基于Fli1调节的补肾益精法对硬皮病小鼠EndMT的影响
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摘要
目的研究补肾益精法中药复方通过干预Fli1表达调控TGF-β信号,抑制系统性硬皮病血管内皮细胞内皮间质转化(endothelial to mesenchymal transition,End MT)的机制。方法采用博来霉素构建硬皮病小鼠模型,分为正常组、模型组、黄芪甲苷组、中药复方组、Fli1 siRNA+中药复方组,其中正常组不予造模。分别采用HE、Masson染色检测小鼠皮肤血管与胶原,采用实时荧光定量聚合酶链式反应法检测各组小鼠皮肤组织Fli1、VE-cadherin和FSP-1 mRNA表达水平;采用蛋白印迹法(Western Blot)检测各组小鼠皮肤组织Fli1、VE-cadherin、FSP-1蛋白表达水平。结果成功建立硬皮病小鼠模型,HE染色与Masson染色结果显示:中药复方组显著降低小鼠皮肤厚度(P<0.01),胶原纤维减少,血管增多,提示中药复方能改善小鼠皮肤纤维化程度及保护血管; QPCR结果显示:补肾益精中药复方能提高Fli1 mRNA、VE-cadherin mRNA表达,降低FSP-1 mRNA表达。Western Blot结果显示:补肾益精中药复方能提高Fli1、VE-cadherin蛋白表达,降低FSP-1蛋白表达。结论补肾益精法中药复方通过上调Fli1表达,抑制血管内皮间质转化。
Objective To study the mechanism of tonifying kidney and strengthening essence methods interfering the Fli1's expression to regulate TGF-β signal,and to inhibit systemic scleroderma endothelial to mesenchymal transition( End MT). Methods Bleomycin was used to construct mice model,and they were randomly divided them into 5 groups,normal group,model group,astragaloside IV group,Traditional Chinese Medicine( TCM) formula group and FLi1 si RNA + TCM formula group. And the mice in normal group was not given modeling. Mice's skin blood vessels were detected with HE staining method,and collagen in mice were detected with Masson staining method. Gene expression of Fli1,VE-cadherin and FSP-1 m RNA in skin tissue of each group were detected by quantitative polymerase chain reaction( QPCR)method. Protein expression of Fli1,VE-cadherin and FSP-1 were detected by western blotting( WB).Results Mice model of scleroderma were successfully established. In TCM formula group,the HE and Masson staining results showed that the thickness of skin was reduced( P<0.01),and the number of col-lagenous fibers reduced and the number of vessel increased. These results suggest that TCM formula can reduce the degree dermal fibrosis and protect vascular in model mice. And the QPCR results showed that TCM formula of tonifying kidney and strengthening essence could increase the level of m RNA expression of FLi1 and VE-cadherin and decrease the level of m RNA expression of FSP-1. Western Bolt results showed that TCM formula of tonifying kidney and strengthening essence could increase the level FLi1 and VE-cadherin protein expression and decrease the level of protein expression of FSP-1.Conclusion TCM formula of tonifying kidney and strengthening essence methods can interact the Fli1's expression to inhibit End MT.
Objective To study the mechanism of tonifying kidney and strengthening essence methods interfering the Fli1's expression to regulate TGF-β signal,and to inhibit systemic scleroderma endothelial to mesenchymal transition( End MT). Methods Bleomycin was used to construct mice model,and they were randomly divided them into 5 groups,normal group,model group,astragaloside IV group,Traditional Chinese Medicine( TCM) formula group and FLi1 si RNA + TCM formula group. And the mice in normal group was not given modeling. Mice's skin blood vessels were detected with HE staining method,and collagen in mice were detected with Masson staining method. Gene expression of Fli1,VE-cadherin and FSP-1 m RNA in skin tissue of each group were detected by quantitative polymerase chain reaction( QPCR)method. Protein expression of Fli1,VE-cadherin and FSP-1 were detected by western blotting( WB).Results Mice model of scleroderma were successfully established. In TCM formula group,the HE and Masson staining results showed that the thickness of skin was reduced( P<0.01),and the number of col-lagenous fibers reduced and the number of vessel increased. These results suggest that TCM formula can reduce the degree dermal fibrosis and protect vascular in model mice. And the QPCR results showed that TCM formula of tonifying kidney and strengthening essence could increase the level of m RNA expression of FLi1 and VE-cadherin and decrease the level of m RNA expression of FSP-1. Western Bolt results showed that TCM formula of tonifying kidney and strengthening essence could increase the level FLi1 and VE-cadherin protein expression and decrease the level of protein expression of FSP-1.Conclusion TCM formula of tonifying kidney and strengthening essence methods can interact the Fli1's expression to inhibit End MT.
引文
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[17]张慈安,魏品康,李勇进.痰浊与肿瘤微环境的相关性探讨[J].中西医结合学报,2010,8(3):215-219.
[18]王学敏,崔亚钦,王静,等.六味地黄丸抑制巨噬细胞激活抗肝纤维化作用机制研究[J].南京中医药大学学报,2017,33(1):65-68.
