葡萄子原花青素对高糖条件下载脂蛋白A-Ⅰ合成及表达的影响
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摘要
第一部分葡萄子原花青素抑制糖尿病大鼠主动脉载脂蛋白A-I的表达
背景
世界卫生组织推测2025年成年人中糖尿病患者将达到3亿。若按糖尿病患者人数计算,中国位居印度之后,排名第2位。糖尿病是老年人面临的重大的健康、社会以及经济问题。糖尿病大血管病变是糖尿病患者常见的慢性并发症,也是糖尿病患者致死、致残的主要原因之一,其病理变化过程始于内皮功能不良可发展到动脉粥样斑块形成,发病机制尚不明确,但由慢性高血糖诱导的氧化应激反应可能是重要原因之一。葡萄子原花青素(grape seed proanthocyanidinsextracts,GSPE)是从葡萄籽中提取的多酚类混合物,其原花青素含量超过95%。动物实验表明,GSPE具有抗氧化、抗动脉硬化及保护心血管等作用。并且,GSPE对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠并发的血管病变、心肌病变、肾脏病变及神经病变均具备一定的保护作用。众所周知,载脂蛋白A-I(apolipoprotein A-I,apoA-I)是高密度脂蛋白(high density lipoprotein,HDL)最主要的蛋白成分。研究证明,血浆中HDL的含量与冠状动脉疾病的发生呈负相关性,即HDL含量越高,冠状动脉疾病发病率越低。ApoA-I对冠状动脉保护作用明确。监测GSPE对apoA-I水平的影响以及因此带来的效应、探讨GSPE血管保护机制将有助于发现药物作用靶点,为研究新的糖尿病并发症防治药物提供理论依据。
目的
大量研究证实,葡萄子原花青素(GSPE)有强大的抗氧化和抗衰老功能。本实验观察葡萄子原花青素对链脲佐菌素(STZ)诱导的糖尿病大鼠血浆和主动脉组织内载脂蛋白A-I(apoA-I)的影响。
方法
55只雄性Wistar大鼠随机分成对照组(C组,n=15)、糖尿病组(DM组,n=20)和糖尿病GSPE治疗组(DM+GSPE组,n=20)。糖尿病组和糖尿病GSPE治疗组大鼠一次性尾静脉注射0.1%STZ柠檬酸缓冲液55 mg/kg,正常对照组注射同等剂量的柠檬酸缓冲液。注射72h后,取大鼠尾血测定血糖,血糖≥16.7mmol/L为糖尿病模型成功的标准。对DM组每天给予生理盐水灌胃,DM+GSPE组每天给予GSPE(250mg/kg)灌胃。持续观察24周。测定每组动物血浆低密度脂蛋白(LDL)、高密度脂蛋白(HDL)和载脂蛋白A-I(apoA-I)的水平。24周后处死动物,迅速分离胸主动脉,并用冰磷酸盐缓冲液彻底清洗去除血液成分,之后立刻置于液氮中冻存。胸主动脉自根部切成1cm左右的小段,在10%的甲醛中固定,石蜡包埋,切成4μm厚的切片,行苏木素-曙红染色,在200倍的光镜下观察。应用荧光差异凝胶电泳技术观察经GSPE治疗后糖尿病大鼠主动脉差异表达蛋白,并应用质谱分析鉴定该差异蛋白点。鉴定后应用Western blot和免疫荧光技术验证经GSPE治疗后该差异蛋白apoA-I在糖尿病大鼠主动脉中的表达。
结果
糖尿病组大鼠血浆LDL、HDL和apoA-I水平均高于正常对照组,经GSPE治疗后LDL、HDL和apoA-I水平下降至接近正常。糖尿病组大鼠主动脉内皮细胞肥大,结构紊乱,平滑肌细胞增生,内弹力板破坏,管腔变小,经GSPE治疗后,上述变化均未出现,血管结构接近对照组。荧光差异凝胶电泳和质谱分析发现糖尿病大鼠主动脉apoA-I经GSPE治疗后差异表达(DM组/C组:-1.68;DM+GSPE组/DM组:1.77)。之后的Western blot和免疫荧光实验对该差异蛋白进行了验证,结果发现糖尿病组大鼠主动脉组织中的apoA-I水平最高(p＜0.05),GSPE能有效地降低这种表达(p＜0.05)。
结论
1.GSPE有助于缓解糖尿病的大血管病变;
2.GSPE可以调节apoA-I在主动脉组织的表达水平,并且该调节可能是其实现对血管保护作用的机制之一;
