从bFGF/Akt/Caspase通路探讨生肌象皮膏促进糖尿病大鼠溃疡愈合的机制
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摘要
目的:通过观察2型糖尿病Sprague Dawley(SD)大鼠溃疡创面肉芽组织中碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF),丝氨酸/苏氨酸蛋白激酶(serine/threonine kinase,Akt),半胱氨酸天冬氨酸酶-9(cystein-asparate protease-9,Caspase-9)的表达,探讨生肌象皮膏促进糖尿病大鼠溃疡愈合的作用机制。方法:制备2型糖尿病SD大鼠溃疡模型,随机分为糖尿病生肌象皮膏组、糖尿病凡士林组、糖尿病生理盐水组,正常组大鼠溃疡模型成模即为模型组。糖尿病生肌象皮膏组用生肌象皮膏纱条外敷,糖尿病凡士林组用凡士林纱条外敷,糖尿病生理盐水组和模型组用生理盐水纱条外敷伤口。各组分别于在造模后第1,3,7,14,21天观察创面愈合情况,取溃疡创面肉芽组织备用。采用苏木素-伊红(hematoxylineosin staining,HE)染色法于光镜下观察创面肉芽组织形态学变化,实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Western blot)检测创面中bFGF的表达,酶联免疫吸附法(ELISA)测定创面p-Akt含量,免疫组织化学法检测创面中Caspase-9表达情况。结果:与糖尿病凡士林组、糖尿病生理盐水组比较,生肌象皮膏显著上调了溃疡创面肉芽组织中bFGF表达水平(P<0.05),增加了p-AKT的含量(P<0.05),同时抑制了Caspase-9的表达(P<0.05)。结论:生肌象皮膏可通过bFGF/Akt/Caspase通路,促进糖尿病难愈创面愈合。
Objective: To observe the expressions of basic fibroblast growth factor(bFGF),serine/threonine kinase(Akt) and cystein-asparate protease-9(Caspase-9) in diabetic rat ulcer granulation tissues,in order to investigate the mechanism of Shengji Xiangpi mastic in promoting the ulcer healing in diabetic rats.Method: The ulcer model of type 2 diabetic SD rats was established,and randomly divided into normal saline group,vaseline group and Shengji Xiangpi mastic group.The ulcer model of normal rats was set up as the blank control group.The rats in Shengji Xiangpi mastic group were applied with Shengji Xiangpi mastic and covered with gauze,those in vaseline group were applied with vaseline and covered with gauze,and those in normal saline group and blank control group were treated with physiological saline.The wound healing conditions in each group were observed on 1,3,7,14,21 d after modeling,and ulcer tissues were collected.Hematoxylin-eosin(HE) staining was performed to observe the morphological changes in wound granulation tissues.Real-time PCR and Western blot were used to analyze the expression of bFGF,emzyme-linked immunosorbent assay(ELISA) was used to analyze the expression of p-Akt,and immunohistochemistry was used to analyze the expression of Caspase-9.Result:Compared with vaseline group and saline group,Shengji Xiangpi mastic significantly up-regulated bFGF expression in ulcer granulation tissues of diabetic rats,increased p-Akt content,and inhibited the expression of Caspase-9.Conclusion: Shengji Xiangpi mastic can promote ulcer healing in diabetic rats through bFGF/Akt/Caspase pathway.
Objective: To observe the expressions of basic fibroblast growth factor(bFGF),serine/threonine kinase(Akt) and cystein-asparate protease-9(Caspase-9) in diabetic rat ulcer granulation tissues,in order to investigate the mechanism of Shengji Xiangpi mastic in promoting the ulcer healing in diabetic rats.Method: The ulcer model of type 2 diabetic SD rats was established,and randomly divided into normal saline group,vaseline group and Shengji Xiangpi mastic group.The ulcer model of normal rats was set up as the blank control group.The rats in Shengji Xiangpi mastic group were applied with Shengji Xiangpi mastic and covered with gauze,those in vaseline group were applied with vaseline and covered with gauze,and those in normal saline group and blank control group were treated with physiological saline.The wound healing conditions in each group were observed on 1,3,7,14,21 d after modeling,and ulcer tissues were collected.Hematoxylin-eosin(HE) staining was performed to observe the morphological changes in wound granulation tissues.Real-time PCR and Western blot were used to analyze the expression of bFGF,emzyme-linked immunosorbent assay(ELISA) was used to analyze the expression of p-Akt,and immunohistochemistry was used to analyze the expression of Caspase-9.Result:Compared with vaseline group and saline group,Shengji Xiangpi mastic significantly up-regulated bFGF expression in ulcer granulation tissues of diabetic rats,increased p-Akt content,and inhibited the expression of Caspase-9.Conclusion: Shengji Xiangpi mastic can promote ulcer healing in diabetic rats through bFGF/Akt/Caspase pathway.
