地黄抑制脂肪细胞分化和饮食性肥胖的研究
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摘要
肥胖是由于能量物质摄入超过消耗,体内脂肪积聚过多而造成的疾病。肥胖不仅降低患者的生活质量,而且与高血压、糖尿病、冠心病、高血脂、性功能障碍以及肿瘤等多种疾病密切相关。所以,防治肥胖的保健食品和药物的研制,不仅可以减少肥胖人数,还防治肥胖相关疾病。
地黄(Rehmannia glutinosa Libosch)为常用中药,始载于神农本草经,列位上品,有强身、清热凉血,养阴生津等作用。近年来的研究表明,地黄提取物及其不同制剂具有止血、抗衰老、抗炎和利尿等作用。
依据地黄强身补肾的作用,我们研究了地黄对脂肪细胞分化和饮食性肥胖的作用,分离获得地黄中抑制脂肪细胞分化的有效组分地黄寡糖,并阐明了地黄提取液抑制脂肪细胞分化的分子机制,观察了地黄提取液中一个组分甜菜碱对脂肪细胞分化和饮食性肥胖的抑制作用。
一、地黄提取液抑制脂肪细胞分化和大鼠饮食性肥胖的研究
我们发现,在3T3-L1前脂肪细胞诱导分化时加入不同浓度的地黄提取液(RE),对脂肪细胞的终末分化有不同程度的抑制作用,Western Blot试验发现地黄提取液抑制脂肪细胞分化的关键转录因子C/EBPβ、C/EBPα和脂肪细胞特征性蛋白422/aP2的表达。C/EBPβ蛋白质有两种表达形式,18kD为具有DNA结合活性的蛋白质,38kD为具有DNA结合活性和转录激活作用的蛋白质,地黄提取液只对其中的38kD的蛋白质的表达有抑制作用,而对18kD的蛋白质的表达无抑制作用。C/EBPα蛋白质也具有两种表达形式,30kD和42kD的蛋白质都具有DNA结合活性和转录激活活性,地黄提取液对30kD和42kD蛋白质的表达都有抑制作用。
应用血清药理学的方法研究单味中药地黄对脂肪细胞分化的影响,结果发现,相对于对照组(空白)血清,给药组血清分别在1.25%和2.5%的浓度可以明显抑制3T3-L1前脂肪细胞的分化,进一步证明了地黄提取液经过体内代谢后仍然具有抑制脂肪细胞分化的活性。
我们用高脂饮食喂饲幼年SD大鼠成功制作了饮食性肥胖模型。通过对大鼠灌胃给药,发现灌胃组体重明显低于同期喂饲高脂饮食阳性对照组,体重指数和脂肪组织的重量也比后者低,同时地黄提取液可以降低大鼠的血清甘油三脂和血清低密度脂蛋白的浓度,增加血清高密度脂蛋白的浓度,以上变化都具有统计学差异。
二、地黄提取液抑制脂肪细胞分化的有效组分的研究
我们应用LC/MS分离地黄提取液抑制脂肪细胞分化有效组分,采用活性跟踪逐步寻找抑制脂肪细胞分化活性最强的组分。首先利用C18色谱柱将地黄提取液根据极性分成18个组分,其中抑制脂肪细胞分化作用最强的组分为Hx,利用制备级别的色谱柱大量制备Hx,用Waters公司的Atlantis HOLIC制备级色谱柱对Hx进一步的分离,并鉴定各组分对脂肪细胞分化的抑制作用,发现其中的三个组分合并在一起产生的抑制分化的活性达到Hx的50%。这三个组分主要为寡糖类物质,用KS-802色谱柱分离鉴定这一混合物,结果显示这一活性组分主要由单糖、单糖醇、双糖和三糖组成。
三、地黄抑制脂肪细胞分化过程中有丝分裂及其分子机制的研究
在3T3-L1前脂肪细胞的分化过程中存在细胞分裂和分化偶联现象,当前脂肪细胞生长到接触抑制状态,激素和胎牛血清能诱导前脂肪细胞重新进入细胞周期,经过两轮分裂,称为mitotic clonal expansion(MCE),进入终末分化期,形成成熟脂肪细胞。我们的研究显示:地黄提取液抑制脂肪细胞分化的时间窗是诱导后0～16小时。细胞计数表明地黄提取液阻止3T3-L1前脂肪细胞分化过程中细胞数量的增加。流式细胞仪试验显示地黄提取液抑制细胞从G1期进入S期。Western Blot试验显示地黄提取液抑制cdk2蛋白质的表达。免疫荧光试验发现地黄提取液可以阻止3T3-L1前脂肪细胞分化过程中C/EBPs定位到着丝粒。因此,地黄提取液抑制脂肪细胞分化的机制是:通过抑制MCE和C/EBPβ获得DNA结合活性抑制脂肪细胞的分化。
四、甜菜碱抑制脂肪细胞分化和饮食性肥胖的研究
在分析Hx组成的实验中,Atlantis HILIC Silica分析级色谱柱可以将Hx至少分为6个组分,而其中一个保留时间为8.90min组分的质谱图与甜菜碱标准品的质谱图极为相似,提示Hx中含有甜菜碱、甜菜碱类似物或由甜菜碱形成的盐类。利用脂肪细胞分化模型和高脂饮食诱导的肥胖大鼠模型,我们证明甜菜碱能抑制脂肪细胞分化和脂肪细胞分化过程中关键转录因子的表达,并能抑制饮食性肥胖的发生。
离体脂肪细胞分化模型和整体饮食性肥胖动物模型均显示地黄提取液和Hx组分中的甜菜碱均能抑制脂肪细胞分化和抑制大鼠饮食性肥胖,Hx中的寡糖同样具有抑制脂肪细胞分化的能力。因此,我们利用现代分子生物学和细胞生物学从离体到整体证明了经典中药地黄抑制肥胖的作用,并研究了有效成分及其作用的分子机制。
Obesity is characterized by an increase in the size of the adipocytes as a result of lipid accumulation and an increase in the number of adipocytes.It is considered to be one of the most important risk factors for noninsulin-dependent diabetes mellitus(NIDDM),diabetes,cardiovascular diseases and cerebrovascular diseases.The research on obesity is very popular in current medical research area because of its high incidence and profound social effects.
Rehmannia Glutinosa is a Traditional Chinese Medicine(TCM),which has been used for enhancing physical strength.The chemical compositions of Rehmannia have been studied since 1970's.Tomoda reported the classical method to separate the water-soluble constituents of Rehmannia.Ever since then,it was reported that iridoids,sugars and amino acids have been isolated from Rehmannia.
