老鹰茶总黄酮中黄酮类单体对TGF-β1诱导的肝星状细胞增殖的影响及部分机制研究
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摘要
肝纤维化是一种严重危害人类健康的疾病,是细胞外基质(Extracellur matrix, ECM)合成和降解失衡,引起过多的ECM在肝脏沉积所致。肝星状细胞(Hepatic stellate cells, HSC)的激活、增生、转化是肝纤维化的中心环节,其中活化的HSC大量增殖和胶原的过度生成是导致ECM大量沉积的重要原因。
老鹰茶,又名豹皮樟,系樟科木姜子属毛豺皮樟(Litsea coreana leve)的叶,其中黄酮类成分是老鹰茶的主要成分。药理学实验证明老鹰茶总黄酮(Total flavonids of Litsea coreana L, TFLC)具有抗氧化、降糖、降脂、抗肝纤维化、抗炎及免疫调节等功效。本课题组前期分离出四个黄酮苷类化合物,分别为:槲皮素-3-O-β-D-葡萄糖苷(I)、槲皮素-3-O-β-D-半乳糖苷(II)、山柰酚-3-O-β-D-葡萄糖苷(Ⅲ)、山柰酚-3-O-β-D-半乳糖苷(IV)。因此,本实验通过探讨黄酮类单体是否能通过调节TGF-β1来改善肝纤维化,从而进一步了解老鹰茶总黄酮抗肝纤维化的作用机制。具体研究内容包括以下两个部分:
1.老鹰茶总黄酮中四种黄酮类单体的提取与分离
以黄酮含量为评价指标,采用L9(33)正交试验对老鹰茶总黄酮水提取工艺进行优化,结果优化的生产工艺为用12倍量水提取3次,每次1小时。本文采用非极性的D101型大孔吸附树脂纯化老鹰茶总黄酮,采用该工艺纯化TFLC后,紫外分光光度法测定总黄酮回收率>90 %,平均纯度为48.59%。本文采用正、反相硅胶色谱法反复对老鹰茶总黄酮进行分离,分别得四种黄酮类化合物,经与已知化合物比较,鉴定为:槲皮素-3-O-β-D-半乳糖苷、槲皮素-3-O-β-D-葡萄糖苷、山柰酚-3-O-β-D-半乳糖苷和山柰酚-3-O-β-D-葡萄糖苷。
2.黄酮类单体对TGF-β1诱导的肝星状细胞增殖的影响及部分机制的研究
本文采取肝脏灌流来分离出正常大鼠的肝星状细胞,并进行培养。此法分离的肝星状细胞得率为2×107/肝,细胞活力及纯度都在90%以上。MTT法检测四种黄酮类单体对HSC增殖的抑制作用,结果发现,四种黄酮类单体在培养24、48及72h时均对HSC的增殖具有不同程度的抑制作用,且山柰酚-3-O-β-D-葡萄糖苷在培养48h时对HSC的增殖的抑制作用最强。RT-PCR法检测山柰酚-3-O-β-D-葡萄糖苷对TGF-β1诱导的HSC中Ⅰ型胶原、Ⅲ型胶原、Smad2、Smad3及Smad7中mRNA的表达的影响,结果发现,能够明显下调HSC的Ⅰ型胶原、Ⅲ型胶原、Smad2及Smad3中mRNA的表达,上调Smad7 mRNA的表达。
Liver fibrosis is a serious public health problem of disease, which mainly result from the unbalance between synthesis and degradation of extracellular matrix (ECM), lead to excessive ECM deposition in the liver. Hepatic stellate cells (HSC) activation, proliferation; transformation is the central link of liver fibrosis, which leads to the important cause of sedimentary ECM is the activation of the proliferation and HSC collagen excessive formation.
Laoying tea(also called Litsea coreana )is leaf of Litsea Coreana Leve which belongs to Neolitsea Lauraceae department,flavonoids is the main component of Laoying tea. It has been reported that the extract of Litsea Coreana L. exerts various pharmacological effects including antioxygen, antihyperlipidemic, antihypertensive, anti-inflammatory anti-hyperlipidemic and immunomodulation. In the pre-study, four effective flavonoids have been separated from the plant. Their structures are identified as quercetin-3-O-β-D-glucoside (I), quercetin-3-O-β-D-galactoside (II), kaempferol-3-O-β- D-glucoside (Ⅲ) and kaempferol-3-O-β-D-galactose (IV). Therefore, this study is to investigate whether the flavonoid monomers through regulating TGF-β1 to improve liver fibrosis, thus further understanding of total flavonids of litsea coreana of hepatic fibrosis antioxidant mechanism. Specific content includes the following two parts:
