CN110025611A - 一种槲皮素对酪氨酸磷酸酶1b抑制中的应用 - Google Patents
一种槲皮素对酪氨酸磷酸酶1b抑制中的应用 Download PDFInfo
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- CN110025611A CN110025611A CN201910195009.5A CN201910195009A CN110025611A CN 110025611 A CN110025611 A CN 110025611A CN 201910195009 A CN201910195009 A CN 201910195009A CN 110025611 A CN110025611 A CN 110025611A
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Abstract
本发明公开了一种槲皮素对酪氨酸磷酸酶1B抑制中的应用，其中槲皮素在抑制胰岛素信号转导通路的负调节酶蛋白质酪氨酸磷酸酶1B中的应用，包括如下步骤：槲皮素的处理：精确称取槲皮素粉末溶于二甲基亚砜，配成贮液，至于4℃下备用；再稀释成不同浓度的溶液；槲皮素抑制：以p‑NPP为底物，向MOPS缓冲液中分别加入PTP1B和上述稀释后的槲皮素溶液，反应总体系为100μl，于37℃反应15min，用NaHCO3终止反应后，在405nm波长处测定其抑制率；本发明对制备治疗及预防与蛋白质酪氨酸磷酸酶1B活力过高或过表达相关疾病如糖尿病的食品、药物或食品及药物组合物具有重要影响。
Description
技术领域
本发明涉及蛋白质去磷酸化技术领域，具体涉及一种槲皮素对酪氨酸磷酸酶1B抑制中的应用。
背景技术
PTP1B是一种非跨膜PTP,主要通过内质网表达其C-末端结构域。PTP1B可以直接与PTK受体结合，包括胰岛素受体、表皮生长因子受体。蛋白酪氨酸磷酸酶PTP1B是葡萄糖体内平衡和能量代谢的主要调节剂，研究表明，体内胰岛素调节失衡的人，其脂肪组织中的PTP1B表达增加。目前为止，研究最多的磷酸酶就是PTP1B，在肝脏和脂肪组织中，PTP1B表达的最为广泛，而且作为胰岛素受体的主要负调节因子，因此，PTP1B在胰岛素抵抗相关疾病中发挥着重要作用。本论文中的△PTP1B的基因序列，共298个氨基酸残基，分子量34674Da。
1988年，E.H.Fischer实验室在人胎盘细胞中分离纯化了第一个蛋白酪氨酸磷酸酶(现称PTP1B)。该酶为一个37KD的胞内酶(无跨膜结构域)，其氨基酸顺序分析表明，它与Ser/Thr型蛋白磷酸酶(如PP-1，PP-2A、2B、2C)无同源性，却与一种称为CD45的蛋白具有很高的相似性。CD45是一类在结构上相关的、高分子量(150～280kD)的跨膜蛋白，具有与受体极为相似的结构特点，在免疫T细胞和B细胞中的含量很高。后来弄清CD45是一种具有受体结构的跨膜蛋白酪氨酸磷酸酶。因此PTP1B和CD45代表了PTP的两种基本类型：胞内型和跨膜受体型。十几年时间过去了，现在已有100多种同功酶相继分离纯化或基因克隆成功。
最近体内和体外的研究都表明PTP1B是一个治疗II型糖尿病和肥胖症的潜在靶标。有证据表明，PTP1B先将被激活的胰岛素受体(IR)去磷酸化，进而阻止了胰岛素的信号转导途径。因此，任何与IR相关的PTP1B表达水平和活力的变化都可能会影响胰岛素信号转导，并可能导致胰岛素阻抗的发生。在PTP1B基因敲除小鼠的骨骼肌和肝脏中，胰岛素受体的自身磷酸化增加，而且对胰岛素的敏感性也有提高。无PTP1B小鼠的肥胖倾向明显降低，而且免患饮食诱导性肥胖症。同时也发现，PTP1B功能缺乏的小鼠发育良好，且具有正常的繁殖能力，癌症的发生率也未见提高；因此筛选PTP1B的特异性抑制剂有望提高机体对胰岛素的敏感性，应用于PTP1B的催化结构域(△PTP1B，分子量34674Da)为靶标，通过体外酶反应动力学实验，测定槲皮素对蛋白质酪氨酸磷酸酶活性水平的影响。