3.apoA-I表达数量或修饰的变化可能是糖尿病大鼠主动脉病变发病机制之一,但仍需今后的实验进一步加以验证。
第二部分葡萄子原花青素对实验性高糖条件下HepG2细胞表达载脂蛋白A-I的影响
背景
载脂蛋白A I(apoA-I)是高密度脂蛋白(HDL)的主要成分之一,占HDL中蛋白质含量的50%左右,介导了许多重要的生理功能。它可以与血管壁上的ATP结合转运体Al(ABCAl)结合,诱导游离胆固醇从血管内皮中的巨噬细胞或其他表皮组织中向外流出,是诱导胆固醇逆向转运(RCT)的起始阶段,随后能激活结合于HDL的卵磷脂胆固醇酰基转移酶(LCAT),将游离胆固醇酯化,再从HDL的表面转移至其内核,进而向肝脏运输,是体内逆向胆固醇转运的关键作用因子。最近研究表明,它还具有其他生化作用,包括抗氧化作用、抗炎、抗内毒素作用等。因此,促进apoA-I的表达有助于缓解动脉硬化的进程。
目的
葡萄子原花青素(GSPE)是从葡萄籽中提取的天然抗氧化物质。本研究以体外培养的人肝癌细胞系HepG2为研究对象,分别从蛋白和基因水平观察GSPE是否能诱导HepG2的apoA-I表达。
方法
首先用10%灭活新生牛血清的DMEM培养基常规培养肝癌细胞株HepG2并传代,然后用高糖及低糖无血清培养基,添加不同浓度(2.5、5及10μg/ml)GSPE与HepG2细胞孵育24 h,期间用SRB测定GSPE对HepG2细胞增殖的影响,Western Blot测定细胞内apoA-I蛋白水平,RT-PCR测定apoA-ImRNA变化。观察GSPE是否促进实验性高糖条件下HepG2细胞apoA-I的高表达。
结果
1.本实验用高糖和低糖培养基孵育HepG2细胞,结果发现,用高糖培养基孵育的HepG2细胞产生的apoA-I不论从蛋白还是基因水平均明显低于用低糖培养基者(p＜0.05)。
2.在添加GSPE与无血清高糖培养基共同孵育HepG2细胞时,SRB检测细胞存活率发现,GSPE浓度在20μg/ml以上时,孵育4小时,即出现了HepG2细胞的明显凋亡;而在GSPE浓度低于10μg/ml的各组,共同孵育24小时,HepG2细胞无明显凋亡,细胞存活率均在70%以上,因此,实验最终设立了2.5、5、10μg/ml GSPE干预组作为观察对象。
3.经GSPE孵育后,不论从基因还是蛋白水平,HepG2细胞表达apoA-I均较单纯高糖孵育增高,差异有统计学意义(p＜0.05),且该种促进作用与GSPE浓度相关,2.5μg/ml时促进作用为最佳。
结论
1.20μg/ml以上浓度的GSPE在体外对无血清高糖培养基孵育的HepG2细胞存活率有明显抑制;
2.实验性高糖状态可以从蛋白和基因水平抑制apoA-I的表达;
3.高糖环境下,10μg/ml以下浓度的GSPE能从蛋白和基因水平促进apoA-I的表达,并且该种促进作用与GSPE浓度相关,当GSPE浓度为2.5μg/ml时作用为最佳。
Part one Inhibitory effect of grape seed proanthocyanidins extracts on the levels of apolipoprotein A-I of both plasma and aortic tissue in streptozocin induced diabetic rats
Background
Macroangiopathy is a major complication of both typeⅠand typeⅡdiabetes,and its main mechanism is not so clear.The pathologic changes of diabetic macroangiopathy can be from endothelial dysfunction to formation of atherosclerotic plaque,in which oxidative stress induced by chronic hyperglycemia may play a key role.
Proanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.
Previous studies have proved that plasma apolipoprotein A-I(apoA-I),the major apolipoprotein of high density lipoprotein(HDL),by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells, is a protective factor of cardiovascular disease.And there is a negative relationship between the level of apoA-I and cardiovascular diseases.But neither the influence of GSPE on the levels of apoA-I of both plasma and aortic tissue in diabetic models nor the influence of fluctuation of levels of apoA-I on diabetic aortic angiopathy remained unknown.Our previous experiments showed that GSPE played a protective role in cardiomyocyte,nervous system,etc.In this study,we aimed to examine the effects of GSPE on the levels of apoA-I of plasma and aortic tissue in streptozocin (STZ) induced diabetic rats and to evaluate the relationship between GSPE and diabetic macro-angiopathy,which may be helpful to find a new drug target point.
Objective
In this study,we investigated the effect of grape seed proanthocyanidins extracts (GSPE),which have been proved to have anti-oxidative and anti-aging functions,on the levels of apolipoprotein A-I(apoA-I) of both plasma and aortic tissue in a rat model of diabetes.
Methods
Fifty-five rats were randomly divided into three groups:group C(control,n=15); group DM(diabetes,n=20);group DM+GSPE(GSPE administrated diabetes,n=20). GSPE(250 mg/kg body weight/d),were administrated to diabetic rats induced by STZ intravenous injection via tail veins,for 24 weeks.Plasma low density lipoprotein(LDL),high density lipoprotein(HDL)and apoA-I were determined in each group.Aftern all animals were sacrificed after 24 weeks,the aortas were excised about 1cm long from the end,fixed in 10%formaldehyde,and embedded in paraffin and cut into 4μm-thick sections for light microscopy.Then they were stained with hematoxylin-eosin under a light microscope at a magnification of 200×.Beside this, difference gel electrophoresis technique was used to explore differential expression protein after treatment with GSPE in diabetic rats.Western blot and immunofluorescence techniques were used to validate the protein expression levels of apoA-I in aortic tissue.
Results
In untreated diabetic rats,the levels of LDL,HDL and apoA-I were higher than those in group C.With the treatment of GSPE,the levels of LDL,HDL and apoA-I decreased to normal values similar to those of control rats.In the diabetic rats, hypertrophy and disarray of endothelial cells,proliferation of smooth muscle cells were observed in aortic wall,and the internal elastic laminae were impaired.The lumina of aorta became smaller.After treatment of GSPE light microscopic findings were similar to those of the control rats.ApoA-I was found differential expressed in DIGE(DM group/C group:-1.68;DM+GSPE group/DM group:1.77).In the following Western blot and immunofluorescence experiment,the expression of apoA- I in aortic tissue was found to be the highest(p<0.05) in group DM,GSPE significantly reduced this expression(p<0.05).
Conclusions
In conclusion,GSPE can downregulate the expression of apoA-I in both plasma and aortic tissue,which may be contributed to the beneficial influences of GSPE on the diabetic aortic angiopathy.
Part two Effects of Grape seed proanthocyanidins extracts on apolipoprotein A-I expression in HepG2 cells under the experimental conditions of high glucose
Background
ApoA-I,which constitutes 70%of the apolipoprotein content of HDL,acts as acceptor for the transfer of phospholipids and free cholesterol from peripheral tissues and transports cholesterol in the liver and other tissues for excretion and steroidogenesis.In particular,apoA-I interacts with the ATP-binding cassette transporter A1 and accepts free cholesterol,and acts as a cofactor of the HDL-associated enzyme lecithin cholesterol acyl transferase(LCAT),thereby mediating the delivery of cholesterol ester to the liver.This process is important in reducing the accumulation of foam cells in the arterial walls.Furthermore apoA-I exerts antioxidant properties by removing lipid hydroperoxides from low-density lipoprotein(LDL),thereby reducing the oxidation of LDL and consequently the atherogenicity of these lipoproteins.Therefore,apoA-I also shows anti-inflammatory actions through the inhibition of the expression of adhesion molecules in endothelial cells and reducing the recruitment of monocytes in the arterial walls.It may be helpful to promote the expression of apoA-I to prevent the pathogenesis of diabetic angiopathy.