According to its effect of enhancing physical strength,we investigated the effect of Rehmannia extract(RE) on the differentiation of preadipocyte and HFD induced obesity,the effective component and molecular mechanism of RE to inhibit the differentiation of preadipocyte,the effect of betaine,a component of Rehmannia,on the differentiation of preadipocyte and adipogenesis.
PartⅠEffect of RE on the differentiation of preadipocyte and HFD induced fat rat model
In our experiment,different concentration of RE was added to the 3T3-L1 cell culture medium to observe the effect of RE on the differentiation of preadipocyte.Our findings show that RE inhibits the differentiation of preadipocyte in a dose dependent manner.At the molecular level,RE inhibits the expressions of key transcriptional regulator C/EBPβand C/EBPα,and adipocyte phenotype protein 422/aP2.C/EBPβhas two kinds of isoform.The 38kD form of C/EBPβhas DNA binding activity and transcriptional active activity,while the 18kD form only has DNA binding activity.Our results showed that the expression of 18kD was not affected by RE while the 38kD form was nearly inhibited completely by RE.Furthermore,both the expression level of 30kD form and 42kD form of C/EBPαwas inhibited by RE.
Serum pharmacology of traditional Chinese medicine(TCM) has been used to study the TCM pharmacology in vitro since 1980's.In our research, rats were orally administrated with RE or water twice a day and lasted for 3 days,one hour after the last administration,blood was separated and serum was got,after inactivated,the serum was added to the cell culture system to test the effect of RE contained serum on the preadipocyte differentiation.The results showed that RE contained serum could inhibit adipocyte differentiation compared with the control serum at a dose dependent manner.
Then we established HFD induced fat rats model and tested the effect of RE on the HFD induced obesity.The results showed that rats fed with HFD plus RE had reduced body weight,body weight index,weight of fat tissue,TG level,and LDL level,and increased level of HDL.These results suggest that RE prevented the development of HFD-induced adipogenesis in vivo.
PartⅡEffective component of RE to inhibit the differentiation of preadipocyte
Having confirmed that RE could inhibit preadipocyte differentiation,we used LC-MS to investigate the effective component involved in RE to inhibit preadipocyte differentiation.Among 18 fractions,HPLC identified a fraction named Hx that contains 50%of the RE activity.Then Hx was separated by Atlantis HILIC Silica column,which is a kind of hydrophilic column.A combined fraction named Hx-9a which had nearly half activity of Hx was got.KS-802 sugar column was used to separate Hx-9a and mass spectrometry showed that this fraction contains a series of carbohydrate,which including monosaccharide,sugar alcohol,disaccharide and trisaccharide.
