CN104292103B - 一种抑制脂肪细胞3t3-l1分化的化合物及其应用 - Google Patents
一种抑制脂肪细胞3t3-l1分化的化合物及其应用 Download PDFInfo
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Abstract
提供一种抑制脂肪细胞3T3-L1分化的化合物,该化合物分离自药用植物腺花香茶菜(Isodon?adenantha),是一个二萜化合物,以及该化合物在制备抑制脂肪细胞分化的药物中和减肥药物和功能食品中的应用。已报道的文献显示该化合物具有促进白血病细胞的分化和改善自身免疫疾病的活性,本发明发现其可以显著抑制脂肪细胞3T3-L1的分化。
Description
技术领域:
本发明属于药物技术领域,具体涉及一种具有抑制脂肪细胞3T3-L1分化的化合物,及其在制药中的应用。
技术背景:
脂肪细胞是终末分化细胞,本身不能通过分裂增殖,主要是由前脂肪细胞在多种激素的作用下细胞内与脂肪生成相关的基因表达,细胞的形态也发生一定的变化,并最终形成成熟的脂肪细胞,细胞内可见明显的脂肪累积,细胞变大变圆。脂肪细胞的主要功能是储存多余脂质,在机体糖供应不足时脂肪细胞中的脂肪氧化供能。现在的研究表明脂肪细胞不仅是能量储存库,而且是一个重要的内分泌器官,可以大量分泌包括肿瘤坏死因子α、瘦素(Leptin)、抵抗素(Resistin)、脂联素(Adiponectin)、内脏脂肪素(Visfatin)和Chemerin等细胞因子和激素,其具有调节机体新陈代谢和炎症等供能。
现代社会,随着社会物质财富的日益积累,人们的生活水平有了极大的提高,肥胖已经成为一个严重影响人类健康分问题,据世界卫生组织的统计,在2008年发达国家的人口中超重或肥胖的人数超过30%,不科学的生活习惯和不合理的饮食结构导致的肥胖越来越多,而肥胖是引起II型糖尿病,高血压,高血脂及动脉粥样硬化等代谢疾病的主要诱因,因此肥胖已成为威胁人类健康的一个重要问题。肥胖主要是脂肪细胞数量增多和单个脂肪细胞的体积增大所致,脂肪细胞体积增大是因为细胞内累积了过多的脂肪,脂肪细胞数量增多是由前脂肪细胞分化而来,因此抑制脂肪细胞的分化可以成为一种潜在抑制肥胖的手段,从而降低了各种由于肥胖引起的代谢疾病的发病率。
腺花素是一种植物中天然存在的化合物,具有较高的安全性。以往的研究中显示与腺花素类似的化合物如毛萼乙素、冬凌草甲素在肿瘤、炎症等方面疾病的防治中具有广阔的前景。现有技术中没有腺花素抑制脂肪细胞分化的活性的报到。
发明内容:
本发明的目的在于提供一种可显著抑制脂肪细胞3T3-L1分化的化合物,以及该化合物在制备抑制脂肪细胞分化的药物中和减肥药物和功能食品中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
结构式(I)所示的抑制脂肪细胞3T3-L1分化的化合物,
所述的结构式(I)的化合物在制备减肥药物中的应用。
所述的结构式(I)的化合物在制备功能食品中的应用。
所述的结构式(I)的化合物在制备抑制脂肪细胞分化的药物中的应用。
所述的结构式(I)的化合物在制备抑制脂肪细胞分化的食品中的应用。
本发明在长期从事脂肪细胞分化调控的基础上,以脂肪细胞分化模型筛选大量化合物得到上述具有抑制脂肪细胞分化活性的化合物。
本发明的化合物是从传统的药用植物腺花香茶菜中分离得的来,制备方法均为公认的技术,可在各种文献中查阅。
本发明中的化合物腺花素可以通过抑制脂肪细胞分化中相关的转录因子如C/EBPβ和PPARγ的表达,从而显著抑制了脂肪细胞的分化。
