桃仁对糖尿病大血管纤维化大鼠蛋白激酶B信号通路的影响
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摘要
目的:观察中药桃仁对2型糖尿病大血管纤维化大鼠模型中蛋白激酶B(AKT)信号通路的影响。方法:雄性SD大鼠200只,随机选取30只为空白对照组,余170只采用高脂高糖饲料喂养-腹腔注射链脲佐菌素(STZ)-高脂高糖饲料持续喂养的方法制备2型糖尿病大血管纤维化大鼠模型。造模成功后分为空白对照组(n=29)、模型对照组(n=34)、早期干预组(n=34)、低剂量组(n=32)和高剂量组(n=32)。空白对照组不予特殊处理,早期干预组和低剂量组予桃仁颗粒剂水溶液10 mL/(kg·d)灌胃,高剂量组予桃仁颗粒剂水溶液20 mL/(kg·d)灌胃,模型对照组给予10 mL/(kg·d)生理盐水灌胃。干预7周后分别从各组随机选取5只大鼠处死、取材。实时荧光定量PCR(QPCR)检测AKT mRNA表达;免疫组织化学、Western blotting检测股动脉组织AKT信号通路关键信号分子AKT、磷酸化AKT(p-AKT)的表达。结果:免疫组化显示,空白对照组大鼠血管内皮细胞及中膜平滑肌细胞中有散在黄色物质,呈弱阳性改变,模型对照组呈强阳性反应,早期干预组和高剂量组呈弱阳性反应,低剂量组呈阳性反应。QPCR检测,与空白对照组(1.05±0.05)比较,模型对照组(5.68±0.61)、药物干预组(4.27±0.32、5.33±0.60、4.72±0.28)AKTmRNA表达上调(P<0.05或P<0.01);与模型对照组比较,药物干预组AKT mRNA表达均上调,且以早期干预组(4.27±0.32)和高剂量组(4.72±0.28)为著(P<0.01);Western blotting检测,与空白对照组(0.16±0.01、0.10±0.03)比较,模型对照组(0.38±0.03、0.21±0.02)、药物干预组(0.27±0.04、0.18±0.01;0.30±0.05、0.17±0.01;0.28±0.03、0.19±0.02)AKT、p-AKT表达均显著上调(P<0.01);与模型对照组比较,药物干预组AKT、p-AKT表达均上调(P<0.01或P<0.05);药物干预组两两比较显示,AKT、p-AKT表达无差异(P>0.05)。结论:中药桃仁可以抑制糖尿病大鼠大血管纤维化,其机制可能和抑制AKT信号通路有关。
Objective To observe the effect of Peach Kernel Granule on protein kinase B(AKT)signaling pathway in type 2 diabetic rats with macrovascular fibrosis. Methods There were 200 male SD rats, 30 of which were randomly selected as blank control group. The remaining 170 rats were given high fat and sugar diet-intraperitoneal injection of streptozotocin(STZ)-continuous feeding with high fat and sugar diet to prepare macrovascular fibrosis model of type 2 diabetes mellitus. Thirty-nine rats were excluded from the loss of death during feeding and modeling, and were eventually divided into blank control group(n = 29), model control group(n = 34), early intervention group(n = 34), low dose group(n = 32) and high dose group(n = 32). The blank control group was not given special treatment. The model control group was given 10 mL/(kg·d) normal saline by gavage, the early intervention group and the low dose group were given 10 mL/(kg·d) Peach kernel granule solution by gavage, and the high dose group was given 20 mL/(kg·d) Peach Kernel Granule solution by gavage. The early intervention group began to be intervened until the model of type 2 diabetes mellitus was established. The other groups began to be intervened after the model of type 2 diabetes mellitus macrovascular fibrosis rats was established. The intervention lasted for 7 weeks.At last, five rats were randomly selected from each group for experiment. Real-time fluorescence quantitative polymerase chain reaction(QPCR) was used to detect the expression of AKT. Immunohistochemistry and Western blotting were used to detect the expression of AKT and phosphorylated AKT in femoral artery tissue.Results Immunohistochemistry showed that yellow substances were scattered in vascular endothelial cells and smooth muscle cells of rats in blank control group, showing weak positive changes, strong positive reactions in model control group, weak positive reactions in early intervention group and high dose group, and positive reactions in low dose group. Compared with the blank control group(1.05 ± 0.05), the expression of mAKT in the model control group(5.68 ± 0.61), and drug intervention groups(4.27 ± 0.32, 5.33 ± 0.60, 4.72 ±0.28) was up-regulated(P < 0.05 or P < 0.01) Compared with the model control group, the expression of mAKT in the drug intervention groups was up-regulated, especially in the early intervention group(4.27 ± 0.32) and the high dose group(4.72 ± 0.28)(P < 0.01). Compared with the blank control group(0.16 ± 0.01, 0.10 ±0.03), the expression of AKT and p-AKT in the model control group(0.38 ± 0.03, 0.21 ± 0.02), and the drug intervention groups(0.27 ± 0.04, 0.18 ± 0.01; 0.30 ± 0.05, 0.17 ± 0.01; 0.28 ± 0.03, 0.19 ± 0.02) was signi?cantly increased(P<0.01). Compared with the model control group, the expression of AKT and p-AKT in the drug intervention group was significantly increased(P < 0.01 or P < 0.05). The expression of AKT and p-AKT showed no difference between the two groups(P > 0.05). Conclusion Peach kernel can inhibit macrovascular?brosis in diabetic rats, and its mechanism may be related to the inhibition of AKT signaling pathway.
