阿格列汀通过miR-497-5p/GPLD1信号通路抑制高糖诱导的心肌成纤维细胞纤维化的研究

目的 探究阿格列汀对高糖诱导的小鼠心肌成纤维细胞（mouse cardiac fibroblasts,MCFs）纤维化的影响及其作用机制。 方法 使用5.50、25.00 mmol/L葡萄糖处理MCFs细胞，设为低糖组和高塘组。阿格列汀（0.05、0.10、0.20 μmol/L）治疗高糖诱导的MCFs细胞6 h，筛选最适浓度0.10 μmol/L。使用脂质体法将mimics-NC组（转染mimics-NC）、miR-497-5p组（转染miR-497-5p）、anti-NC组（转染anti-NC）、anti-miR-497-5p组（转染anti-miR-497-5p）、si-NC组（转染si-NC）、si-糖基磷脂酰肌醇特异性磷脂酶D（glycosylphosphatidylinositol specific phospholipase D，GPLD1）组（转染si-GPLD1）、阿格列汀+mimics-NC组（转染mimics-NC）、阿格列汀+miR-497-5p组（转染miR-497-5p）、阿格列汀+miR-497-5p+pcDNA组（共转染miR-497-5p和pcDNA）、阿格列汀+miR-497-5p+pcDNA-GPLD1组（共转染miR-497-5p和pcDNA-GPLD1）转染至MCFs细胞，并用25.00 mmol/L葡萄糖或0.10 μmol/L的阿格列汀处理。蛋白免疫印迹（Western blotting，WB）实验、实时荧光定量聚合酶链式反应（real time fluorescence quantitative polymerase chain reaction，RT-qPCR）检测各组细胞中α-平滑肌肌动蛋白（alpha-smooth muscle actin，α-SMA）、胶原Ⅰ（collagenⅠ，ColⅠ）、ColⅢ的蛋白表达及 miR-497-5p、GPLD1的表达。双荧光素酶报告基因检测实验检测细胞的荧光活性。 结果 与NG组相比，HG组MCFs细胞中α-SMA、ColⅠ、ColⅢ的蛋白表达显著升高，miR-497-5p显著降低（P＜0.05），而阿格列汀（0.10、0.20 μmol/L）呈浓度依赖性显著抑制高糖诱导的MCFs细胞中α-SMA、ColⅠ、ColⅢ蛋白升高和miR-497-5p的降低。过表达miR-497-5p后，高糖诱导的MCFs细胞中α-SMA、ColⅠ、ColⅢ蛋白表达均明显降低，并且miR-497-5p抑制野生型GPLD1细胞的荧光活性，负向调控GPLD1蛋白表达。敲减GPLD1具有与过表达miR-497-5p相似的抑制高糖诱导MCFs细胞α-SMA、ColⅠ、ColⅢ蛋白表达的作用。过表达GPLD1则能够显著削弱阿格列汀和过表达miR-497-5p对高糖诱导MCFs细胞α-SMA、ColⅠ、ColⅢ表达的抑制作用。 结论 阿格列汀可抑制高糖诱导的心肌成纤维细胞的纤维化，其机制与调控功能miR-497-5p/GPLD1信号通路有关。

Anagliptin suppresses high glucose-induced fibrosis of myocardial fibroblasts in diabetic cardiomyopathy through miR-497-5p/GPLD1 signaling pathway

Objective To investigate the effect of anagliptin on fibrosis of myocardial fibroblasts(MCFs) induced by high glucose and its mechanism of action.Methods MCFs were treated with 5.50 and 25.00 mmol/L glucose respectively and divided into low glucose group and high glucose group. Anagliptin(0.05, 0.10, 0.20 μmol/L) was used for the treatment of MCFs induced by high glucose for 6 h, and the optimal concentration screened was 0.10 μmol/L. The mimics-NC group(transfected mimics-NC), miR-497-5p group(transfected miR-497-5p), anti-NC group(transfected anti-NC), anti-miR-497-5p group(transfected anti-miR-497-5p), si-NC group(transfected si-NC), si-glycosylphosphatidylinositol specific phospholipase D1(GPLD1) group(transfected si-GPLD1), anagliptin + mimics-NC group(transfected mimics-NC), anagliptin+miR-497-5p group(transfected miR-497-5p), anagliptin + mir-497-5p + pcDNA group(co-transfected miR-497-5p and pcDNA), anagliptin + miR-497-5p + pcDNA-GPLD1 group(co-transfected miR-497-5p and pcDNA-GPLD1) were transfected into MCFs and treated with 25.00 mmol/L glucose or 0.10 μmol/L anagliptin. Western blotting(WB) assay, and real time fluorescence quantitative polymerase chain reaction(RT-qPCR) were used to detect the protein expression of alpha smooth muscle actin(α-SMΑ), collagen Ⅰ(Col Ⅰ), collagen Ⅲ(Col Ⅲ) and the expression of miR-497-5p and GPLD1 in cells of each group. Double luciferase reporter gene assay was used to detect the fluorescence activity of cells.Results Compared with NG group, the protein expression of α-SMA, Col Ⅰ and Col Ⅲ increased significantly, while miR-497-5p decreased significantly in HG group(P＜0.05). Anagliptin(0.10, 0.20 μmol/L) significantly inhibited the increase in α-SMA, Col Ⅰ and Col Ⅲ protein and the decrease in mir-497-5p in high glucose-induced MCFs in a concentration-dependent manner. After overexpression of miR-497-5p, the expression of α-SMA, Col Ⅰ and Col Ⅲ proteins decreased significantly in high glucose-induced MCFs. miR-497-5p inhibited the fluorescence activity of wild-type GPLD1 cells and negatively regulated the protein expression of GPLD1. Knockdown of GPLD1 inhibited high glucose-induced effect of α-SMA, Col Ⅰ and Col Ⅲ protein expression in MCFs, similar to overexpression of miR-497-5p. Overexpression of GPLD1 could significantly weaken the effect of anagliptin and overexpression of miR-497-5p on inhibition of α-SMA, Col Ⅰ and Col Ⅲ expression in high glucose-induced MCFs.Conclusion Anagliptin could inhibit the fibrosis of MCFs induced by high glucose, and its mechanism may be related to the regulation of miR-497-5p/GPLD1 signaling pathway.