密蒙花方防治糖尿病视网膜病变VEGF信号转导机制研究

【作者】 吴正正；

【导师】 高健生； 接传红；

【作者基本信息】 中国中医科学院 ， 中西医结合临床， 2008， 博士

【摘要】 目的从体内动物实验和体外细胞培养两个方面,系统观察中药复方密蒙花方对缺氧状态下人血管内皮细胞增殖的作用以及对糖尿病（diabetes mellitus,DM）大鼠视网膜病变的作用,并从信号转导机制方面深入研究其具体作用机制,为密蒙花方用于临床防治糖尿病视网膜病变（diabetic retinopathy,DR）提供实验依据。方法一、体内动物实验研究:选用链脲佐菌素（streptozocin,STZ）诱导的糖尿病大鼠作为动物模型,以羟苯磺酸钙胶囊（昊畅）作为阳性对照药。密蒙花方煎剂分为低、中、高三种不同浓度。糖尿病大鼠成模后分别以前述不同药物灌胃4个月。进行以下几方面的研究:1.STZ性DM大鼠一般状况观察:饮食、尿量、体重变化等。2.STZ性DM大鼠血液指标检测:血糖、糖化血红蛋白（glycosylated hemoglobin,GHb）、全血黏度、血浆黏度、甘油三酯（Triglyceride,TG）、胆固醇（Cholestrerol,CHO）等。3.HE染色观察STZ性DM大鼠胰腺病理形态学变化。4.眼前节照相系统观察STZ性DM大鼠晶状体混浊情况。5.HE染色观察STZ性DM大鼠视网膜病理形态学变化。6.免疫组织化学法检测STZ性DM大鼠视网膜ICAM-1、E-selectin蛋白表达情况。7.视网膜血管消化铺片PAS染色观察STZ性DM大鼠视网膜毛细血管病理变化。8.RT-PCR法检测STZ性DM大鼠视网膜VEGF mRNA、Flt-1 mRNA、Flk-1/KDR mRNA的变化情况。9.Western blot法检测STZ性DM大鼠视网膜VEGF、Flt-1、Flk-1/KDR蛋白磷酸化情况。二、体外细胞培养研究:采用人脐静脉血管内皮细胞（Human Umbilical Vascular Endothelial Cell,HUVEC）进行体外培养,利用100μmol/L二氯化钴（Cocl₂）模拟缺氧状态,同时分别将10、20、40mg/ml密蒙花方水提液作用于缺氧状态下的HUVEC 24h,进行以下几方面的研究:1.采用WST-8法检测密蒙花方对缺氧状态下HUVEC增殖的影响。2.采用流式细胞术（flow cytometry,FCM）检测密蒙花方对缺氧状态下HUVEC细胞周期,以及细胞内增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）蛋白表达的影响。3.采用逆转录聚合酶链反应（Reverse Transcription Polymerase Chain Reaction,RT-PCR）检测密蒙花方对缺氧状态下HUVEC细胞内VEGFmRNA、fms样酪氨酸激酶受体1（fms-like tyrosine kinase-1,Flt-1）mRNA、胎肝激酶-1/激酶插入区受体（fetal liverkinase-1/kinase-insert domain-containing receptor,Flk-1/KDR）mRNA的影响。4.采用免疫印迹法（Western blot）检测密蒙花方对缺氧状态下HUVEC细胞内VEGF、Flt-1、Flk-1/KDR蛋白磷酸化水平的影响。5.采用免疫组织化学方法检测密蒙花方对缺氧状态下HUVEC细胞内ICAM-1、E-selectin、vWF蛋白表达的影响。结果一、体内动物实验研究:用药4月后:1.一般状况:STZ诱导的DM大鼠高血糖状态持久稳定,表现为明显的“三多一少”典型症状。密蒙花方中剂量组体重高于DM模型组,具有统计学意义（p＜0.05）。2.血糖:DM模型组空腹血糖显著高于正常对照组（p＜0.01）。密蒙花方中、高剂量组空腹血糖较DM模型组降低,具有统计学意义（p＜0.01）。3.糖化血红蛋白:DM模型组糖化血红蛋白显著高于正常对照组,密蒙花方中、高剂量组糖化血红蛋白较DM模型组降低,具有统计学意义（p＜0.01）。4.血液流变学:DM模型组全血黏度、血浆黏度较正常对照组明显增高（p＜0.01）,昊畅组及密蒙花方中、高剂量组全血黏度、血浆黏度均较DM模型组降低（p＜0.01）。密蒙花方中、高剂量组与昊畅组比较无统计学意义（p＞0.05）。密蒙花方低剂量组全血黏度较DM模型组降低（p＜0.01）,但高于昊畅组（p＜0.05）。密蒙花方低剂量组血浆黏度与DM模型组比较无统计学意义（p＞0.05）。5.血脂:DM模型组血清TG、CHO均显著高于正常对照组（p＜0.01）;昊畅组与DM模型组比较,血清TG含量无统计学意义（p＞0.05）,血清CHO含量昊畅组较DM模型组降低（p＜0.01）;密蒙花方各剂量组与DM模型组比较,血清TG、CHO均明显降低（p＜0.01）;密蒙花方中、高剂量组间比较无统计学意义（p＞0.05）,均较昊畅组及密蒙花方低剂量组进一步降低（p＜0.05）。6.胰腺形态学变化:DM模型组大鼠见典型的DM病理改变。密蒙花方中、高剂量组胰腺病理改变较DM模型组减轻。7.白内障:造模4月后,与正常对照组比较,其它5组均发生不同程度的白内障（p＜0.05）。密蒙花方中剂量组大鼠白内障程度较DM模型组明显减轻,差异具有统计学意义（p＜0.05）。8.视网膜形态学变化:DM模型组视网膜各层出现典型的DM病理学改变,密蒙花方中剂量组视网膜病理学改变较DM模型组减轻。9.免疫组化检测结果显示:DM模型组大鼠视网膜ICAM-1、E-selectin表达较正常组明显增多（p＜0.01）;昊畅组及各密蒙花方组ICAM-1、E-selectin表达均较模型组有不同程度的减少,差异具有统计学意义（p＜0.01）。与昊畅组比较,密蒙花方中剂量组ICAM-1、E-selectin表达降低,差异具有统计学意义。10.视网膜血管消化铺片PAS染色形态学观察:DM模型组视网膜毛细血管出现典型的病理学改变,密蒙花方中剂量组视网膜毛细血管病理学改变较DM模型组减轻。11.RT-PCR检测结果显示:①DM模型组VEGFmRNA、Flk-1/KDRmRNA表达较正常对照组升高（p＜0.01）,Flt-1mRNA表达与正常对照组比较无统计学差异;②昊畅组VEGF mRNA、Flk-1/KDR mRNA表达较DM模型组降低,差异具有统计学意义（p＜0.05）,Flt-1 mRNA表达与DM模型组比较无统计学意义。③各密蒙花方组VEGFmRNA、Flk-1/KDR mRNA表达较DM模型组降低（p＜0.01）,中、高剂量密蒙花方组Flt-1mRNA表达较DM模型组升高（p＜0.01）,中、高剂量组间比较,差异无统计学意义。12.Western blot检测结果显示:①DM模型组VEGF、Flk-1/KDR磷酸化水平较正常对照组升高,Flt-1磷酸化水平较正常对照组降低。②与DM模型组相比,昊畅组及各密蒙花方组VEGF、Flk-1/KDR磷酸化水平降低,Flt-1磷酸化水平升高。③密蒙花方组间比较,随着密蒙花方浓度的增高,VEGF、Flk-1/KDR蛋白磷酸化水平逐渐降低,Flt-1蛋白磷酸化水平逐渐增高。二、体外细胞培养研究:HUVEC贴壁生长,呈铺路石状镶嵌排列。缺氧组细胞增殖旺盛,排列紧密。密蒙花方组细胞生长迟缓,细胞间隙增大,可见漂浮死细胞。