Objective
Proanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.In this study,we investigated the effect of grape seed proanthocyanidins extracts(GSPE), which have been proved to have anti-oxidative and anti-aging functions,on the expression of apoA-I at both protein and gene levels of HepG2 cells in vitro under the experimental conditions of high glucose.
Methods
Cell viability was measured by SRB.The apoA-I mRNA expression was assayed by RT-PCR.The apoA-I protein level was assayed by Western blot.Firstly,HepG2 cells were incubated in 10%inactivated newborn calf serum in DMEM medium.Next, cells were incubated with high-sugar and sugar serum-free medium,and added different concentration of GSPE(2.5、5 and 10μg/ml) for more than 24 hours,and thereafter,investigated whether GSPE can promote more apoA-I expression in HepG2 cells under the experimental conditions of high glucose.
Results
1.In this experiment,HepG2 cells were incubated with high-sugar and sugar serum-free medium,and the HepG2 cells incubated with high-sugar medium produced less apoA-I no matter at protein or gene level.The difference was significant(p<0.05).
2.When HepG2 cells were incubated with GSPE at concentration of 20μg/ml or above for about 4 hours,cell viability measured by SRB was lower than 50%. However,cell viability of HepG2 cells incubated with GSPE at concentration of 10μg/ml or below was higher than 70%for more than 24 hours.Therefore,we chose the HepG2 cells incubated with GSPE concentration of 2.5、5、10μg/ml to observe the effect of GSPE on the expression of apoA-I.
3.After incubated with GSPE,the apoA-I expression of HepG2 cells were significantly elevated at both protein and gene levels compared to that of high sugar control(p<0.05).Moreover,this action of GSPE showed dose dependent,and the dose of 2.5μg/ml was optimal.
Conclusions
1.GSPE could inhibit HepG2 cell survival when HepG2 cells were incubated with high-sugar serum-free medium and GSPE at concentration of higher than 20μg/ml.
2.In HepG2 cells,endogenous apoA-I was significantly suppressed following 24h of exposure to high concentrations of glucose.
3.GSPE could promote expression of apoA-I dose dependently at both protein and gene levels when its concentration was lower than 10μg/ml.
背景