Objective To observe the effect of Peach Kernel Granule on protein kinase B(AKT)signaling pathway in type 2 diabetic rats with macrovascular fibrosis. Methods There were 200 male SD rats, 30 of which were randomly selected as blank control group. The remaining 170 rats were given high fat and sugar diet-intraperitoneal injection of streptozotocin(STZ)-continuous feeding with high fat and sugar diet to prepare macrovascular fibrosis model of type 2 diabetes mellitus. Thirty-nine rats were excluded from the loss of death during feeding and modeling, and were eventually divided into blank control group(n = 29), model control group(n = 34), early intervention group(n = 34), low dose group(n = 32) and high dose group(n = 32). The blank control group was not given special treatment. The model control group was given 10 mL/(kg·d) normal saline by gavage, the early intervention group and the low dose group were given 10 mL/(kg·d) Peach kernel granule solution by gavage, and the high dose group was given 20 mL/(kg·d) Peach Kernel Granule solution by gavage. The early intervention group began to be intervened until the model of type 2 diabetes mellitus was established. The other groups began to be intervened after the model of type 2 diabetes mellitus macrovascular fibrosis rats was established. The intervention lasted for 7 weeks.At last, five rats were randomly selected from each group for experiment. Real-time fluorescence quantitative polymerase chain reaction(QPCR) was used to detect the expression of AKT. Immunohistochemistry and Western blotting were used to detect the expression of AKT and phosphorylated AKT in femoral artery tissue.Results Immunohistochemistry showed that yellow substances were scattered in vascular endothelial cells and smooth muscle cells of rats in blank control group, showing weak positive changes, strong positive reactions in model control group, weak positive reactions in early intervention group and high dose group, and positive reactions in low dose group. Compared with the blank control group(1.05 ± 0.05), the expression of mAKT in the model control group(5.68 ± 0.61), and drug intervention groups(4.27 ± 0.32, 5.33 ± 0.60, 4.72 ±0.28) was up-regulated(P < 0.05 or P < 0.01) Compared with the model control group, the expression of mAKT in the drug intervention groups was up-regulated, especially in the early intervention group(4.27 ± 0.32) and the high dose group(4.72 ± 0.28)(P < 0.01). Compared with the blank control group(0.16 ± 0.01, 0.10 ±0.03), the expression of AKT and p-AKT in the model control group(0.38 ± 0.03, 0.21 ± 0.02), and the drug intervention groups(0.27 ± 0.04, 0.18 ± 0.01; 0.30 ± 0.05, 0.17 ± 0.01; 0.28 ± 0.03, 0.19 ± 0.02) was signi?cantly increased(P<0.01). Compared with the model control group, the expression of AKT and p-AKT in the drug intervention group was significantly increased(P < 0.01 or P < 0.05). The expression of AKT and p-AKT showed no difference between the two groups(P > 0.05). Conclusion Peach kernel can inhibit macrovascular?brosis in diabetic rats, and its mechanism may be related to the inhibition of AKT signaling pathway.
引文
[1]中华医学会糖尿病学分会.中国2型糖尿病防治指南(2017年版)[J].中华糖尿病杂志,2018,10(1):26-34.