1.WST-8法检测显示:①缺氧组较正常组吸光度值增高,具有统计学意义（p＜0.01）;②各密蒙花方组吸光度值较缺氧组降低（p＜0.01）,并且呈浓度依赖关系（p＜0.01）,以密蒙花方高浓度组吸光度值最低。2.FCM检测细胞周期结果显示:①缺氧组较正常组G₀/G₁期细胞比例减少,S期细胞比例增高,差异具有统计学意义（p＜0.01）;②各密蒙花方组均较缺氧组G₀/G₁期细胞比例增高,S期细胞比例减少（p＜0.01）,组间具有浓度依赖关系（p＜0.01）;③密蒙花方组在G₀/G₁期前还出现了指示细胞凋亡的亚G₁期凋亡峰,表明密蒙花方对缺氧状态下的HUVEC具有促凋亡作用。3.FCM检测细胞内PCNA结果显示:①缺氧组细胞核内PCNA蛋白表达率比正常组显著增高（p＜0.01）;②各密蒙花方组细胞核内PCNA蛋白表达率比缺氧组显著降低（p＜0.01）;③HUVEC核内PCNA蛋白表达率随密蒙花方浓度增加而降低,呈明显的浓度依赖关系（p＜0.01）。4.RT-PCR检测结果显示:①缺氧组VEGFmRNA、Flk-1/KDRmRNA表达较正常组升高（p＜0.01）,Flt-1mRNA表达较正常组降低（p＜0.01）;②密蒙花方组VEGFmRNA、Flk-1/KDR mRNA表达均较缺氧组降低,Flt-1mRNA表达较缺氧组增高,差异具有统计学意义（p＜0.01）。③各密蒙花方组间比较,随着密蒙花方浓度的增高,VEGFmRNA、Flk-1/KDRmRNA表达逐渐降低,Flt-1mRNA表达逐渐增高。5.Western blot检测结果显示:①缺氧组VEGF、Flk-1/KDR蛋白磷酸化水平较正常组升高,Flt-1蛋白磷酸化水平较正常组降低。②与缺氧组比较,各密蒙花方组VEGF、Flk-1/KDR磷酸化水平降低,Flt-1磷酸化水平升高。6.免疫组化检测结果显示:①缺氧组细胞间质ICAM-1、E-selectin、vWF表达较正常组增高（p＜0.01）:②各密蒙花方组ICAM-1、E-selectin、vWF表达较缺氧组降低（p＜0.01）。③密蒙花方组间比较,密蒙花方高浓度组较低浓度组ICAM-1、E-selectin蛋白表达降低,具有统计学意义（p＜0.01）,密蒙花方各组间vWF差异无统计学意义（p＞0.05）。结论一、体内动物实验研究:1.STZ诱导的DM大鼠是一种相对简单易行、可靠的模型制备方法。2.密蒙花方能够改善STZ性DM大鼠的一般状况,在一定程度上降低血糖、血脂、血液流变学水平,延缓白内障的发生。3.密蒙花方中剂量能在一定程度上减轻STZ性DM大鼠视网膜及视网膜毛细血管的病理改变。4.密蒙花方改善大鼠视网膜及视网膜毛细血管病理改变的作用与减少视网膜ICAM-1、E-selectin表达有关。5.密蒙花方改善大鼠视网膜及视网膜毛细血管病理改变的作用与下调视网膜内VEGF、Flk-1/KDRmRNA及蛋白磷酸化水平,上调视网膜内Flt-1 mRNA及蛋白磷酸化水平,干预VEGF-VEGFR信号转导通路有关。二、体外细胞培养研究:1.密蒙花方可有效抑制缺氧状态下HUVEC增殖。2.密蒙花方抑制HUVEC增殖的作用与阻滞细胞由G₁期进入S期,促进细胞凋亡有关。3.密蒙花方抑制HUVEC增殖与降低细胞核内PCNA蛋白表达,使DNA合成受阻有关。4.下调细胞内VEGF、Flk-1/KDR mRNA及蛋白磷酸化水平,上调细胞内Flt-1 mRNA及蛋白磷酸化水平,干预细胞内VEGF-VEGFR信号转导通路,是密蒙花方抑制缺氧状态下HUVEC增殖的作用机制之一。5.密蒙花方可通过减少HUVEC细胞间质ICAM-1、E-selectin、vWF表达以减少细胞损伤,改善细胞功能。更多还原

【Abstract】 Objective:To investigate the inhibition effect of Mimeng Flower decoction on the proliferation of Human Umbilical Vascular Endothelial Cell（HUVEC） induced by chronic hypoxia and to study the effect of Mimeng Flower decoction to diabetic retinopathy of rat,and further to study their mechanism of signal transduction,in order to provide experimental basis for Mimeng Flower decoction used in clinic to preventing and curing diabetic retinophty（DR）.Methods:Animal model study:Used diabetic rats induced by streptozocin（STZ） as animal model. Used calcium dobesilate as positive control medicine.Mimeng Flower decoction had three different concentration which were low dosage,median dosage and high dosage.The following studies were made when the animals were administered for 4 months.1.To observe the general status of diabetic rats induced by STZ,such as food,urinary produc-tion,weight,et al.2.To test the blood item of diabetic rats induced by STZ,such as blood glucose, glycosylated hemoglobin（GHb）,the whole blood viscosity,plasma viscosity, Triglyceride（TG）,Cholestrerol（CHO）and so on.3.Pathological changes of pancreases of diabetic rats were observed with hematoxylin and eosin（HE） stained method.4.Opacity degree of lens of diabetic rats were observed with slip lamp and photo analyzed system.5.Pathological changes of retinals in diabetic rats were observed with hematoxylin and eosin（HE） stained method.6.Through immunohistochemistry method to detect the protein expression of ICAM-1、E-selectin in retinal