世界卫生组织推测2025年成年人中糖尿病患者将达到3亿。若按糖尿病患者人数计算,中国位居印度之后,排名第2位。糖尿病是老年人面临的重大的健康、社会以及经济问题。糖尿病大血管病变是糖尿病患者常见的慢性并发症,也是糖尿病患者致死、致残的主要原因之一,其病理变化过程始于内皮功能不良可发展到动脉粥样斑块形成,发病机制尚不明确,但由慢性高血糖诱导的氧化应激反应可能是重要原因之一。葡萄子原花青素(grape seed proanthocyanidinsextracts,GSPE)是从葡萄籽中提取的多酚类混合物,其原花青素含量超过95%。动物实验表明,GSPE具有抗氧化、抗动脉硬化及保护心血管等作用。并且,GSPE对链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠并发的血管病变、心肌病变、肾脏病变及神经病变均具备一定的保护作用。众所周知,载脂蛋白A-I(apolipoprotein A-I,apoA-I)是高密度脂蛋白(high density lipoprotein,HDL)最主要的蛋白成分。研究证明,血浆中HDL的含量与冠状动脉疾病的发生呈负相关性,即HDL含量越高,冠状动脉疾病发病率越低。ApoA-I对冠状动脉保护作用明确。监测GSPE对apoA-I水平的影响以及因此带来的效应、探讨GSPE血管保护机制将有助于发现药物作用靶点,为研究新的糖尿病并发症防治药物提供理论依据。
目的
大量研究证实,葡萄子原花青素(GSPE)有强大的抗氧化和抗衰老功能。本实验观察葡萄子原花青素对链脲佐菌素(STZ)诱导的糖尿病大鼠血浆和主动脉组织内载脂蛋白A-I(apoA-I)的影响。
方法
55只雄性Wistar大鼠随机分成对照组(C组,n=15)、糖尿病组(DM组,n=20)和糖尿病GSPE治疗组(DM+GSPE组,n=20)。糖尿病组和糖尿病GSPE治疗组大鼠一次性尾静脉注射0.1%STZ柠檬酸缓冲液55 mg/kg,正常对照组注射同等剂量的柠檬酸缓冲液。注射72h后,取大鼠尾血测定血糖,血糖≥16.7mmol/L为糖尿病模型成功的标准。对DM组每天给予生理盐水灌胃,DM+GSPE组每天给予GSPE(250mg/kg)灌胃。持续观察24周。测定每组动物血浆低密度脂蛋白(LDL)、高密度脂蛋白(HDL)和载脂蛋白A-I(apoA-I)的水平。24周后处死动物,迅速分离胸主动脉,并用冰磷酸盐缓冲液彻底清洗去除血液成分,之后立刻置于液氮中冻存。胸主动脉自根部切成1cm左右的小段,在10%的甲醛中固定,石蜡包埋,切成4μm厚的切片,行苏木素-曙红染色,在200倍的光镜下观察。应用荧光差异凝胶电泳技术观察经GSPE治疗后糖尿病大鼠主动脉差异表达蛋白,并应用质谱分析鉴定该差异蛋白点。鉴定后应用Western blot和免疫荧光技术验证经GSPE治疗后该差异蛋白apoA-I在糖尿病大鼠主动脉中的表达。
结果
糖尿病组大鼠血浆LDL、HDL和apoA-I水平均高于正常对照组,经GSPE治疗后LDL、HDL和apoA-I水平下降至接近正常。糖尿病组大鼠主动脉内皮细胞肥大,结构紊乱,平滑肌细胞增生,内弹力板破坏,管腔变小,经GSPE治疗后,上述变化均未出现,血管结构接近对照组。荧光差异凝胶电泳和质谱分析发现糖尿病大鼠主动脉apoA-I经GSPE治疗后差异表达(DM组/C组:-1.68;DM+GSPE组/DM组:1.77)。之后的Western blot和免疫荧光实验对该差异蛋白进行了验证,结果发现糖尿病组大鼠主动脉组织中的apoA-I水平最高(p＜0.05),GSPE能有效地降低这种表达(p＜0.05)。
结论
1.GSPE有助于缓解糖尿病的大血管病变;
2.GSPE可以调节apoA-I在主动脉组织的表达水平,并且该调节可能是其实现对血管保护作用的机制之一;
3.apoA-I表达数量或修饰的变化可能是糖尿病大鼠主动脉病变发病机制之一,但仍需今后的实验进一步加以验证。
第二部分葡萄子原花青素对实验性高糖条件下HepG2细胞表达载脂蛋白A-I的影响
背景
载脂蛋白A I(apoA-I)是高密度脂蛋白(HDL)的主要成分之一,占HDL中蛋白质含量的50%左右,介导了许多重要的生理功能。它可以与血管壁上的ATP结合转运体Al(ABCAl)结合,诱导游离胆固醇从血管内皮中的巨噬细胞或其他表皮组织中向外流出,是诱导胆固醇逆向转运(RCT)的起始阶段,随后能激活结合于HDL的卵磷脂胆固醇酰基转移酶(LCAT),将游离胆固醇酯化,再从HDL的表面转移至其内核,进而向肝脏运输,是体内逆向胆固醇转运的关键作用因子。最近研究表明,它还具有其他生化作用,包括抗氧化作用、抗炎、抗内毒素作用等。因此,促进apoA-I的表达有助于缓解动脉硬化的进程。
目的
葡萄子原花青素(GSPE)是从葡萄籽中提取的天然抗氧化物质。本研究以体外培养的人肝癌细胞系HepG2为研究对象,分别从蛋白和基因水平观察GSPE是否能诱导HepG2的apoA-I表达。
方法
首先用10%灭活新生牛血清的DMEM培养基常规培养肝癌细胞株HepG2并传代,然后用高糖及低糖无血清培养基,添加不同浓度(2.5、5及10μg/ml)GSPE与HepG2细胞孵育24 h,期间用SRB测定GSPE对HepG2细胞增殖的影响,Western Blot测定细胞内apoA-I蛋白水平,RT-PCR测定apoA-ImRNA变化。观察GSPE是否促进实验性高糖条件下HepG2细胞apoA-I的高表达。
结果
