| 王天宇,丁君灿,程心怡,胡鹏飞.炙甘草汤通过miR-181a-5p靶向SPHK2调控心肌纤维化改善糖尿病心肌病[J].浙江中西医结合杂志,2024,34(1): |
| 炙甘草汤通过miR-181a-5p靶向SPHK2调控心肌纤维化改善糖尿病心肌病 |
| Zhigancao Decoction improves diabetic cardiomyopathy by regulating myocardial fibrosis by targeting SPHK2 with miR-181a-5p |
| 投稿时间：2023-04-14 修订日期：2023-09-25 |
| DOI： |
| 中文关键词: 糖尿病心肌病 心肌成纤维细胞 炙甘草汤 miR-181a-5p SPHK2 |
| 英文关键词:Diabetic Cardiomyopathy Cardiac Fibroblasts Zhigancao Decoction miR-181a-5p SPHK2 |
| 基金项目:浙江省中医药科技计划项目(2022ZA082),浙江省医药卫生科技计划项目(2020KY205) |
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| 中文摘要: |
| 目的 探究炙甘草汤对糖尿病心肌病（DCM）模型大鼠心肌纤维化的影响以及调控机制。方法 雄性Sprague Dawley（SD）大鼠30只，10只大鼠作为对照组；20只通过高糖高脂饮食联合腹腔注射链脲佐菌素（STZ）构建DCM模型大鼠，造模后随机按随机数字表法分成 DCM组、炙甘草汤组，每组10只。炙甘草汤组予2mL/100g炙甘草汤（1g/mL）灌胃，对照组及DCM组予等量生理盐水灌胃，连续给药4周，每天1次。灌胃给药结束后，STZ注射后12周， 苏木素-伊红（HE）染色观察DCM模型大鼠心肌组织的病理改变。马松（Masson）染色观察DCM模型大鼠的心肌纤维化情况。提取大鼠乳鼠心肌成纤维细胞（CFs）随机按随机数字表法分为 对照组、高糖组、炙甘草汤组、miRNA-NC组、miR-181a-5p模拟物组、miR-181a-5p拮抗剂组、炙甘草汤+miRNA-NC组和炙甘草汤+miR-181a-5p拮抗剂组并进行相应处理。通过高糖（30mmol/L）诱导 CFs 增殖模型。蛋白免疫印迹法（Western blot）检测大鼠心肌组织和CFs中的α-SMA、Collagen I、Collagen III和SPHK2蛋白表达。实时荧光定量聚合酶链式反应（qRT-PCR）检测miR-181a-5p、Collagen I和Collagen III的相对表达水平。细胞计数试剂盒8（CCK-8）检测CFs的细胞活力。TargetScan数据库预测miR-181a-5p与SPHK2的结合序列。双荧光素酶报告基因实验验证miR-181a-5p与SPHK2的靶向关系。结果 与对照组比较，DCM组大鼠血糖水平显著升高[（30.40±3.48）mmol/L比（5.08±0.17）mmol/L，P＜0.001]，心肌细胞肥大、排列紊乱、成纤维细胞增生伴炎症细胞浸润，炙甘草汤组大鼠上述情况均得到改善。与对照组比较，DCM组大鼠心肌组织α-SMA、Collagen I、Collagen III、SPHK2蛋白水平升高，miR-181a-5p水平显著下降[（0.45±0.20）比（1.00±0.12），P＜0.001]；而炙甘草汤喂养能够逆转上述表达水平变化。与对照组相比比较，高糖组CFs的细胞活力[（194.03±25.59）%比（100.00±11.00）%，P＜0.001]、Collagen I [（3.73±0.81）比（1.00±0.17），P＜0.001]、Collagen III [（3.93±0.97）比（1.00±0.24），P＜0.001]、α-SMA蛋白、SPHK2蛋白水平升高，miR-181a-5p水平显著下降[（0.41±0.18）比（1.00±0.21），P＜0.001]；与高糖组相比比较，炙甘草汤组CFs的细胞活力[（147.63±25.80）%比（194.03±25.59）%，P＜0.01]、Collagen I [（1.91±0.58）比（3.73±0.81），P＜0.001]、Collagen III [（2.02±0.45）比（3.93±0.97），P＜0.01]、α-SMA蛋白、SPHK2蛋白水平降低，miR-181a-5p水平显著升高[（0.69±0.17）比（0.41±0.18），P＜0.01]。与miRNA-NC相比比较，CFs转染miR-181a-5p模拟物后miR-181a-5p表达显著升高[（2.82±0.86）比（1.00±0.28），P＜0.001]，SPHK2表达下降；而转染miR-181a-5p拮抗剂后miR-181a-5p表达显著下降[（0.29±0.09）比（1.00±0.28），P＜0.001]，SPHK2表达升高。TargetScan数据库分析发现miR-181a-5p与SPHK2的3’UTR存在结合序列。双荧光素酶报告基因实验证实miR-181a-5p和SPHK2存在靶向作用。与炙甘草汤+miRNA-NC组相比比较，炙甘草汤+miR-181a-5p拮抗剂组CFs Collagen I [（2.95±0.67）比（1.89±0.26），P＜0.01]、Collagen III [（2.99±0.74）比（1.85±0.63），P＜0.01]、细胞活力[（78.57±10.64）%比（59.75±8.08）%，P＜0.01]、α-SMA蛋白、SPHK2蛋白水平升高，miR-181a-5p水平显著降低[（0.77±0.17）比（2.89±0.57），P＜0.001]。结论 炙甘草汤具有改善DCM的作用，其作用机制是通过miR-181a-5p靶向SPHK2调控心肌纤维化来实现的。 |
| 英文摘要: |
| Objective To investigate the effects of Zhigancao Decoction on myocardial fibrosis in rats with diabetic cardiomyopathy（DCM）model and the pharmacological regulation mechanism. Methods There were 30 male Sprague Dawley（SD）rats, 10 rats were used as the control group, 20 rats were used to construct DCM model rats by combining high glucose and high fat diet with intraperitoneal injection of streptozotocin（STZ）, and then they were divided into the DCM group and the Zhigancao Decoction group according to the method of randomized numerical table. The Zhigancao Decoction group was given 2mL/100g of Zhigancao Decoction（1g/mL）by gastric gavage, while the control group and the DCM group were given equal amounts of saline by gastric gavage, and the drug was administered once a day for 4 weeks. At the end of gavage, hematoxylin-eosin （HE）staining was used to observe the pathological changes in the myocardial tissue of DCM model rats. Masson staining was used to observe myocardial fibrosis in DCM model rats. The extracted rat cardiac fibroblasts（CFs）were