PartⅢRE inhibited the mitotic clonal expansion during differentiation of preadipocyte and its molecular mechanism
In order to explain why RE inhibited the differentiation,we investigated the molecular mechanism by which the differentiation of preadipocyte was inhibited.Treatment of growth-arrested 3T3-L1 preadipocytes with differentiation inducers initiates mitotic clonal expansion(MCE),which is characterized by synchronous re-entry of the cells into the cell cycle.Our results showed that the time window for RE to inhibit the differentiation is between 0～16 hours after induction.We provided evidence that the inhibition of adipogenesis by RE occurs with the cell number not increasing.The expression level of cdk2 was down regulated by Rehmannia.after induction of differentiation,C/EBPβin RE-treated cells exhibits loss acquisition of DNA-binding activity and centromeric localization as observed with 3T3-L1 adipocytes.Rehmannia inhibited the differentiation of preadipocyte by impaired cell cycle and the expression and localization to the centromers of C/EBPβ.
PartⅣBetaine inhibits the differentiation of preadipocyte and adipogenesis
In the process of using Atlantis HILIC silica column to separate Hx,the mass spectrum of one fraction was especially similar with that of betaine.It suggested that betaine or betaine like compound or betaine related compound might be involved in Hx.And we proved that betaine inhibits preadipocyte differentiation in vitro and prevents HFD induced body and fat tissue weight increase as well.At the molecular level,we provide evidence that betaine inhibits expression of the key adipocyte differentiation regulators C/EBPβ, C/EBPαand the terminal marker protein 422/aP2 during the differentiation of preadipocyte to adipocyte.Using HFD induced fat rats model,we confirmed that betaine could prevent the HFD induced obesity.
Taken together our results suggest that RE and betaine function to inhibit preadipocyte differentiation in vivo and adipogenesis in vitro.The oligosaccharide involved in Hx could inhibit the differentiation of preadipocyte as well.Therefore,we used modern molecular biology technology and cellular biology to test that the traditional Chinese medicine Rehmannia could inhibit adipocyte differentiation and adipogenesis and its effective component and molecular mechanism to inhibit the differentiation of preadipocyte.
地黄(Rehmannia glutinosa Libosch)为常用中药,始载于神农本草经,列位上品,有强身、清热凉血,养阴生津等作用。近年来的研究表明,地黄提取物及其不同制剂具有止血、抗衰老、抗炎和利尿等作用。
依据地黄强身补肾的作用,我们研究了地黄对脂肪细胞分化和饮食性肥胖的作用,分离获得地黄中抑制脂肪细胞分化的有效组分地黄寡糖,并阐明了地黄提取液抑制脂肪细胞分化的分子机制,观察了地黄提取液中一个组分甜菜碱对脂肪细胞分化和饮食性肥胖的抑制作用。
一、地黄提取液抑制脂肪细胞分化和大鼠饮食性肥胖的研究