1.本实验用高糖和低糖培养基孵育HepG2细胞,结果发现,用高糖培养基孵育的HepG2细胞产生的apoA-I不论从蛋白还是基因水平均明显低于用低糖培养基者(p＜0.05)。
2.在添加GSPE与无血清高糖培养基共同孵育HepG2细胞时,SRB检测细胞存活率发现,GSPE浓度在20μg/ml以上时,孵育4小时,即出现了HepG2细胞的明显凋亡;而在GSPE浓度低于10μg/ml的各组,共同孵育24小时,HepG2细胞无明显凋亡,细胞存活率均在70%以上,因此,实验最终设立了2.5、5、10μg/ml GSPE干预组作为观察对象。
3.经GSPE孵育后,不论从基因还是蛋白水平,HepG2细胞表达apoA-I均较单纯高糖孵育增高,差异有统计学意义(p＜0.05),且该种促进作用与GSPE浓度相关,2.5μg/ml时促进作用为最佳。
结论
1.20μg/ml以上浓度的GSPE在体外对无血清高糖培养基孵育的HepG2细胞存活率有明显抑制;
2.实验性高糖状态可以从蛋白和基因水平抑制apoA-I的表达;
3.高糖环境下,10μg/ml以下浓度的GSPE能从蛋白和基因水平促进apoA-I的表达,并且该种促进作用与GSPE浓度相关,当GSPE浓度为2.5μg/ml时作用为最佳。
Part one Inhibitory effect of grape seed proanthocyanidins extracts on the levels of apolipoprotein A-I of both plasma and aortic tissue in streptozocin induced diabetic rats
Background
Macroangiopathy is a major complication of both typeⅠand typeⅡdiabetes,and its main mechanism is not so clear.The pathologic changes of diabetic macroangiopathy can be from endothelial dysfunction to formation of atherosclerotic plaque,in which oxidative stress induced by chronic hyperglycemia may play a key role.
Proanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.
Previous studies have proved that plasma apolipoprotein A-I(apoA-I),the major apolipoprotein of high density lipoprotein(HDL),by removing cholesterol and cholesteryl ester hydroperoxides from oxidized low density lipoprotein and foam cells, is a protective factor of cardiovascular disease.And there is a negative relationship between the level of apoA-I and cardiovascular diseases.But neither the influence of GSPE on the levels of apoA-I of both plasma and aortic tissue in diabetic models nor the influence of fluctuation of levels of apoA-I on diabetic aortic angiopathy remained unknown.Our previous experiments showed that GSPE played a protective role in cardiomyocyte,nervous system,etc.In this study,we aimed to examine the effects of GSPE on the levels of apoA-I of plasma and aortic tissue in streptozocin (STZ) induced diabetic rats and to evaluate the relationship between GSPE and diabetic macro-angiopathy,which may be helpful to find a new drug target point.
Objective
In this study,we investigated the effect of grape seed proanthocyanidins extracts (GSPE),which have been proved to have anti-oxidative and anti-aging functions,on the levels of apolipoprotein A-I(apoA-I) of both plasma and aortic tissue in a rat model of diabetes.