of diabetic rats induced by STZ.7.The pathological changes of retinal capillary vessels were observed in stretched preparation of retinal vessels digested stained with periodic acid-Schiff（PAS）.8.Through RT-PCR method to detect the expression of VEGFmRNA、Flt-1mRNA、Flk-1/KDRmRNA in retinal of diabetic rats induced by STZ.9.Through Western blot method to detect the phosphorylation level of VEGF、Flt-1、Flk-1/KDR in retinal of diabetic rats induced by STZ. Cell culture in vitro study:HUVEC were cultured in vitro.Used 100μmol/L Cocl₂ to set up the cell chemistry hypoxia model,in the same time,10、20、40mg/ml Mimeng Flower decoction extract were added to the HUVEC separately,the following studies were made after 24h incubation.1.Through WST-8 method to check up the inhibition effect of Mimeng Flower decoction on the proliferation of HUVEC induced by chronic hypoxia.2.The changes of cell cycle and proliferating cell nuclear antigen（PCNA） expression of HUVEC regulated by Mimeng Flower decoction were measured with flow cytometry （FCM）.3.Through RT-PCR method to detect the expression of VEGFmRNA、Flt-1mRNA、Flk-1/KDRmRNA in HUVEC regulated by Mimeng Flower decoction.4.Through Western blot method to detect the phosphorylation level of VEGF、Flt-1、Flk-1/KDR in HUVEC regulated by Mimeng Flower decoction.5.Through immunohistochemistry method to detect the protein expression of ICAM-1、E-selectin、vWF regulated by Mimeng Flower decoction.ResultsAnimal model study（after 4 months）:1.General status:STZ induced diabetic rats had lasting and stable hyperglycaemia, displayingwith the obvious signs of "polydipsia,polyphagia,hyperdiuresis and loss of weight".Median dosage of Mimeng Flower decoction can increase the body weight of rats compared with DM model group（p＜0.05）.2.Blood glucose:Compared with normal group,the blood glucose of the rats increased mark-edly in DM model group（p＜0.01）.Compared with model group,the blood glucose of the rats reduced in median and high dosage group after taking 4 month’s medicine.3.GHb:Compared with normal group,GHb of the rats increased markedly in DM model group（p＜0.01）.Compared with model group,GHb of the rats reduced in median and high dosage group（p＜0.01）.4.Blood rheology:Compared with normal group,the whole blood viscosity and plasma viscosity of the rats increased markedly in DM model group（p＜0.01）.Compared with model group,the whole blood viscosity and plasma viscosity of the rats reduced in median and high dosage group and Haochang group（p＜0.01）.There was no remarkable difference between median,high dosage and Haochang group（p＞0.05）.The whole blood viscosity reduced in low dosage group compared with model group（p＜0.01） while increased compared with Haochang group（p＜0.05）.There was no remarkable difference in plasma viscosity between low dosage group and Haochang group（p＞0.05）.5.Serum lipoids:Compared with normal group,TG and CHO of the rats increased markedly in DM model group（p＜0.01）.Compared with model group,CHO reduced in Haochang group（p＜0.01） while there was no difference between model and Haochang group（p＞0.05）.Compared with model