[19]赖虹伊,黄磊,黄仁发.六味地黄汤及其加味治疗肾间质纤维化的研究进展[J].广西中医药大学学报,2016,19(4):80-83.
[20]郝艳鹏,张悦,刘煜敏,等.六味地黄丸含药血清对HK-2细胞TGF-β/Smad信号通路的影响[J].中国病理生理杂志,2011,27(1):149-152.
[21]李万斌,何泽云.六味地黄丸延缓5/6肾切除大鼠肾组织纤维化研究[J].中国药师,2009,12(4):411-413.
[22]余榕捷,蒲含林,周天鸿,等.利用基因芯片研究六味地黄丸对老年大鼠基因表达的影响[J].中国病理生理杂志,2004,(2):117-122.
[23]齐庆,李东海,查旭山,等.补肾益精法对硬皮病成纤维细胞表型的影响及其表观遗传学意义[J].新中医,2011,43(2):134-136.
[2] Asano Y. Future treatments in systemic sclerosis[J]. The Journal of dermatology,2010,37(1):54-70.
[3] Denton CP. Therapeutic targets in systemic sclerosis[J]. Arthritis research&therapy,2007,9(2):S6.
[4]邓铁涛.肺脾肾相关辨治硬皮病[J].中国中医药现代远程教育,2004,2(6):15-16.
[5]齐庆,毛越苹,易娟娟,等.补肾益精法对硬皮病成纤维细胞SMAD3/FLI1平衡的影响[J].四川中医,2012,30(6):43-44.
[6]田永贞.基于补肾益精法复方的黄芪甲苷对硬皮纤维化的作用及机制研究[D].广州:广州中医药大学,2016.
[7]齐庆,陈永贤,李英杰,等.软皮汤对硬皮病小鼠模型皮肤组织中p-SMAD3、FLI1因子表达的影响[J].湖南中医药大学学报,2014,34(9):18-22.
[8]邱路萍,田永贞,张佳林,等.补肾益精法对系统性硬皮病血管内皮间质转化的影响[J].上海中医药大学学报,2017,31(1):63-68.
[9] Bhattacharyya S,Wei J,Varga J. Understanding fibrosis in systemic sclerosis:shifting paradigms, emerging opportunities[J]. Nature Reviews Rheumatology,2012,8(1):42.
[10] Sato Y,Nakanuma Y. Role of endothelial-mesenchymal transition in idiopathic portal hypertension[J]. Histol Histopathol,2013,28(2):145-154.
[11] Arciniegas E,Frid MG,Douglas IS,et al. Perspectives on endothelial-to-mesenchymal transition:potential contribution to vascular remodeling in chronic pulmonary hypertension[J]. Am J Physiol Lung Cell Mol Physiol,2007,293(1):1-8.
[12] Mendoza FA,Piera-Velazquez S,Farber JL,et al. Endothelial cells expressing endothelial and mesenchymal cell gene products in lung tissue from patients with systemic sclerosis-associated interstitial lung disease[J]. Arthritis Rheumatol,2016,68(1):210-217.
[13] Jimenez SA,Piera-Velazquez S. Endothelial to mesenchymal transition(EndoMT)in the pathogenesis of systemic sclerosis-associated pulmonary fibrosis and pulmonary arterial hypertension. myth or reality?[J]. Matrix Biol,2016,51:26-36.
[14] Medici D,Potenta S,Kalluri R. Transforming growth factor-β2promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling[J]. Biochem J,2011,437(3):515-520.
[15] Van Meeteren LA,Ten Dijke P. Regulation of endothelial cell plasticity by TGF-beta[J]. Cell Tissue Res,2012,347(1):177-186.
[16] Kubo M,Czuwara-Ladykowska J,Moussa O,et al. Persistent down-regulation of Fli1,a suppressor of collagen transcription,in fibrotic scleroderma skin[J]. Am J Pathol,2003,163(2):571-581.
[17]张慈安,魏品康,李勇进.痰浊与肿瘤微环境的相关性探讨[J].中西医结合学报,2010,8(3):215-219.
[18]王学敏,崔亚钦,王静,等.六味地黄丸抑制巨噬细胞激活抗肝纤维化作用机制研究[J].南京中医药大学学报,2017,33(1):65-68.
[19]赖虹伊,黄磊,黄仁发.六味地黄汤及其加味治疗肾间质纤维化的研究进展[J].广西中医药大学学报,2016,19(4):80-83.
[20]郝艳鹏,张悦,刘煜敏,等.六味地黄丸含药血清对HK-2细胞TGF-β/Smad信号通路的影响[J].中国病理生理杂志,2011,27(1):149-152.
[21]李万斌,何泽云.六味地黄丸延缓5/6肾切除大鼠肾组织纤维化研究[J].中国药师,2009,12(4):411-413.
[22]余榕捷,蒲含林,周天鸿,等.利用基因芯片研究六味地黄丸对老年大鼠基因表达的影响[J].中国病理生理杂志,2004,(2):117-122.
[23]齐庆,李东海,查旭山,等.补肾益精法对硬皮病成纤维细胞表型的影响及其表观遗传学意义[J].新中医,2011,43(2):134-136.