divided into control group, high glucose group, Zhigancao Decoction group, miRNA-NC group, miR-181a-5p mimics group, miR-181a-5p inhibitors group, Zhigancao Decoction group + miRNA-NC group, and Zhigancao Decoction group + miR-181a-5p inhibitors group by randomized numerical table method and treated accordingly. The CFs proliferation model was induced by high glucose（30mmol/L）. Protein immunoblotting （Western blot）was used to detect α-SMA，Collagen I, Collagen III, and SPHK2 protein expression in rat myocardial tissues and CFs. The relative expression levels of miR-181a-5p, Collagen I and Collagen III were detected by real-time fluorescence quantitative polymerase chain reaction（qRT-PCR）. Cell counting kit 8（CCK-8）was used to detect the cell viability of CFs. The TargetScan database predicted the binding sequence of miR-181a-5p to SPHK2. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-181a-5p and SPHK2. Results Compared with the control group, rats in the DCM group showed significantly higher blood glucose levels（30.40±3.48 vs. 5.08±0.17mmol/L；P<0.001）, cardiomyocyte hypertrophy, disordered arrangement, and fibroblast hyperplasia accompanied by inflammatory cell infiltration, which were improved in the rats in the Zhigancao Decoction group. Compared with the control group, α-SMA, Collagen I, Collagen III, and SPHK2 protein levels in myocardial tissues of rats in the DCM group were elevated，and miR-181a-5p levels were significantly decreased（0.45±0.20 vs. 1.00±0.12；P<0.001）, whereas feeding with Zhigancao Decoction was able to reverse the changes in these expression levels. Compared with the control group, the cell viability of CFs in the high glucose group（194.03±25.59 vs. 100.00±11.00%；P<0.001）, Collagen I（3.73±0.81 vs. 1.00±0.17；P<0.001）, Collagen III（3.93±0.97 vs. 1.00±0.24；P<0.001）, α-SMA protein, SPHK2 protein levels were elevated, and miR-181a-5p levels were significantly decreased（0.41±0.18 vs. 1.00±0.21；P<0.001）； the cell viability of CFs in the Zhigancao Decoction group was significantly higher compared with that of the high glucose group（147.63±25.80 vs. 194.03±25.59%；P<0.01）, Collagen I （1.91±0.58 vs. 3.73±0.81；P<0.001）, Collagen III（2.02±0.45 vs. 3.93±0.97；P<0.01）, and the levels of α-SMA proteins, SPHK2 proteins, were decreased. miR-181a-5p levels were significantly increased（0.69±0.17 vs. 0.41±0.18；P<0.01）. Compared with miRNA-NC, miR-181a-5p expression was significantly elevated after transfection of CFs with miR-181a-5p mimics（2.82±0.86 vs. 1.00±0.28, P<0.001）, and SPHK2 expression was decreased, whereas miR-181a-5p expression was significantly decreased after transfection with miR-181a-5p inhibitors（0.29±0.09 vs. 1.00±0.28, P<0.001）, and SPHK2 expression was elevated. The TargetScan database analysis revealed the binding sequence between miR-181a-5p and the 3’UTR of SPHK2. The dual luciferase reporter gene assay confirmed the existence of binding between miR-181a-5p and SPHK2. Compared with the Zhigancao Decoction + miRNA-NC group, the CFs in the Zhigancao Decoction + miR-181a-5p inhibitors group Collagen I（2.95±0.67 vs. 1.89±0.26；P<0.01）, Collagen III（2.99±0.74 vs. 1.85±0.63；P<0.01）, cell viability（78.57±10.64 vs. 59.75±8.08%；P<0.01）, α-SMA protein, SPHK2 protein levels were increased, and miR-181a-5p levels were significantly decreased（0.77±0.17 vs. 2.89±0.57；P<0.001）. Conclusion The mechanism of action of Zhigancao Decoction with improved DCM is mediated by miR-181a-5p targeting SPHK2 to regulate myocardial fibrosis. |
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