group,TG and CHO reduced in Mimeng Flower groups （p＜0.01）.There was no difference between median and high dosage group（p＞0.05）. TG and CHO reduced in median and high dosage group compared with low dosage and Haochang group（p＜0.05）.6.Pancreas:Remarkable pathological changes of pancreases of diabetic rats were found in DM model group.In median and high dosage group,the pathological changes of pancreases of diabetic rats reduced.7.Cataract:4 months after making model,compared with normalgroup,remarkable opacity of lens were observed in another five groups（p＜0.05）,compared with model group,the degree of cataract relieved remarkablely in median dosage group（p＜0.05）.8.Retinal:Remarkable pathological changes of retinal of diabetic rats were found in DM model group.In median dosage group,the pathological changes of retinal of diabetic rats reduced.9.The result of immunohistochemistry test show that the expression of ICAM-1、E-selectin in retinal increased in DM model group compared with blank group（p＜0.01）.Mimeng Flower decoction groups and Haochang group could down regulate the expression of ICAM-1、E-selectin compared with model group（p＜0.01）.In median dosage group the expression of ICAM-1、E-selectin reduced compared with Haochang group.10.The result of PAS stained show that remarkable pathological changes of retinal capillary vessels of diabetic rats were found in DM model group.In median dosage group,the pathological changes of retinal capillary vessels of diabetic rats reduced.11.The result of RT-PCR test show that the expression of VEGFmRNA、Flk-1/KDRmRNA increased in DM model group compared with normal group（p＜0.01）.Mimeng Flower decoction groups could down regulate the expression of VEGFmRNA、Flk-1/KDRmRNA and up regulate the expression of Flt-1 mRNA compared with model group.12.The result of Western-blot test show that the phosphorylation level of VEGF、Flk-1/KDR increased while Flt-1 reduced in DM model group compared with normal group.Mimeng Flower decoction groups and Haochang group could down regulate the phosphorylation level of VEGF、Flk-1/KDR and up regulate the phosphorylation level of Flt-1 compared with DM model group and these effects were dose dependent.Cell culture in vitro study:Cells grew more rapidly in hypoxia group than in Mimeng Flower decoction groups.1.The results of WST-8 test show that the hypoxia group can increase the optical density （OD） values compared with blank group（p＜0.01）;Mimeng Flower decoction groups can reduce the OD values obviously compared with hypoxia group（p＜0.01） and these effects were dose dependent;The high dose of Mimeng Flower decoction group’s OD values were the lowest.2.The results of FCM test show that cells in G₀/G₁ phase reduced while cells in S phase increased in hypoxia group compared with blank group（p＜0.01）.In Mimeng Flower decoction groups,cells in G₀/G₁ phase increased while cells in S phase reduced compared with hypoxia group（p＜0.01） and these effects were dose dependent.Further more, Mimeng Flower decoction can induce HUVEC apoptosis.3.The results of FCM test show that the expession of PCNA