引文
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[14]朱金墙,宋宛珊,马妍,等.PI3K/Akt信号通路与血管性痴呆的关系及中药干预作用研究进展[J].中国实验方剂学杂志,2016,22(9):223-229.
[15]Almhanna K,Strosberg J,Malafa M.Targeting Akt protein kinase in gastric cancer[J].Anticancer Res,2011,31(12):4387-4392.
[16]WANG Z G,WANG Y,YE J M,et al.bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway[J].J Cell Mol Med,2015,19(3):595-607.
[17]Walther S,Awad S,Lonchyna V A,et al.NFAT transcription factor regulation by urocortinⅡin cardiac myocytes and heart failure[J].Am J Physiol-Heart C,2014,306(6):H856-H866.
[18]Sangawa A,Shintani M,Yamao N,et al.Phosphorylation status of Akt and Caspase-9 in gastric and colorectal carcinomas[J].Int J Clin Exp Patho,2014,7(6):3312-3317.
[2]中华医学会糖尿病学分会.中国2型糖尿病防治指南(2013年版)[J].中华内分泌代谢杂志,2014,30(10):26-89.
[3]Inamura K,Matsuzaki Y,Uematsu N,et al.Rapid inhibition of MAPK signaling and anti-proliferation effect via JAK/STAT signaling by interferon-αin hepatocellular carcinoma cell lines[J].Bba-Mol Cell Res,2005,1745(3):401-410.
[4]Agarwal E,Brattain M G,Chowdhury S.Cell survival and metastasis regulation by Akt signaling in colorectal cancer[J].Cell Signal,2013,25(8):1711-1719.
[5]李巧芬,常柏,李晓军,等.生肌象皮膏对糖尿病大鼠难治性溃疡愈合速度的影响[J].中国现代医生,2008,46(5):96-97.
[6]李巧芬,李春深,李云平,等.生肌象皮膏对糖尿病大鼠溃疡肉芽组织中白介素-2与肿瘤坏死因子-α的影响[J].中国实验方剂学杂志,2013,19(18):279-282.
[7]李巧芬,常柏.生肌象皮膏对2型糖尿病大鼠溃疡肉芽组织的胰岛素样生长因子-1及受体的影响[J].天津中医药,2012,29(6):570-572.
[8]宋绍华,何黎升,金岩,等.猪糖尿病皮肤溃疡模型建立的实验研究[J].中国组织工程研究,2004,8(36):8217-8219.
[9]关小宏,李宝军,肖黎,等.糖尿病足流行病学及糖尿病足截肢(趾)的临床情况分析[J].中华损伤与修复杂志:电子版,2012,7(4):406-408.
[10]Goldberg S R,Diegelmann R F.Wound healing primer[J].Surg Clin N Am,2010,90(6):1133-1146.
[11]Sasaki A T,Chun C,Takeda K,et al.Localized Ras signaling at the leading edge regulates PI3K,cell polarity,and directional cell movement[J].J Cell Biol,2004,167(3):505-518.
[12]Sepe L,Ferrari M C,Cantarella C,et al.Ras activated ERK and PI3K pathways differentially affect directional movement of cultured fibroblasts[J].Cell Physiol Biochem,2013,31(1):123-142.
[13]林敏,李平,钱其军.bFGF激活PI3K信号通路调控大鼠胚胎干细胞自我更新[J].中国细胞生物学学报,2016,38(5):550-556.
[14]朱金墙,宋宛珊,马妍,等.PI3K/Akt信号通路与血管性痴呆的关系及中药干预作用研究进展[J].中国实验方剂学杂志,2016,22(9):223-229.
[15]Almhanna K,Strosberg J,Malafa M.Targeting Akt protein kinase in gastric cancer[J].Anticancer Res,2011,31(12):4387-4392.
[16]WANG Z G,WANG Y,YE J M,et al.bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway[J].J Cell Mol Med,2015,19(3):595-607.
[17]Walther S,Awad S,Lonchyna V A,et al.NFAT transcription factor regulation by urocortinⅡin cardiac myocytes and heart failure[J].Am J Physiol-Heart C,2014,306(6):H856-H866.
[18]Sangawa A,Shintani M,Yamao N,et al.Phosphorylation status of Akt and Caspase-9 in gastric and colorectal carcinomas[J].Int J Clin Exp Patho,2014,7(6):3312-3317.