1. Studies on the extraction and separation of four flavonoids monomer from Litsea Coreana L.
With the flavonoids content as a evaluation index, orthogonal design L9(33) is applied to optimize the water extraction technique for total flavonoids of Litsea Coreana L. The best extraction technology is that 12BV water, 1 hour of reflux, and 3 times of extraction. The D101 macroporous resin (non-polar type) is the suitable medium to prepare TFLC. After adsorption on macroporous resin and desorption by ethanol, the content of total flavonoids in Litsea Coreana L is 48.59%, and the recovery rate is > 90 %. In this paper, four flavonoids are separated from TFLC by the normal-phase and reversed-phase silica gel column chromatography. Compared with the known compounds, they are identified as: quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, and kaempferol-3-O-β-D-glucoside.
2. Studies on the effects and mechanisms of flavonoids monomer on the proliferation of hepatic stellate cells induced by TGF-β1
The article adopts the liver perfusion to isolate the normal rat hepatic stellate cells, and culture it. This separation of hepatic stellate cell ratio of 2 x 107 / liver, the cell vitality and purity is more than 90%. By MTT method for detecting four flavonoids monomer proliferation of inhibition on HSC, the result shows that, after cultivating 24、48、72hour, four kinds of flavonoids monomer have varying levels of inhibition on the proliferation of HSC, but kaempferol-3-O-β-D-glucoside have the strongest effective at 48h. Reverse transcription - polymerase chain reaction (RT-PCR) analysis of kaempferol-3-O-β-D-glucoside on HSC which was induced by TGF-β1 was detected in collagenⅠ,collagenⅢ,Smad2, Smad3 and Smad7 receptor mRNA expression in the impact. it can obviously down-regulate collagenⅠ, collagenⅢ, Smad2 and Smad3 receptor mRNA expression,and it can up-regulate Smad7 mRNA expression.
老鹰茶,又名豹皮樟,系樟科木姜子属毛豺皮樟(Litsea coreana leve)的叶,其中黄酮类成分是老鹰茶的主要成分。药理学实验证明老鹰茶总黄酮(Total flavonids of Litsea coreana L, TFLC)具有抗氧化、降糖、降脂、抗肝纤维化、抗炎及免疫调节等功效。本课题组前期分离出四个黄酮苷类化合物,分别为:槲皮素-3-O-β-D-葡萄糖苷(I)、槲皮素-3-O-β-D-半乳糖苷(II)、山柰酚-3-O-β-D-葡萄糖苷(Ⅲ)、山柰酚-3-O-β-D-半乳糖苷(IV)。因此,本实验通过探讨黄酮类单体是否能通过调节TGF-β1来改善肝纤维化,从而进一步了解老鹰茶总黄酮抗肝纤维化的作用机制。具体研究内容包括以下两个部分:
1.老鹰茶总黄酮中四种黄酮类单体的提取与分离
以黄酮含量为评价指标,采用L9(33)正交试验对老鹰茶总黄酮水提取工艺进行优化,结果优化的生产工艺为用12倍量水提取3次,每次1小时。本文采用非极性的D101型大孔吸附树脂纯化老鹰茶总黄酮,采用该工艺纯化TFLC后,紫外分光光度法测定总黄酮回收率>90 %,平均纯度为48.59%。本文采用正、反相硅胶色谱法反复对老鹰茶总黄酮进行分离,分别得四种黄酮类化合物,经与已知化合物比较,鉴定为:槲皮素-3-O-β-D-半乳糖苷、槲皮素-3-O-β-D-葡萄糖苷、山柰酚-3-O-β-D-半乳糖苷和山柰酚-3-O-β-D-葡萄糖苷。
2.黄酮类单体对TGF-β1诱导的肝星状细胞增殖的影响及部分机制的研究
本文采取肝脏灌流来分离出正常大鼠的肝星状细胞,并进行培养。此法分离的肝星状细胞得率为2×107/肝,细胞活力及纯度都在90%以上。MTT法检测四种黄酮类单体对HSC增殖的抑制作用,结果发现,四种黄酮类单体在培养24、48及72h时均对HSC的增殖具有不同程度的抑制作用,且山柰酚-3-O-β-D-葡萄糖苷在培养48h时对HSC的增殖的抑制作用最强。RT-PCR法检测山柰酚-3-O-β-D-葡萄糖苷对TGF-β1诱导的HSC中Ⅰ型胶原、Ⅲ型胶原、Smad2、Smad3及Smad7中mRNA的表达的影响,结果发现,能够明显下调HSC的Ⅰ型胶原、Ⅲ型胶原、Smad2及Smad3中mRNA的表达,上调Smad7 mRNA的表达。
Liver fibrosis is a serious public health problem of disease, which mainly result from the unbalance between synthesis and degradation of extracellular matrix (ECM), lead to excessive ECM deposition in the liver. Hepatic stellate cells (HSC) activation, proliferation; transformation is the central link of liver fibrosis, which leads to the important cause of sedimentary ECM is the activation of the proliferation and HSC collagen excessive formation.