我们发现,在3T3-L1前脂肪细胞诱导分化时加入不同浓度的地黄提取液(RE),对脂肪细胞的终末分化有不同程度的抑制作用,Western Blot试验发现地黄提取液抑制脂肪细胞分化的关键转录因子C/EBPβ、C/EBPα和脂肪细胞特征性蛋白422/aP2的表达。C/EBPβ蛋白质有两种表达形式,18kD为具有DNA结合活性的蛋白质,38kD为具有DNA结合活性和转录激活作用的蛋白质,地黄提取液只对其中的38kD的蛋白质的表达有抑制作用,而对18kD的蛋白质的表达无抑制作用。C/EBPα蛋白质也具有两种表达形式,30kD和42kD的蛋白质都具有DNA结合活性和转录激活活性,地黄提取液对30kD和42kD蛋白质的表达都有抑制作用。
应用血清药理学的方法研究单味中药地黄对脂肪细胞分化的影响,结果发现,相对于对照组(空白)血清,给药组血清分别在1.25%和2.5%的浓度可以明显抑制3T3-L1前脂肪细胞的分化,进一步证明了地黄提取液经过体内代谢后仍然具有抑制脂肪细胞分化的活性。
我们用高脂饮食喂饲幼年SD大鼠成功制作了饮食性肥胖模型。通过对大鼠灌胃给药,发现灌胃组体重明显低于同期喂饲高脂饮食阳性对照组,体重指数和脂肪组织的重量也比后者低,同时地黄提取液可以降低大鼠的血清甘油三脂和血清低密度脂蛋白的浓度,增加血清高密度脂蛋白的浓度,以上变化都具有统计学差异。
二、地黄提取液抑制脂肪细胞分化的有效组分的研究
我们应用LC/MS分离地黄提取液抑制脂肪细胞分化有效组分,采用活性跟踪逐步寻找抑制脂肪细胞分化活性最强的组分。首先利用C18色谱柱将地黄提取液根据极性分成18个组分,其中抑制脂肪细胞分化作用最强的组分为Hx,利用制备级别的色谱柱大量制备Hx,用Waters公司的Atlantis HOLIC制备级色谱柱对Hx进一步的分离,并鉴定各组分对脂肪细胞分化的抑制作用,发现其中的三个组分合并在一起产生的抑制分化的活性达到Hx的50%。这三个组分主要为寡糖类物质,用KS-802色谱柱分离鉴定这一混合物,结果显示这一活性组分主要由单糖、单糖醇、双糖和三糖组成。
三、地黄抑制脂肪细胞分化过程中有丝分裂及其分子机制的研究
在3T3-L1前脂肪细胞的分化过程中存在细胞分裂和分化偶联现象,当前脂肪细胞生长到接触抑制状态,激素和胎牛血清能诱导前脂肪细胞重新进入细胞周期,经过两轮分裂,称为mitotic clonal expansion(MCE),进入终末分化期,形成成熟脂肪细胞。我们的研究显示:地黄提取液抑制脂肪细胞分化的时间窗是诱导后0～16小时。细胞计数表明地黄提取液阻止3T3-L1前脂肪细胞分化过程中细胞数量的增加。流式细胞仪试验显示地黄提取液抑制细胞从G1期进入S期。Western Blot试验显示地黄提取液抑制cdk2蛋白质的表达。免疫荧光试验发现地黄提取液可以阻止3T3-L1前脂肪细胞分化过程中C/EBPs定位到着丝粒。因此,地黄提取液抑制脂肪细胞分化的机制是:通过抑制MCE和C/EBPβ获得DNA结合活性抑制脂肪细胞的分化。
四、甜菜碱抑制脂肪细胞分化和饮食性肥胖的研究
在分析Hx组成的实验中,Atlantis HILIC Silica分析级色谱柱可以将Hx至少分为6个组分,而其中一个保留时间为8.90min组分的质谱图与甜菜碱标准品的质谱图极为相似,提示Hx中含有甜菜碱、甜菜碱类似物或由甜菜碱形成的盐类。利用脂肪细胞分化模型和高脂饮食诱导的肥胖大鼠模型,我们证明甜菜碱能抑制脂肪细胞分化和脂肪细胞分化过程中关键转录因子的表达,并能抑制饮食性肥胖的发生。
离体脂肪细胞分化模型和整体饮食性肥胖动物模型均显示地黄提取液和Hx组分中的甜菜碱均能抑制脂肪细胞分化和抑制大鼠饮食性肥胖,Hx中的寡糖同样具有抑制脂肪细胞分化的能力。因此,我们利用现代分子生物学和细胞生物学从离体到整体证明了经典中药地黄抑制肥胖的作用,并研究了有效成分及其作用的分子机制。
Obesity is characterized by an increase in the size of the adipocytes as a result of lipid accumulation and an increase in the number of adipocytes.It is considered to be one of the most important risk factors for noninsulin-dependent diabetes mellitus(NIDDM),diabetes,cardiovascular diseases and cerebrovascular diseases.The research on obesity is very popular in current medical research area because of its high incidence and profound social effects.
Rehmannia Glutinosa is a Traditional Chinese Medicine(TCM),which has been used for enhancing physical strength.The chemical compositions of Rehmannia have been studied since 1970's.Tomoda reported the classical method to separate the water-soluble constituents of Rehmannia.Ever since then,it was reported that iridoids,sugars and amino acids have been isolated from Rehmannia.
According to its effect of enhancing physical strength,we investigated the effect of Rehmannia extract(RE) on the differentiation of preadipocyte and HFD induced obesity,the effective component and molecular mechanism of RE to inhibit the differentiation of preadipocyte,the effect of betaine,a component of Rehmannia,on the differentiation of preadipocyte and adipogenesis.