[2]Tunon J,Ruiz-Ortega M,Egido J.Regulation of matrix proteins and impact onvascular structure[J].Curr Hypertens Rep,2000,2(1):106-113.
[3]张庚扬,范英昌,金树梅,等.糖尿病足辨证分型与病理学相关性研究[J].天津中医药,2006,23(2):115-117.
[4]谷雪,付文娟,孙芳初,等.PI3K/Akt信号通路与放射性肝损伤的关系研究[J].解放军预防医学杂志,2018,36(12):1581-1584.
[5]王乙安,陈静,赵恒,等.PI3K/AKT/Survivin通路在大鼠腹膜纤维化中的作用机制[J].广东医学,2018,39(21):3165-3169.
[6]Yu Y,Lyons TJ.Alethal tetrad in diabetes:hyperglycemia,dyslipidemia,oxidative stress,and endothelial dysfunction[J].Am J Med Sci,2005,330(5):227-232.
[7]Coelho RP,Yuelling LM,Fuss B,et al.Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes[J].Glia,2009,57(16):1754-1764.·作者须知·参考文献著参考文献著录格式按GB/T 7714-2005《文后参考文献著录序用阿拉伯数字标出,并将序号置于方括号中,排列于文后。日文汉字请按日文规定书写,勿与我国汉字及简化字混淆。同后以“,等”表示(英文文献以“,et al”表示),不同作者姓名之外文参考文献著录参照医学期刊编辑国际委员会制定的格式名称必须用缩写,缩写名以《Index Medicus》中的格式为准。作式,示例如下:[1]张晖,孔棣,吴咸中,等.人参皂甙Rh2致MCF-7/AD261-264.
[2]Fu X,Qu Z,Sheng Z.Potentiality of mesenchymal stem204-208.著作的引用方式可采用如下两种方式:a.主编,著作名,第名,第几版,出版地,出版者,出版年,页码。示例如下:[8]严冬梅,李敏军,刘友平,等.内质网应激下肝癌细胞Akt和ERK通路间的cross-talk研究[J].中国现代医学杂志,2011,21(20):2361-2364.
[9]董洪亮,刘乃国,苗双,等.1,25-(OH)_2D_3对肺纤维化大鼠中PI3K、AKT、m TOR表达的影响及其机制研究[J].中国现代医学杂志,2018,19(10):1-6.
[10]蔡凤桃,张学荣,孙林,等.眼镜蛇毒NGF通过PI3K/Akt促人肝星状细胞凋亡的机制研究[J].中国药理学通报,2018,01(19):23-27.
[11]付岩,杨文成,魏世杰,等.苦参碱调控p-Akt/Akt蛋白表达抑制大鼠心肌肥厚的作用[J].中国医院药学杂志,2014,07(07):505-509.
[12]徐阳,王军.桃核承气汤对大鼠糖尿病大血管Toll样受体表达的影响[J].中国中西医结合外科杂志,2017,23(01):60-64.
[13]徐阳,王军,武海阔.桃核承气汤对糖尿病大鼠大血管纤维病变Toll样受体通路作用研究[J].天津中医药,2016,33(05):299-302.
[14]张晓平,陈建明,强世平,等.山桃仁水煎提取物对肝纤维化小鼠血清Ⅰ、Ⅲ型前胶原的降解作用[J].福建中医药,2002,33(04):36-37.
[15]李雪梅,冯琴,彭景华,等.苦杏仁苷对二甲基亚硝胺诱导的大鼠肝纤维化的防治作用[J].中西医结合肝病杂志,2011,21(04):221-223+259.
[16]贾磊,陈德兴,文小平.基于关联规则的王清任桃仁用药配伍挖掘[J].上海中医药大学学报,2011,25(4):86-88.格式及要求则》执行。采用顺序编码制著录,依照其在文中出现的先后顺用文献(包括文字和表达的原意)务请作者与原文核对无误。一文献作者不超过3人时全部著录,超过3人者只著录前3人,用“,”隔开。中文期刊用全名,后接出版年,卷(期):起止页。准(http://www.nlm.nih.gov/bsd/uniform_requirements.html),期刊姓名一律姓在前、名在后,国外作者的名字采用首字母缩写形凋亡的实验研究[J].中国中西医结合外科杂志,2007,17(3):ells in regeneration of sweat glands[J].J Surg Res,2006,136(2):
[2]Tunon J,Ruiz-Ortega M,Egido J.Regulation of matrix proteins and impact onvascular structure[J].Curr Hypertens Rep,2000,2(1):106-113.
