六味地黄方含药血清及其入血成分对大鼠前脂肪细胞增殖、分化的影响
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摘要
目的探讨六味地黄方含药血清及其入血成分对大鼠前脂肪细胞内环腺苷酸、环鸟苷酸及其增殖、分化、分化过程中过氧化物酶增殖物活化受体PPARγ2 mRNA表达的影响。
方法一、采用RP—HPLC法对六味地黄方含药血清中3入血成分进行鉴认及含量测定。二、培养大鼠皮下脂肪的前脂肪细胞,在其增殖与分化的不同阶段分别加六味地黄含药血清及其入血3成分,观察其对大鼠前脂肪细胞的增殖,分化过程中的磷酸甘油脱氢酶(GPDH)和细胞内脂肪积聚的作用。三、采用竞争性蛋白结合分析方法测定大鼠前脂肪细胞的cAMP、cGMP含量。四:应用半定量RT—PCR技术,观察不同浓度六味地黄超微含药血清对前脂肪细胞分化过程中PPARγ2 mRNA表达的影响。
结果实验发现六味地黄方(汤、丸、超微)含药血清中含有3个入血成分:马钱苷、芍药苷、丹皮酚,它们在阴虚病理状态下血清中3种成分的含量比正常状态下的要高,且马钱苷、芍药苷、丹皮酚的含量都以超微最高,其次为丸剂、汤剂。六味地黄方对犬鼠前脂肪细胞的增殖有促进作用,并通过降低PPARγ基因的表达来抑制其分化过程中的GPDH的升高,对分化过程中的脂肪积聚有明显的促进分解作用,使前脂肪细胞分化为成熟脂肪细胞的过程受到抑制,其主要有效成分是马钱苷、芍药苷、丹皮酚,但其抑制作用以芍药苷最佳,促进作用以马钱苷最佳;该方还能降低前脂肪细胞内cAMP的水平,其主要有效成分是马钱苷。此外,六味地黄方汤、丸、超微三剂型中,超微饮片不但很好地保留了传统饮片的上述作用,而且作用强于六味地黄汤剂、丸剂。
结论首先,六味地黄方对大鼠前脂肪细胞增殖、分化的作用,可望在分子水平与脂肪细胞相关疾病的基因治疗上提供较特异的靶点药物,有助于进一步探讨六味地黄方对人体脂肪代谢的影响;同时表明该方滋阴作用的机制之一可能是通过其含药血清中的主要成分马钱苷降低前脂肪细胞内cAMP浓度,作用于cAMP受体,以芍药苷、丹皮酚、马钱苷为主要有效成分促进该细胞的增殖并抑制其分化,使细胞功能也随之发生明显变化,进而调节基础代谢之一的脂肪代谢从而缓解阴虚症状;其次,在阴虚病理状态下机体对该方有效成分的吸收要高于正常状态,这对阐明六味地黄丸入血成分中各有效成分滋阴作用的机理,揭示其起滋阴作用的主要成分,从“证”的动物模型来研究中药复方的有效成分及不同剂型对药效的影响,具有一定的深远意义。此外,六味地黄方汤、丸、超微三剂型上述作用的比较研究,对研究各剂型因素对药效的影响及控制药物制剂的内在质量,超微饮片与传统饮片作用机理的对比研究向科学、规范方向发展有着较大的实用价值。
[Objective] To confer the effects of Liuweidihuang drug—containing serum and its original constituents migrating to blood on the cAMP cGMP proliferation differentiation and the gene expression of peroxisome proliferator—activated receptor-γ2 (PPAR_(γ2))of rat preadipocyte cells.
[Methods] Firstly, the three original constituents migrating to blood were identified and determined by RP—HPLC. Secondly, Liuwei dihuang drug—containing serum and its three original constituents migrating to blood were added to the culture media of rat preadipocytes. The proliferation of cells, the expression of GPDH and lipid droplet accumulation in cytoplasm were observed and recorded. Thirdly, The changes of cAMP and cGMP contents were determined by using competitive protein binding assay, The last, the effects of Liuweidihuang drug—containing serum on the PPAR_(γ2) gene and protein expression were assayed by semiquantitative RT-PCR
[Results ]Loganin. paeoniflorin and paeonol were identified as the original constituents Liuweidihuang decoction migrating to blood. in the situation of yin-asthenia, the contents were more than normal. Compared with pills and decoction, they were the highest in micro-powder. Liuwei dihuang drug—containing serum and its three original constituents migrating to blood stimulated rat preadipocyte proliferation、inhibited GPDH up-regulation during differentiation、inhibited lipid accumulation in cell differentiation、decreased the amount of cAMP and restrained the expression of PPAR_(γ2) mRNA in rat preadipocyte cells, loganin was one of the main constituents which decreased the amount of cAMP. micro-powder not only kept the effects of traditional decoction pieces, but also was better than pills and decoction.
[Conclusion] Firstly ,the effects of Liuweidihuang prescription on the proliferation and differentiation of rat preadipocyte may supply especial drugs on molecule level and gene treatment of adipocyte illness. However the mechanism of it nourishing yin may through decreasing the amount of cAMP, affecting cAMP receptor, stimulating rat preadipocyte proliferation and inhibiting its' differentiation with Loganin. paeoniflorin , paeonol etc to improve the function of cells. Secondly, the absorption of active constituents were more than normal in yin-asthenia condition, it may create deep significance in clarifying the mechanism of Liuwei dihuang prescription affecting yin-asthenia and in researching the influence of different compound form of prepared drugs. Thirdly, the results of researching these three forms of prepared drugs above may bring valuable influence in controlling the quality and effects of Liuweidihuang prescription and researching the mechanism of micro-powder and traditionalChinese herbal medicine.