本发明中的化合物腺花素在细胞分化的最后可以使ERK的磷酸化水平持续保持在较高的水平,而ERK的磷酸化水平在分化过程中持续升高会抑制脂肪细胞的分化。
肥胖可引起高血糖、高血脂、糖尿病和心血管疾病等。肥胖主要是体内累积的脂肪过多,脂肪主要储存在脂肪细胞中,而脂肪细胞是由前脂肪细胞分化而来。在体外实验中,本发明使用小鼠前脂肪细胞3T3-L1作为实验材料,通过传统的“激素鸡尾酒法”使其分化形成成熟的脂肪细胞,成熟的脂肪细胞与前脂肪细胞相比,形态变大变圆,在显微镜下细胞中可见明显的脂肪滴。在分化过程中本发明加入腺花素发现可以显著地抑制3T3-L1细胞的分化,显示其能成为抑制肥胖的药物。在已报道的腺花素的活性中,腺花素可以促进白血病细胞的分化和改善自身免疫,没有腺花素抑制脂肪细胞分化的活性的报道。本发明的腺花素安全低毒,药理作用强,有很好的药用前景。
附图说明:
图1为人工诱导3T3-L1脂肪细胞分化过程示意图;
图2为腺花素抑制3T3-L1脂肪细胞分化的浓度梯度实验,腺花素对3T3-L1脂肪细胞分化抑制的结果;图2A为腺花素抑制脂肪细胞分化的浓度梯度实验的油红O染色照片;将图2A的油红O染色照片进行数据量化后得到腺花素抑制脂肪细胞分化的剂量效应关系如图2B示。
图3为腺花素抑制3T3-L1脂肪细胞分化中的两个关键转录因子C/EBPβ和PPARγ表达的结果;图3A为western检测腺花素对脂肪细胞分化中重要的转录因子C/EBPβ和PPARγ表达的影响,如3B和图3C分别为C/EBPβ和PPARγ的western结果的量化并作T检验。
图4为腺花素使ERK的磷酸化水平提高;图4A为western检测腺花素MAPK/ERK蛋白表达水平磷酸化水平的影响,如4B和图4C分别为p-MAPK/ERK和总的MAPK/ERK的western结果的量化并作T检验。
具体实施方式:
下面结合附图,用本发明的实施例来进一步说明本发明的实质性内容,但并不以此来限定本发明。
实施例1:
本发明化合物腺花素,结构式如下式(I)所示,制备方法请参考如下献中所记载的方法。
Yun-LongXu,Han-DongSun,De-ZuWang,StructureofAdenanthin,TakashiIwashita,HajimeKomura,MutsuoKozuka,KeizoNaya,IsaoKubo,TetrahedronLetters,1987,28(5),499-502.
本发明化合物(I)腺花素对3T3-L1脂肪细胞分化影响实验:
实验仪器:普通倒置光学显微镜(Leica,德国),超纯水系统,超净工作台(Thermo,美国),低温保存箱(海尔,中国),全自动高压灭菌锅(Hirayama,日本),细胞二氧化碳培养箱(Thermo,美国),移液枪(Eppendorf,德国),酶标仪(PerkinElmer,美国),电子天平(DenverInstrument,美国),超低温冰箱(海尔,中国),针头过滤器(Millex-HV,美国),通风橱窗(昆明天悦),相机(三星)。
实验试剂:DMEM培养基(Hyclone,美国),小牛血清(Gibco,美国),胎牛血清(BiologicalIndustries,以色列),IBMX(Sigma,美国),地塞米松(Adamas),罗格列酮(Sigma,美国),胰岛素(Roche,瑞士),链霉素/青霉素(BiologicalIndustries,以色列),0.25%胰酶(BiologicalIndustries,以色列),6孔板(NEST),腺花素,DMSO(Sigma,美国),PBS,细胞冻存管(Thermo,美国),油红O(Sigma,美国),10%多聚甲醛,无水异丙醇,60%异丙醇,超纯水,滤纸。