Laoying tea(also called Litsea coreana )is leaf of Litsea Coreana Leve which belongs to Neolitsea Lauraceae department,flavonoids is the main component of Laoying tea. It has been reported that the extract of Litsea Coreana L. exerts various pharmacological effects including antioxygen, antihyperlipidemic, antihypertensive, anti-inflammatory anti-hyperlipidemic and immunomodulation. In the pre-study, four effective flavonoids have been separated from the plant. Their structures are identified as quercetin-3-O-β-D-glucoside (I), quercetin-3-O-β-D-galactoside (II), kaempferol-3-O-β- D-glucoside (Ⅲ) and kaempferol-3-O-β-D-galactose (IV). Therefore, this study is to investigate whether the flavonoid monomers through regulating TGF-β1 to improve liver fibrosis, thus further understanding of total flavonids of litsea coreana of hepatic fibrosis antioxidant mechanism. Specific content includes the following two parts:
1. Studies on the extraction and separation of four flavonoids monomer from Litsea Coreana L.
With the flavonoids content as a evaluation index, orthogonal design L9(33) is applied to optimize the water extraction technique for total flavonoids of Litsea Coreana L. The best extraction technology is that 12BV water, 1 hour of reflux, and 3 times of extraction. The D101 macroporous resin (non-polar type) is the suitable medium to prepare TFLC. After adsorption on macroporous resin and desorption by ethanol, the content of total flavonoids in Litsea Coreana L is 48.59%, and the recovery rate is > 90 %. In this paper, four flavonoids are separated from TFLC by the normal-phase and reversed-phase silica gel column chromatography. Compared with the known compounds, they are identified as: quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, and kaempferol-3-O-β-D-glucoside.
2. Studies on the effects and mechanisms of flavonoids monomer on the proliferation of hepatic stellate cells induced by TGF-β1
The article adopts the liver perfusion to isolate the normal rat hepatic stellate cells, and culture it. This separation of hepatic stellate cell ratio of 2 x 107 / liver, the cell vitality and purity is more than 90%. By MTT method for detecting four flavonoids monomer proliferation of inhibition on HSC, the result shows that, after cultivating 24、48、72hour, four kinds of flavonoids monomer have varying levels of inhibition on the proliferation of HSC, but kaempferol-3-O-β-D-glucoside have the strongest effective at 48h. Reverse transcription - polymerase chain reaction (RT-PCR) analysis of kaempferol-3-O-β-D-glucoside on HSC which was induced by TGF-β1 was detected in collagenⅠ,collagenⅢ,Smad2, Smad3 and Smad7 receptor mRNA expression in the impact. it can obviously down-regulate collagenⅠ, collagenⅢ, Smad2 and Smad3 receptor mRNA expression,and it can up-regulate Smad7 mRNA expression.
引文
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2. Lamireau T, Desmouliere A, Bioulac-Sage P, et al. Mechanisms of hepatic fibrogenesis[J]. Arch Pediatr, 2002, 9(4):392-405.
3. Friedman SL. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies[J]. N Engl J Med.1993 Jun 24;328(25):1828-35
4.陈俊,余光开.转化生长因子β1与肝纤维化关系的研究进展[J].西南军医, 2008,10(6):132-4.
5. Long JY,Wang GN,Matsuura,et al. Activation of Smad transcriptional activity by protein inhibitor of activated STAT3(PLAS3). Proc Natl Acad Sci USA,2004,101(1):99-104.
6.叶辉.老鹰茶中三种黄酮类物质抗脂质过氧化作用初探[J].中药材, 2004, 27(2): 113- 5.
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