PartⅠEffect of RE on the differentiation of preadipocyte and HFD induced fat rat model
In our experiment,different concentration of RE was added to the 3T3-L1 cell culture medium to observe the effect of RE on the differentiation of preadipocyte.Our findings show that RE inhibits the differentiation of preadipocyte in a dose dependent manner.At the molecular level,RE inhibits the expressions of key transcriptional regulator C/EBPβand C/EBPα,and adipocyte phenotype protein 422/aP2.C/EBPβhas two kinds of isoform.The 38kD form of C/EBPβhas DNA binding activity and transcriptional active activity,while the 18kD form only has DNA binding activity.Our results showed that the expression of 18kD was not affected by RE while the 38kD form was nearly inhibited completely by RE.Furthermore,both the expression level of 30kD form and 42kD form of C/EBPαwas inhibited by RE.
Serum pharmacology of traditional Chinese medicine(TCM) has been used to study the TCM pharmacology in vitro since 1980's.In our research, rats were orally administrated with RE or water twice a day and lasted for 3 days,one hour after the last administration,blood was separated and serum was got,after inactivated,the serum was added to the cell culture system to test the effect of RE contained serum on the preadipocyte differentiation.The results showed that RE contained serum could inhibit adipocyte differentiation compared with the control serum at a dose dependent manner.
Then we established HFD induced fat rats model and tested the effect of RE on the HFD induced obesity.The results showed that rats fed with HFD plus RE had reduced body weight,body weight index,weight of fat tissue,TG level,and LDL level,and increased level of HDL.These results suggest that RE prevented the development of HFD-induced adipogenesis in vivo.
PartⅡEffective component of RE to inhibit the differentiation of preadipocyte
Having confirmed that RE could inhibit preadipocyte differentiation,we used LC-MS to investigate the effective component involved in RE to inhibit preadipocyte differentiation.Among 18 fractions,HPLC identified a fraction named Hx that contains 50%of the RE activity.Then Hx was separated by Atlantis HILIC Silica column,which is a kind of hydrophilic column.A combined fraction named Hx-9a which had nearly half activity of Hx was got.KS-802 sugar column was used to separate Hx-9a and mass spectrometry showed that this fraction contains a series of carbohydrate,which including monosaccharide,sugar alcohol,disaccharide and trisaccharide.
PartⅢRE inhibited the mitotic clonal expansion during differentiation of preadipocyte and its molecular mechanism
In order to explain why RE inhibited the differentiation,we investigated the molecular mechanism by which the differentiation of preadipocyte was inhibited.Treatment of growth-arrested 3T3-L1 preadipocytes with differentiation inducers initiates mitotic clonal expansion(MCE),which is characterized by synchronous re-entry of the cells into the cell cycle.Our results showed that the time window for RE to inhibit the differentiation is between 0～16 hours after induction.We provided evidence that the inhibition of adipogenesis by RE occurs with the cell number not increasing.The expression level of cdk2 was down regulated by Rehmannia.after induction of differentiation,C/EBPβin RE-treated cells exhibits loss acquisition of DNA-binding activity and centromeric localization as observed with 3T3-L1 adipocytes.Rehmannia inhibited the differentiation of preadipocyte by impaired cell cycle and the expression and localization to the centromers of C/EBPβ.
PartⅣBetaine inhibits the differentiation of preadipocyte and adipogenesis
In the process of using Atlantis HILIC silica column to separate Hx,the mass spectrum of one fraction was especially similar with that of betaine.It suggested that betaine or betaine like compound or betaine related compound might be involved in Hx.And we proved that betaine inhibits preadipocyte differentiation in vitro and prevents HFD induced body and fat tissue weight increase as well.At the molecular level,we provide evidence that betaine inhibits expression of the key adipocyte differentiation regulators C/EBPβ, C/EBPαand the terminal marker protein 422/aP2 during the differentiation of preadipocyte to adipocyte.Using HFD induced fat rats model,we confirmed that betaine could prevent the HFD induced obesity.
Taken together our results suggest that RE and betaine function to inhibit preadipocyte differentiation in vivo and adipogenesis in vitro.The oligosaccharide involved in Hx could inhibit the differentiation of preadipocyte as well.Therefore,we used modern molecular biology technology and cellular biology to test that the traditional Chinese medicine Rehmannia could inhibit adipocyte differentiation and adipogenesis and its effective component and molecular mechanism to inhibit the differentiation of preadipocyte.
引文
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