实验方法:3T3-L1小鼠成纤维细胞购自美国模式培养物集存库(ATCC,CL-173TM)。先将冻存的在液氮罐中的细胞取出,在37℃的水浴锅中融化,融化时轻轻震荡冻存管以加速融化;3T3-L1细胞接种到含有DMEM+10%CS+1%P/S培养基的10cm培养皿中,在37℃10%CO2培养箱中培养,每隔两天换一次培养基,长到60%的汇合度时传代,加入0.25%的胰酶消化3min,然后接种到6孔板中,两天换一次培养基,直到细胞长到100%的汇合度,然后在day-2的时候加入腺花素;在day0时加入分化培养基(DMEM+10%FBS+1%P/S),其中每10ml培养基加入20μlIBMX,10μl胰岛素,10μlDex,1μl罗格列酮,同时加入腺花素;在day3时换成只含胰岛素的培养基,同时加入腺花素,实验中腺花素的浓度梯度是0.01、0.1、0.5、1、3和4μM,过程如图1所示。
在分化的day4时用甲醛固定然后用油红O染色在592nm处检测其吸光值,结果如图2所示。由图片(图2A)可以看出,随着腺花素浓度的逐渐升高,油红O染色变浅,即表示脂肪细胞的分化受到抑制。并同时对各个浓度进行数据统计处理拟合出腺花素对3T3-L1脂肪细胞分化的抑制效果的浓度梯度图(图2B),从图可以看出腺花素在3μM已达到最佳的抑制效果。
实施例2:
本发明化合物(I)腺花素对3T3-L1脂肪细胞分化中的转录因子C/EBPβ和PPARγ的影响实验及对MAPK/ERK信号通路的影响实验:
实验仪器:普通倒置光学显微镜(Leica,德国),超纯水系统,超净工作台(THERMO,美国),低温保存箱(海尔,中国),全自动高压灭菌锅(Hirayama,日本),细胞二氧化碳培养箱(Thermo,美国),移液枪(Eppendorf,德国),酶标仪(PerkinElmer,美国),电子天平(DenverInstrument,美国),超低温冰箱(海尔,中国)针头过滤器(Millex-HV,美国),通风橱窗(昆明天悦),蛋白电泳仪(Bio-Rad),蛋白显色设备。
实验试剂:DMEM培养基(Hyclone,美国),小牛血清(Gibco,美国),胎牛血清(BiologicalIndustries,以色列),IBMX(Sigma,美国),地塞米松(Adamas),罗格列酮(Sigma,美国),胰岛素(Roche,瑞士),链霉素/青霉素(BiologicalIndustries,以色列),0.25%胰酶(BiologicalIndustries,以色列),6孔板(NEST),腺花素,DMSO(Sigma,美国),PBS,细胞冻存管(Thermo,美国),油红O(Sigma,美国),10%多聚甲醛,无水异丙醇,60%异丙醇,超纯水,显影液(Kodak),定影液(Kodak),电泳缓冲液,转膜缓冲液,无水乙醇,感光显影胶片(Kodak),细胞裂解液,PVDF膜(Millipore)。
实验方法:
a:收蛋白:细胞分化完成后用预冷的PBS洗一次,6孔板每孔加入200μl的细胞裂解液,用枪头把细胞刮下来,轻轻吹打,置于冰面上,裂解30分钟,每隔5分钟左右吹打一次。裂解结束后回收到1.5ml的EP管中,每管加入50ml的5×loadingbuffer,然后在100℃的金属浴中加热10分钟,结束后-80℃保存。
b:制胶:按照以下的配方配(两块胶)制10%的胶。注意制的胶块中不能有气泡。
9ml的分离胶:
组装好制胶的模板后再配混合液。各组分混合后一定要混合均匀,迅速注入到模板之间,上层用ddH2O封闭,室温下约30分钟即可凝固,气温太低凝固需要的时间长,一般凝固后可以看见明显的界限,由此判断是否凝固。
