7.43，MMP-9/β-肌动蛋白：145.65±11.56 vs. 74.63±10.55，LC3B-II/β-肌动蛋白：150.15±13.28 vs. 22.98±2.87，p62/SQSTM1/β-肌动蛋白：130.48±10.74 vs. 49.90±2.27，均P<0.05）。
miR-1基因沉默抑制高糖诱导的心肌纤维化，其机制可能与上调p-AMPK从而恢复自噬通量有关。