发明内容
针对上述存在的问题，本发明提出了一种槲皮素对酪氨酸磷酸酶1B抑制中的的应用。
为了实现上述的目的，本发明采用以下的技术方案：
一种槲皮素对酪氨酸磷酸酶1B抑制中的应用。
优选的，所述槲皮素在抑制胰岛素信号转导通路的负调节酶蛋白质酪氨酸磷酸酶1B中的应用，包括以下步骤：
(1)槲皮素的处理：精确称取槲皮素粉末溶于二甲基亚砜，配成贮液，至于4℃下备用；再稀释成不同浓度的溶液；
(2)槲皮素抑制：以p-NPP为底物，向MOPS缓冲液中分别加入PTP1B和上述稀释后的槲皮素溶液，反应总体系为100μl，于37℃反应15min，用NaHCO3终止反应后，在405nm波长处测定其抑制率。
优选的，所述MOPS缓冲液由DTT溶液、NaCl溶液、EDTA溶液、BSA溶液组成。
优选的，所述抑制率为＝100％×(A0－A)/A0；
其中A0：未加入槲皮素实验体系的光吸收值；
A：加入槲皮素实验体系的光吸收值。
优选的，其特征在于，所述槲皮素购自于阿拉丁试剂公司，纯度≥98.5％。
由于采用上述的技术方案，本发明的有益效果是：
(1)PTP1B作为胰岛素受体的主要负调节因子，而人体肝脏和脂肪组织中大量存在PTP1B，本发明对制备治疗及预防与蛋白质酪氨酸磷酸酶1B(PTP1B)活力过高或过表达相关疾病如糖尿病的食品、药物或食品及药物组合物具有重要影响；
(2)本发明槲皮素对酪氨酸磷酸酶PTP1B的抑制作用以及对胰岛素信号通路的影响，对研制治疗饮食诱导性肥胖症以及癌症的药物或者方法具有重要意义。
附图说明
图1：槲皮素对PTP1B的抑制类型及IC50；
图2：HepG2细胞中不同槲皮素浓度诱导的总蛋白磷酸化水平；
图3：HepG2细胞中不同槲皮素浓度诱导的IR磷酸化；
图4：HepG2细胞中由不同槲皮素浓度诱导的IRS磷酸化；
图5：HepG2细胞中由不同槲皮素浓度诱导的ERK磷酸化。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。基于本发明的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
槲皮素在抑制胰岛素信号转导通路的负调节酶蛋白质酪氨酸磷酸酶1B中的应用。
槲皮素的处理：精确称取槲皮素粉末溶于二甲基亚砜，配成5000μM的贮液，至于4℃备用。再稀释成3500μM、1500μM、1000μM、500μM、400μM、200μM、100μM、50μM、25μM、10μM、5μM、1μM、0.5μM、0μM的溶液；
槲皮素抑制效果的测定：以p-NPP(20mM)为底物，向MOPS缓冲液(25mM，pH＝7.0，含1mM DTT，0.1M NaCl，1mM EDTA，1mg/ml BSA)中分别加入10μlΔPTP1B(10μg/ml)及10μl不同稀释浓度的槲皮素溶液，反应总体系为100μl，于37℃反应15min，用100μl 0.2M NaHCO3终止反应后，在405nm波长处测定其光吸收值，以未加入槲皮素的实验体系为空白对照，其光吸收值为A0，其余加入槲皮素的各实验体系的光吸收值为A，则不同浓度槲皮素对ΔPTP1B的抑制率＝100％×(A0－A)/A0；