Methods
Fifty-five rats were randomly divided into three groups:group C(control,n=15); group DM(diabetes,n=20);group DM+GSPE(GSPE administrated diabetes,n=20). GSPE(250 mg/kg body weight/d),were administrated to diabetic rats induced by STZ intravenous injection via tail veins,for 24 weeks.Plasma low density lipoprotein(LDL),high density lipoprotein(HDL)and apoA-I were determined in each group.Aftern all animals were sacrificed after 24 weeks,the aortas were excised about 1cm long from the end,fixed in 10%formaldehyde,and embedded in paraffin and cut into 4μm-thick sections for light microscopy.Then they were stained with hematoxylin-eosin under a light microscope at a magnification of 200×.Beside this, difference gel electrophoresis technique was used to explore differential expression protein after treatment with GSPE in diabetic rats.Western blot and immunofluorescence techniques were used to validate the protein expression levels of apoA-I in aortic tissue.
Results
In untreated diabetic rats,the levels of LDL,HDL and apoA-I were higher than those in group C.With the treatment of GSPE,the levels of LDL,HDL and apoA-I decreased to normal values similar to those of control rats.In the diabetic rats, hypertrophy and disarray of endothelial cells,proliferation of smooth muscle cells were observed in aortic wall,and the internal elastic laminae were impaired.The lumina of aorta became smaller.After treatment of GSPE light microscopic findings were similar to those of the control rats.ApoA-I was found differential expressed in DIGE(DM group/C group:-1.68;DM+GSPE group/DM group:1.77).In the following Western blot and immunofluorescence experiment,the expression of apoA- I in aortic tissue was found to be the highest(p<0.05) in group DM,GSPE significantly reduced this expression(p<0.05).
Conclusions
In conclusion,GSPE can downregulate the expression of apoA-I in both plasma and aortic tissue,which may be contributed to the beneficial influences of GSPE on the diabetic aortic angiopathy.
Part two Effects of Grape seed proanthocyanidins extracts on apolipoprotein A-I expression in HepG2 cells under the experimental conditions of high glucose
Background
ApoA-I,which constitutes 70%of the apolipoprotein content of HDL,acts as acceptor for the transfer of phospholipids and free cholesterol from peripheral tissues and transports cholesterol in the liver and other tissues for excretion and steroidogenesis.In particular,apoA-I interacts with the ATP-binding cassette transporter A1 and accepts free cholesterol,and acts as a cofactor of the HDL-associated enzyme lecithin cholesterol acyl transferase(LCAT),thereby mediating the delivery of cholesterol ester to the liver.This process is important in reducing the accumulation of foam cells in the arterial walls.Furthermore apoA-I exerts antioxidant properties by removing lipid hydroperoxides from low-density lipoprotein(LDL),thereby reducing the oxidation of LDL and consequently the atherogenicity of these lipoproteins.Therefore,apoA-I also shows anti-inflammatory actions through the inhibition of the expression of adhesion molecules in endothelial cells and reducing the recruitment of monocytes in the arterial walls.It may be helpful to promote the expression of apoA-I to prevent the pathogenesis of diabetic angiopathy.
Objective
Proanthocyanidins are naturally occurring polyphenolic compounds widely available in fruits,vegetables,nuts,seeds,flowers and bark.Grape seed proanthocyanidins extracts(GSPE),a combination of biologically active polyphenolic flavonoids including oligomeric proanthocyanidins,have been demonstrated to exert a novel spectrum of biological,pharmacological,therapeutic, and chemoprotective properties against oxygen free radicals and oxidative stress.In this study,we investigated the effect of grape seed proanthocyanidins extracts(GSPE), which have been proved to have anti-oxidative and anti-aging functions,on the expression of apoA-I at both protein and gene levels of HepG2 cells in vitro under the experimental conditions of high glucose.
Methods
Cell viability was measured by SRB.The apoA-I mRNA expression was assayed by RT-PCR.The apoA-I protein level was assayed by Western blot.Firstly,HepG2 cells were incubated in 10%inactivated newborn calf serum in DMEM medium.Next, cells were incubated with high-sugar and sugar serum-free medium,and added different concentration of GSPE(2.5、5 and 10μg/ml) for more than 24 hours,and thereafter,investigated whether GSPE can promote more apoA-I expression in HepG2 cells under the experimental conditions of high glucose.
