复元醒脑汤对糖尿病脑梗死大鼠BMECs的影响及miRNA-320的调控机制研究
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摘要
[目的]在细胞水平观察复元醒脑汤对糖尿病脑梗死大鼠缺血脑组织脑微血管内皮细胞(BMECs)的影响,从微小RNA-320(miRNA-320)调控途径探讨复元醒脑汤治疗糖尿病脑梗死的作用机制。[方法]进行复元醒脑汤大鼠灌胃,制备含药血清和对照血清。对糖尿病脑梗死大鼠的缺血脑组织进行BMECs的分离培养,分别加入对照血清、含药血清、转染miRNA-320 inhibitor、中药血清混合转染miRNA-320 inhibitor;实时荧光定量PCR(qPCR)法检测各组BMECs的miRNA-320、胰岛素样生长因子-1(IGF-1)和胰岛素样生长因子受体-1(IGF-1R)基因表达水平,通过甲臜化合物(MTS)检测复元醒脑汤对BMECs增殖能力的影响;通过Transwell实验观察复元醒脑汤对BMECs迁移能力的影响;观察复元醒脑汤对BMECs小管形成能力的影响。[结果]与对照血清组相比,含药血清组、miRNA-320 inhibitor组、含药血清混合miRNA-320 inhibitor组均能使BMECs的增殖、迁移以及小管形成能力显著提高(P<0.01),同时显著降低了miRNA-320的表达水平,显著提高了IGF-1的表达水平。[结论]复元醒脑汤能够抑制糖尿病脑梗死大鼠缺血脑组织中miRNA-320表达,增加IGF-1的表达,进而促进BMECs的增殖、迁移及成管能力。
[Objective] To explore the function of Fuyuan Xingnao Decoction on the BMECs of diabetic cerebral infarction and to explore specific mechanisms for the treatment of diabetic cerebral infarction from the microRNA-320 regulation pathways. [Methods] Rats were fed with Fuyuan Xingnao Decoction or PBS to prepare medicated serum and control serum. BMECs separated from diabetic rats 'cerebral infarction tissue. The control serum, medicated serum, microRNA inhibitors, medicated serum mixed microRNA inhibitors were added in to BMECs respectively. The gene expression of microRNA-320, IGF-1 and IGF-1 R was observed with the method of qPCR. After the treatment of Fuyuan Xingnao Decoction, the MTS test was used to detect BMECs proliferation ability; Trans-well assay was used to observe BMECs migration ability; and tubule formation ability was also observed. [Results] Comparing with the control serum group,microRNA-320 inhibitor group, medicated serum group, medicated serum mixed with inhibitor control group all can improve the capacity of proliferation, migration and tubular formation in BMECs(P <0.01), decrease the expression of microRNA-320 and improve the expression of IGF-1. [Conclusions] Fuyuan Xingnao Decoction increased the protein secretion of IGF-1 by inhibiting the expression of microR-320, thus enhanced the capacity of proliferation, migration and tubular formation in BMECs.
[Objective] To explore the function of Fuyuan Xingnao Decoction on the BMECs of diabetic cerebral infarction and to explore specific mechanisms for the treatment of diabetic cerebral infarction from the microRNA-320 regulation pathways. [Methods] Rats were fed with Fuyuan Xingnao Decoction or PBS to prepare medicated serum and control serum. BMECs separated from diabetic rats 'cerebral infarction tissue. The control serum, medicated serum, microRNA inhibitors, medicated serum mixed microRNA inhibitors were added in to BMECs respectively. The gene expression of microRNA-320, IGF-1 and IGF-1 R was observed with the method of qPCR. After the treatment of Fuyuan Xingnao Decoction, the MTS test was used to detect BMECs proliferation ability; Trans-well assay was used to observe BMECs migration ability; and tubule formation ability was also observed. [Results] Comparing with the control serum group,microRNA-320 inhibitor group, medicated serum group, medicated serum mixed with inhibitor control group all can improve the capacity of proliferation, migration and tubular formation in BMECs(P <0.01), decrease the expression of microRNA-320 and improve the expression of IGF-1. [Conclusions] Fuyuan Xingnao Decoction increased the protein secretion of IGF-1 by inhibiting the expression of microR-320, thus enhanced the capacity of proliferation, migration and tubular formation in BMECs.
引文
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[14]耿介立,张瑛,苗玲.血浆胰岛索样生长因子-1水平变化对急性缺血性脑卒中患者预后的影响[J].中国现代神经疾病杂志,2006,6(6):463-466.
[15]De Smedt A,Brouns R,Uyttenboogaart M,et al.Insulin-Like growth factor I serum levels influence ischemic stroke outcome[J].Stroke,2011,42(8):2180-2185.