[3]张庚扬,范英昌,金树梅,等.糖尿病足辨证分型与病理学相关性研究[J].天津中医药,2006,23(2):115-117.
[4]谷雪,付文娟,孙芳初,等.PI3K/Akt信号通路与放射性肝损伤的关系研究[J].解放军预防医学杂志,2018,36(12):1581-1584.
[5]王乙安,陈静,赵恒,等.PI3K/AKT/Survivin通路在大鼠腹膜纤维化中的作用机制[J].广东医学,2018,39(21):3165-3169.
[6]Yu Y,Lyons TJ.Alethal tetrad in diabetes:hyperglycemia,dyslipidemia,oxidative stress,and endothelial dysfunction[J].Am J Med Sci,2005,330(5):227-232.
[7]Coelho RP,Yuelling LM,Fuss B,et al.Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes[J].Glia,2009,57(16):1754-1764.·作者须知·参考文献著参考文献著录格式按GB/T 7714-2005《文后参考文献著录序用阿拉伯数字标出,并将序号置于方括号中,排列于文后。日文汉字请按日文规定书写,勿与我国汉字及简化字混淆。同后以“,等”表示(英文文献以“,et al”表示),不同作者姓名之外文参考文献著录参照医学期刊编辑国际委员会制定的格式名称必须用缩写,缩写名以《Index Medicus》中的格式为准。作式,示例如下:[1]张晖,孔棣,吴咸中,等.人参皂甙Rh2致MCF-7/AD261-264.
[2]Fu X,Qu Z,Sheng Z.Potentiality of mesenchymal stem204-208.著作的引用方式可采用如下两种方式:a.主编,著作名,第名,第几版,出版地,出版者,出版年,页码。示例如下:[8]严冬梅,李敏军,刘友平,等.内质网应激下肝癌细胞Akt和ERK通路间的cross-talk研究[J].中国现代医学杂志,2011,21(20):2361-2364.
[9]董洪亮,刘乃国,苗双,等.1,25-(OH)_2D_3对肺纤维化大鼠中PI3K、AKT、m TOR表达的影响及其机制研究[J].中国现代医学杂志,2018,19(10):1-6.
[10]蔡凤桃,张学荣,孙林,等.眼镜蛇毒NGF通过PI3K/Akt促人肝星状细胞凋亡的机制研究[J].中国药理学通报,2018,01(19):23-27.
[11]付岩,杨文成,魏世杰,等.苦参碱调控p-Akt/Akt蛋白表达抑制大鼠心肌肥厚的作用[J].中国医院药学杂志,2014,07(07):505-509.
[12]徐阳,王军.桃核承气汤对大鼠糖尿病大血管Toll样受体表达的影响[J].中国中西医结合外科杂志,2017,23(01):60-64.
[13]徐阳,王军,武海阔.桃核承气汤对糖尿病大鼠大血管纤维病变Toll样受体通路作用研究[J].天津中医药,2016,33(05):299-302.
[14]张晓平,陈建明,强世平,等.山桃仁水煎提取物对肝纤维化小鼠血清Ⅰ、Ⅲ型前胶原的降解作用[J].福建中医药,2002,33(04):36-37.
[15]李雪梅,冯琴,彭景华,等.苦杏仁苷对二甲基亚硝胺诱导的大鼠肝纤维化的防治作用[J].中西医结合肝病杂志,2011,21(04):221-223+259.
[16]贾磊,陈德兴,文小平.基于关联规则的王清任桃仁用药配伍挖掘[J].上海中医药大学学报,2011,25(4):86-88.格式及要求则》执行。采用顺序编码制著录,依照其在文中出现的先后顺用文献(包括文字和表达的原意)务请作者与原文核对无误。一文献作者不超过3人时全部著录,超过3人者只著录前3人,用“,”隔开。中文期刊用全名,后接出版年,卷(期):起止页。准(http://www.nlm.nih.gov/bsd/uniform_requirements.html),期刊姓名一律姓在前、名在后,国外作者的名字采用首字母缩写形凋亡的实验研究[J].中国中西医结合外科杂志,2007,17(3):ells in regeneration of sweat glands[J].J Surg Res,2006,136(2):