Results
1.In this experiment,HepG2 cells were incubated with high-sugar and sugar serum-free medium,and the HepG2 cells incubated with high-sugar medium produced less apoA-I no matter at protein or gene level.The difference was significant(p<0.05).
2.When HepG2 cells were incubated with GSPE at concentration of 20μg/ml or above for about 4 hours,cell viability measured by SRB was lower than 50%. However,cell viability of HepG2 cells incubated with GSPE at concentration of 10μg/ml or below was higher than 70%for more than 24 hours.Therefore,we chose the HepG2 cells incubated with GSPE concentration of 2.5、5、10μg/ml to observe the effect of GSPE on the expression of apoA-I.
3.After incubated with GSPE,the apoA-I expression of HepG2 cells were significantly elevated at both protein and gene levels compared to that of high sugar control(p<0.05).Moreover,this action of GSPE showed dose dependent,and the dose of 2.5μg/ml was optimal.
Conclusions
1.GSPE could inhibit HepG2 cell survival when HepG2 cells were incubated with high-sugar serum-free medium and GSPE at concentration of higher than 20μg/ml.
2.In HepG2 cells,endogenous apoA-I was significantly suppressed following 24h of exposure to high concentrations of glucose.
3.GSPE could promote expression of apoA-I dose dependently at both protein and gene levels when its concentration was lower than 10μg/ml.
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1. Brewer HB, Jr. High-density lipoproteins: a new potential therapeutic target for the prevention of cardiovascular disease. Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):387-391.
2. Franceschini G. Epidemiologic evidence for high-density lipoprotein cholesterol as a risk factor for coronary artery disease. Am J Cardiol. 2001 Dec 20; 88(12A):9N-13N.
3. Van Lenten BJ, Navab M, Shih D, et al. The role of high-density lipoproteins in oxidation and inflammation. Trends Cardiovasc Med. 2001 Apr-May; 11(3-4): 155-161.
4. Shoulders CC, Kornblihtt AR, Munro BS, et al. Gene structure of human apolipoprotein Al. Nucleic Acids Res. 1983 May 11;11(9):2827-2837.
5. Taylor AH, Fox-Robichaud AE, Egan C, et al. Oestradiol decreases rat apolipoprotein AI transcription via promoter site B. J Mol Endocrinol. 2000 Oct;25(2):207-219.
6. Taylor AH, Wishart P, Lawless DE, et al. Identification of functional positive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI promoter. Biochemistry. 1996 Jun 25;35(25):8281-8288.
7. Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology. 2000 Aug 7; 148(2-3): 187-197.
8. Puiggros F, Llopiz N, Ardevol A, et al. Grape seed procyanidins prevent oxidative injury by modulating the expression of antioxidant enzyme systems. J Agric Food Chem. 2005 Jul 27;53(15):6080-6086.
9. Koga T, Moro K, Nakamori K, et al. Increase of antioxidative potential of rat plasma by oral administration of proanthocyanidin-rich extract from grape seeds. J Agric Food Chem. 1999 May;47(5): 1892-1897.
10. Carini M, Stefani R, Aldini G, et al. Procyanidins from Vitis vinifera seeds inhibit the respiratory burst of activated human neutrophils and lysosomal enzyme release. Planta Med. 2001 Nov;67(8):714-717.
11. Nuttall SL, Kendall MJ, Bombardelli E, et al. An evaluation of the antioxidant activity of a standardized grape seed extract, Leucoselect. J Clin Pharm Ther. 1998 Oct;23(5):385-389.
12. Zhang FL, Gao HQ, Wu JM, et al. Selective inhibition by grape seed proanthocyanidin extracts of cell adhesion molecule expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2006 Aug;48(2):47-53.
13. Zhang FL, Gao HQ, Shen L. Inhibitory effect of GSPE on RAGE expression induced by advanced glycation end products in endothelial cells. J Cardiovasc Pharmacol. 2007 Oct;50(4):434-440.
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