increased in hypoxia group compared with blank group（p＜0.01）.Mimeng Flower decoction could down regulate the expression of PCNA significantly compared with hypoxia group and expression of PCNA showed dose-dependent in Mimeng Flower decoction groups（p＜0.01）.4.The results of RT-PCR test show that the expression of VEGFmRNA、Flk-1/KDRmRNA increased while Flt-1mRNA reduced in hypoxia group compared with blank group（p＜0.01）.Mimeng Flower decoction could down regulate the expression of VEGFmRNA、Flk-1/KDRmRNA and up regulate the expression of Flt-1mRNA compared with hypoxia group（p＜0.01） and these effects were dose dependent.5.The results of Western-blot test show that the phosphorylation level of VEGF、Flk-1/KDR increased while Flt-1 reduced in hypoxia group compared with blank group.Mimeng Flower decoction could down regulate the phosphorylation level of VEGF、Flk-1/KDR and up regulate the phosphorylation level of Flt-1 compared with hypoxia group. 6.The results of immunohistochemistry test show that the expression of ICAM-1、E-selectin、vWF increased in hypoxia group compared with blank group（p＜0.01）. Mimeng Flower decoction could down regulate the expression of ICAM-1、E-selectin、vWF compared with hypoxia group（p＜0.01） and the expression of ICAM-1、E-selectin reduced in high dose compared with low dose group（p＜0.01）.ConclusionAnimal model study:1.STZ induced diabetic rat is a simple,easy and reliable animal model.2.Mimeng Flower decoction has the function of improving the general status,reducing the level of blood glucose,GHb,Blood rheology and Serum lipoids and inhibiting the development of cataract of DM rats induced by STZ to a certain.3.Median dosage of Mimeng Flower decoction can reduce the the pathological changes of retinal and retinal capillary vessels in diabetic rats to a certain.4.The effect of improving the pathological changes of retinal and retinal capillary vessels may be contributed to downregulating ICAM-1、E-selectin expression in retinal.5.Downregulating the protein phosphorylation level and mRNA expression of VEGF、Flk-1/KDR mRNA and upregulating the protein phosphorylation level and mRNA expression of Flt-1 mRNA may play a significant role in improving the pathological changes of retinal and retinal capillary vessels of DM rats.Cell culture in vitro study:1.Mimeng Flower decoction can markly inhibit the HUVEC proliferation in hypoxia state.2.The effect of inhibiting proliferation by Mimeng Flower decoction may be contributed to inducing G₁/S transition arrest and increasing the apoptosis of HUVEC.3.The effect of inhibiting proliferation by Mimeng Flower decoction may be contributed to downregulating PCNA expression and hindering the DNA synthesis of HUVEC.4.Downregulating the protein phosphorylation level and mRNA expression of VEGF、Flk-1/KDR mRNA and upregulating the protein phosphorylation level and mRNA expression of Flt-1 mRNA may play a significant role in inhibiting proliferation of HUVEC by Mimeng Flower decoction.5.Mimeng Flower decoction can improve the cells’ function by reducing the expression of ICAM-1、E-selectin、vWF in HUVEC.更多还原