方法一、采用RP—HPLC法对六味地黄方含药血清中3入血成分进行鉴认及含量测定。二、培养大鼠皮下脂肪的前脂肪细胞,在其增殖与分化的不同阶段分别加六味地黄含药血清及其入血3成分,观察其对大鼠前脂肪细胞的增殖,分化过程中的磷酸甘油脱氢酶(GPDH)和细胞内脂肪积聚的作用。三、采用竞争性蛋白结合分析方法测定大鼠前脂肪细胞的cAMP、cGMP含量。四:应用半定量RT—PCR技术,观察不同浓度六味地黄超微含药血清对前脂肪细胞分化过程中PPARγ2 mRNA表达的影响。
结果实验发现六味地黄方(汤、丸、超微)含药血清中含有3个入血成分:马钱苷、芍药苷、丹皮酚,它们在阴虚病理状态下血清中3种成分的含量比正常状态下的要高,且马钱苷、芍药苷、丹皮酚的含量都以超微最高,其次为丸剂、汤剂。六味地黄方对犬鼠前脂肪细胞的增殖有促进作用,并通过降低PPARγ基因的表达来抑制其分化过程中的GPDH的升高,对分化过程中的脂肪积聚有明显的促进分解作用,使前脂肪细胞分化为成熟脂肪细胞的过程受到抑制,其主要有效成分是马钱苷、芍药苷、丹皮酚,但其抑制作用以芍药苷最佳,促进作用以马钱苷最佳;该方还能降低前脂肪细胞内cAMP的水平,其主要有效成分是马钱苷。此外,六味地黄方汤、丸、超微三剂型中,超微饮片不但很好地保留了传统饮片的上述作用,而且作用强于六味地黄汤剂、丸剂。
结论首先,六味地黄方对大鼠前脂肪细胞增殖、分化的作用,可望在分子水平与脂肪细胞相关疾病的基因治疗上提供较特异的靶点药物,有助于进一步探讨六味地黄方对人体脂肪代谢的影响;同时表明该方滋阴作用的机制之一可能是通过其含药血清中的主要成分马钱苷降低前脂肪细胞内cAMP浓度,作用于cAMP受体,以芍药苷、丹皮酚、马钱苷为主要有效成分促进该细胞的增殖并抑制其分化,使细胞功能也随之发生明显变化,进而调节基础代谢之一的脂肪代谢从而缓解阴虚症状;其次,在阴虚病理状态下机体对该方有效成分的吸收要高于正常状态,这对阐明六味地黄丸入血成分中各有效成分滋阴作用的机理,揭示其起滋阴作用的主要成分,从“证”的动物模型来研究中药复方的有效成分及不同剂型对药效的影响,具有一定的深远意义。此外,六味地黄方汤、丸、超微三剂型上述作用的比较研究,对研究各剂型因素对药效的影响及控制药物制剂的内在质量,超微饮片与传统饮片作用机理的对比研究向科学、规范方向发展有着较大的实用价值。
[Objective] To confer the effects of Liuweidihuang drug—containing serum and its original constituents migrating to blood on the cAMP cGMP proliferation differentiation and the gene expression of peroxisome proliferator—activated receptor-γ2 (PPAR_(γ2))of rat preadipocyte cells.
[Methods] Firstly, the three original constituents migrating to blood were identified and determined by RP—HPLC. Secondly, Liuwei dihuang drug—containing serum and its three original constituents migrating to blood were added to the culture media of rat preadipocytes. The proliferation of cells, the expression of GPDH and lipid droplet accumulation in cytoplasm were observed and recorded. Thirdly, The changes of cAMP and cGMP contents were determined by using competitive protein binding assay, The last, the effects of Liuweidihuang drug—containing serum on the PPAR_(γ2) gene and protein expression were assayed by semiquantitative RT-PCR
[Results ]Loganin. paeoniflorin and paeonol were identified as the original constituents Liuweidihuang decoction migrating to blood. in the situation of yin-asthenia, the contents were more than normal. Compared with pills and decoction, they were the highest in micro-powder. Liuwei dihuang drug—containing serum and its three original constituents migrating to blood stimulated rat preadipocyte proliferation、inhibited GPDH up-regulation during differentiation、inhibited lipid accumulation in cell differentiation、decreased the amount of cAMP and restrained the expression of PPAR_(γ2) mRNA in rat preadipocyte cells, loganin was one of the main constituents which decreased the amount of cAMP. micro-powder not only kept the effects of traditional decoction pieces, but also was better than pills and decoction.
[Conclusion] Firstly ,the effects of Liuweidihuang prescription on the proliferation and differentiation of rat preadipocyte may supply especial drugs on molecule level and gene treatment of adipocyte illness. However the mechanism of it nourishing yin may through decreasing the amount of cAMP, affecting cAMP receptor, stimulating rat preadipocyte proliferation and inhibiting its' differentiation with Loganin. paeoniflorin , paeonol etc to improve the function of cells. Secondly, the absorption of active constituents were more than normal in yin-asthenia condition, it may create deep significance in clarifying the mechanism of Liuwei dihuang prescription affecting yin-asthenia and in researching the influence of different compound form of prepared drugs. Thirdly, the results of researching these three forms of prepared drugs above may bring valuable influence in controlling the quality and effects of Liuweidihuang prescription and researching the mechanism of micro-powder and traditionalChinese herbal medicine.
引文
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