以抑制剂的浓度(μM)为横坐标，以抑制剂对PTP1B的抑制率为纵坐标作图，得到槲皮素对PTP1B的IC50为170.9μM。
对PTP1B抑制作用的测定：使用四种浓度的槲皮素(20μM、100μM、200μM、400μM)。反应体系为100μl，包含70μl的25mM MOPS-NaOH(pH 7.0)缓冲液、1mM EDTA、1mM DTT、1mg/mlASB、0.1M NaCl、40ng PTP1B、10μlp-NPP浓度分别为1.25mM、2.5mM、5mM、10mM、20mM、40mM、80mM。在37℃反应15min后用100μl 0.1M NaHCO3终止反应，用酶标仪在405nm的吸收峰值。空白对照组不加酶。通过双倒数作图法确定槲皮素对PTP1B的抑制类型。
对PTP1B抑制作用的测定方法为：各种物质PTP1B的抑制性的考察是通过测定半数抑制浓度(IC50)来衡量。将抑制组分按梯度进行稀释，将稀释后的各个梯度加入上述反应体系中，于405nm测定在37℃，15min内光吸收的变化值之后，将各个梯度的吸收值与未加抑制组分的相除，得出的数值为抑制百分数，当抑制率达到50％时，所对应的稀释梯度的倍数做为IC50，如图1所示。
不同浓度槲皮素对IR、IRS、ERK的酪氨酸磷酸化水平的影响：HepG2细胞在含有10％胎牛血清的培养基中培养,直到细胞数量为90％左右。将细胞分别用槲皮素10μM、50μM、100μM、200μM的处理。温育30分钟后，将细胞在含有25mM甘油磷酸盐(pH＝7.3)，5mMEDTA，2mM EGTA，5mM巯基乙醇，1％Triton X-100，0.1M NaCl和蛋白酶抑制剂混合物中进行提取。12,000×g离心10分钟后，上清液在10％十二烷基硫酸钠(SDS)聚丙烯酰胺凝胶上分离，然后转移到聚偏氟乙烯膜上用抗磷酸酪氨酸，磷酸-IGF-I受体β(Tyr1131)，胰岛素受体β(Tyr1146)和IRS抗体。使用增强化学发光法进行p-ERK(E-4)：sc-7383检测；如图2、3、4、5所示。
综合上述可得，槲皮素可以抑制胰岛素信号转导通路的负调节酶蛋白质酪氨酸磷酸酶1B的活性，并增加IR、IRS、ERK的酪氨酸磷酸化水平，槲皮素还以剂量依赖的方式促进HepG2细胞中IR和IRS的磷酸化。
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (5)
1.一种槲皮素对酪氨酸磷酸酶1B抑制中的应用。
2.根据权利要求1所述槲皮素对酪氨酸磷酸酶1B抑制中的应用，其特征在于，所述槲皮素在抑制胰岛素信号转导通路的负调节酶蛋白质酪氨酸磷酸酶1B中的应用，包括以下步骤：
(1)槲皮素的处理：精确称取槲皮素粉末溶于二甲基亚砜，配成贮液，至于4℃下备用；再稀释成不同浓度的溶液；
(2)槲皮素抑制：以p-NPP为底物，向MOPS缓冲液中分别加入PTP1B和上述稀释后的槲皮素溶液，反应总体系为100μl，于37℃反应15min，用NaHCO3终止反应后，在405nm波长处测定其抑制率。
3.根据权利要求2所述的应用，其特征在于，所述MOPS缓冲液由DTT溶液、NaCl溶液、EDTA溶液、BSA溶液组成。
4.根据权利要求2所述的应用，其特征在于，所述抑制率为＝100％×(A0－A)/A0；
其中A0：未加入槲皮素实验体系的光吸收值；
A：加入槲皮素实验体系的光吸收值。
5.根据权利要求2所述的应用，其特征在于，所述槲皮素购自于阿拉丁试剂公司，纯度≥98.5％。
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