4ml的分离胶:
将分离胶上层的水封倒掉,把混匀的浓缩胶浇筑到玻璃板之间,插上10个泳道的梳子,大约30分钟之后即可凝固。
c:点样:将胶块组装好,倒入Tris-甘氨酸电泳缓冲液,小心拔掉梳子,每个泳道点样10μl。先在90V的电压下跑完浓缩胶部分,等蛋白marker分开后电压升高到120V跑分离胶部分。等到目的条带全部分开后结束电泳。
d:转膜:电泳结束后小心的取出胶片进行转膜,转膜的缓冲液需要预冷。在60V的电压下持续2小时。具体过程是:
i)剪好合适大小的PVDF膜,在无水甲醇中浸泡30秒到一分钟,然后浸泡在预冷的转膜缓冲液中,并在PVDF膜的一个角做标记,以确认转膜后的正面。
ii)取出电泳结束后的胶块,从浓缩胶和分离胶的接触面小心的切开,分离胶浸泡在转膜缓冲液中,弃去浓缩胶部分。
iii)转膜系统的组装:组装的顺序是黑板→泡沫→滤纸→胶→滤纸→泡沫→白板,小心的合上夹子放入转膜的架子中,注意电极的正确。
iv)转膜:60V持续2小时。转膜的电压和时间根据凝胶的浓度做适当的调整,在转膜的过程中,电泳槽周围用冰块冰敷并且电泳槽内放入冰袋,防止转膜过程中转膜缓冲液温度升高对转膜的效果造成影响。
e:封闭:将需要的条带剪出来在5%的脱脂牛奶中(用TBS-T配制)封闭1小时。
f:孵育一抗:按照说明书的比例用3%的脱脂牛奶配好抗体,在室温下孵育2小时或者4℃过夜孵育。
g:孵育二抗:一抗孵育结束后用TBS-T清洗5次,每次5分钟,然后加入二抗,在室温下孵育1小时。
h:显色:二抗孵育结束后用TBS-T清洗5次,每次5分钟,然后把条带在显色液中孵育30秒到1分钟在暗室中显色。整个过程应注意避光,并且不要弄脏感光胶片。
从western的结果(图3A)可以看出,腺花素显著抑制了与脂肪细胞分化有重要关系的转录因子C/EBPβ和PPARγ的表达水平,数据量化并做T-TEST显示有显著差异(图3B和图3C所示)。
从western的结果(图4A)可以看出,腺花素对总的ERK表达量没有影响,但是却显著提高了ERK的磷酸化水平,数据量化并做T-TEST显示ERK磷酸化水平显著差异(图4B)而总ERK的量无显著变化(图4C)。
实施例3:
胶囊剂的制备:
实施例1所得化合物1000g,药用淀粉100g,混合均匀,用适量乙醇作为粘合剂制粒,干燥,经整粒机整粒,装0#胶囊,每粒0.30g,口服,每次1-2粒,每日两次。
实施例4:
颗粒剂的制备:
实施例1所得化合物100g,药用淀粉1000g,糖粉500g,混合均匀,用适量乙醇作为粘合剂制粒,干燥,经整粒机整粒,分装即得,口服,每次5g,每日两次。
实施例5:
食品的制备:
取实施例1所得化合物以1:7的比例添加到果酱中,可以增加食品的香味,同时也能发挥保健功能。
实施例6:
食品的制备:
取实施例1所得化合物以1:8的比例添加到牛奶中,可以增加食品的香味,同时也能发挥保健功能。
实施例7:
食品的制备:
取实施例1所得化合物以1:8的比例添加到蜂蜜中,可以增加食品的香味,同时也能发挥保健功能。
实施例8:
含有化合物(I)的乳酸菌饮料配方(W%):
按常规制作食品的方法制得本发明的上述饮料。
Claims (3)
1.结构式(I)所示的抑制脂肪细胞3T3-L1分化的化合物在制备减肥药物中的应用,
2.权利要求1所述的结构式(I)的化合物在制备抑制脂肪细胞分化的药物中的应用。
3.权利要求1所述的结构式(I)的化合物在制备抑制脂肪细胞分化的食品中的应用。
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