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[17]吴大方,曹晋,叶欣,等.2型糖尿病患者血清IGF-1水平与颈动脉粥样斑块的相关性研究[J].临床军医杂志,2015,43(4):366-368.
[18]Brussee V,Cunningham FA,Zochddne DW.Direct insulin signaling of neurons reverses diabetic neuropauthy[J].Diabetes,2004,53:1824-1830.
[19]方邦江,周爽,陈浩,等.复元醒脑汤对糖尿病合并缺血性脑损伤大鼠VEGF蛋白的影响[J].上海中医药大学学报,2010,44(5):12-15.
[20]张秀萍,翟凤国,关悦,等.脑缺血-再灌注损伤大鼠皮质微小核糖核酸表达谱的变化[J].中国脑血管病杂志,2012,9(6):307-310.
[2]Chen LL,Liao YF,Zeng TS,et al.Number and function of circulating endothelial progenitor cell in diabetics with different vascular complications[J].Zhonghua Yixue Zazhi,2009,89(18):1234-1239.
[3]Krupinski J,Stroemer P,Slevin M,et al.Three-dimensional structure and survival of newly formed blood vesels after focal cerebral ischemia[J].Neuroreport,2003,14:1171-1176.
[4]方邦江,周爽,陈宝瑾,等.复元醒脑汤对糖尿病并发急性脑梗塞胰岛素抵抗干预作用的临床研究[J].上海中医药杂志,2009,43(5):14-15.
[5]Wang XH,Qian RZ,Zhang W,et al.MicroRNA-320 expression in myocardial microvascular endothelial cells and its relationship with insulin-like growth factor-1 in type 2 diabetic rats[J].Clin Exp Pharmacol Physiol,2009,36(2):181-188.
[6]Shantikumar S,Caporali A,Emanueli C.Role of microRNAs in diabetes and its cardiovascular complications[J].Cardiovasc Res,2012,93(4):583-593.
[7]Feng B,Chakrabarti S.MiR-320 regulates glucose-induced gene expression in diabetes[J].ISRN Endocrinol,2012(8):1-6.
[8]张丹丹,沈静,刘芳.微小RNA与糖尿病血管新生[J].国际内分泌代谢杂志,2013,33(1):40-45.
[9]Lovis P,Roggli E,Laybutt DR,et a1.Alterations in microRNAexpression contribute to fatty acid-induced pancreaticβ-cell dysfunction[J].Diabetes,2008,57(10):2728-2736.
[10]Van RE.The art of microRNA research[J].Circ Res,2011,108(2):219-234.
[11]欧阳平,刘伊丽,许顶立,等.P53、Rb、IGF-1、AT1B基因转移对血管新生内膜增值的影响[J].第一军医大学学报,2001,21(3):173-176.
[12]王洪伟,孟庆海,金澎,等.脑微血管内皮细胞和脑肿瘤干细胞共培养的体外实验研究[J].中华神经外科杂志,2010,26(7):657-660.
[13]Feng R,Loewenstern J,Aggarwal A,et al.Cerebral radiation necrosis:an analysis of clinical and quantitative imaging and volumetric features[J].World Neurosurgery,2018,111:485-494.
[14]耿介立,张瑛,苗玲.血浆胰岛索样生长因子-1水平变化对急性缺血性脑卒中患者预后的影响[J].中国现代神经疾病杂志,2006,6(6):463-466.
[15]De Smedt A,Brouns R,Uyttenboogaart M,et al.Insulin-Like growth factor I serum levels influence ischemic stroke outcome[J].Stroke,2011,42(8):2180-2185.
[16]Moses AC.Insulin resistance and type 2 diabets mellitus:is there a therapeutic role for IGF-1?[J].Endocr Dev,2005,9:121-134.
[17]吴大方,曹晋,叶欣,等.2型糖尿病患者血清IGF-1水平与颈动脉粥样斑块的相关性研究[J].临床军医杂志,2015,43(4):366-368.
[18]Brussee V,Cunningham FA,Zochddne DW.Direct insulin signaling of neurons reverses diabetic neuropauthy[J].Diabetes,2004,53:1824-1830.
[19]方邦江,周爽,陈浩,等.复元醒脑汤对糖尿病合并缺血性脑损伤大鼠VEGF蛋白的影响[J].上海中医药大学学报,2010,44(5):12-15.
[20]张秀萍,翟凤国,关悦,等.脑缺血-再灌注损伤大鼠皮质微小核糖核酸表达谱的变化[J].中国脑血管病杂志,2012,9(6):307-310.