CN116159065A - 雌二醇二丙酸酯在防治二型糖尿病中的新用途 - Google Patents
雌二醇二丙酸酯在防治二型糖尿病中的新用途 Download PDFInfo
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Abstract
本发明提供了一种雌二醇二丙酸酯或其药学上可接受的盐在预防和/或治疗二型糖尿病中的新用途。本发明实验发现EDP主要通过激活AMPK与PKC通路来促进GLUT4表达和易位,促进L6细胞摄取葡萄糖,摄取的效果与胰岛素(insulin)相当。除此之外,EDP促进GLUT4与细胞质膜的融合以及与葡萄糖摄取是钙离子依赖性的。
Description
技术领域
本发明属于医疗领域,具体的说,涉及一种雌二醇二丙酸酯在防治二型糖尿病中的新用途。
背景技术
随着我国经济水平的高速发展,糖尿病(Diabetes Mellitus,DM)是严重危害人类健康的疾病,全球糖尿病患者预计2025年全球糖尿病患者将达3亿人,糖尿病是一种病发率很高的慢性疾病,常常引起糖、蛋白质、脂肪、水和电解质等一系列物质代谢紊乱。研究表明人们摄入了更加充分的营养增加了糖尿病的发生概率,其中二型糖尿病(T2DM)占90%以上。T2DM,又称非胰岛素依赖型糖尿病,体内糖脂代谢异常是T2DM的主要特征。在T2DM发病的过程中,开始患者的胰岛β细胞还有分泌胰岛素的能力,之后患者机体会产生胰岛素抵抗,导致胰岛β细胞代偿性分泌更多胰岛素来维持正常血糖水平。当患者的胰岛β细胞失去代偿能力之后,血糖水平升高,引起T2DM。T2DM的发病机理十分复杂,尚未完全阐明。目前的研究表明T2DM与遗传和外界环境因素有关。T2DM病因多与禀赋不足、饮食失节、运动失调等有关,其主要发病机制有胰岛素抵抗(IR)和胰岛β细胞受损。目前临床已有一系列降糖药物用于改善T2DM患者的血糖水平。目前应用较多的主要有双胍类药物,磺脲类药物和α-糖苷酶抑制剂等药物。而长期服用这些药物有较多副作用,例如肠胃不适和低血糖等。T2DM的特征是慢性高血糖和不同程度的胰岛素抵抗。引起胰岛素抵抗的原因之一是GLUT4蛋白的表达和功能的失调。因此,了解GLUT4的转位和表达机制对防治糖尿病有着极为重要的作用。
雌二醇二丙酸酯(Estradiol Dipropionate,EDP)是一种半合成的甾体雌激素,用于女性更年期症状和低雌激素水平的激素治疗,也用于治疗妇科疾病。已有大量已发表的数据关于雌激素治疗在缓解更年期症状如潮热和失眠以及预防泌尿生殖系统萎缩和骨质疏松方面的价值。雌激素会导致大鼠体内脂肪的消耗,但这是如何实现的尚不清楚。迄今为止,国内外尚未研究或报道EDP的降血糖活性。在本研究中,我们证明了EDP通过PKC和AMPK途径促进GLUT4表达,从而促进GLUT4与质膜融合和葡萄糖摄取。我们还证实,在L6骨骼肌细胞中,EDP诱导的GLUT4与质膜融合和葡萄糖摄取都是Ca2+依赖的。这些研究表明EDP可作为治疗2型糖尿病的潜在药物。
发明内容
本发明提供了一种雌二醇二丙酸酯或其药学上可接受的盐在预防和/或治疗二型糖尿病中的新用途。
本发明还提供了一种雌二醇二丙酸酯或其药学上可接受的盐在制备GLUT4活性促进剂中的用途,且该GLUT4活性促进剂用于治疗和/或预防二型糖尿病。
本发明还提供了一种雌二醇二丙酸酯或其药学上可接受的盐在制备PKC激活剂中的用途,且该PKC激活剂用于治疗和/或预防二型糖尿病。
本发明还提供了一种雌二醇二丙酸酯或其药学上可接受的盐在制备AMPK激活剂中的用途,且该AMPK激活剂用于治疗和/或预防二型糖尿。
本发明还提供了一种用于治疗二型糖尿病的药,该药包含雌二醇二丙酸酯或其药学上可接受的盐和一种或多种药学上可接受的药物载体。
进一步地,该药的剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
进一步地,所述药物载体包括:表面活性剂、润滑剂、吸收促进剂、稀释剂。
进一步地,所述的GLUT4活性促进剂,其剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
进一步地,所述的激活剂,其剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
本发明的有益效果:本发明发现了雌二醇二丙酸酯在防治二型糖尿病中的新用途。实验发现EDP主要通过激活AMPK与PKC通路来促进GLUT4表达和易位,促进L6细胞摄取葡萄糖,摄取的效果与胰岛素(insulin)相当。
附图说明
图1为EDP促进L6细胞的葡萄糖摄取实验,测得的吸光度柱状图;
图2为EDP促进L6细胞GLUT4转运图;
图3为EDP促进L6细胞GLUT4表达图;
图4为EDP依赖于AMPK和PKC信号通路促进GLUT4的表达;
图5为Ca2+参与EDP诱导的GLUT4表达;
图6为Ca2+参与EDP诱导L6细胞对葡萄糖的摄取以及GLUT4-PM融合的过程。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚,下面将对本发明的优选实施例进行详细的说明,以方便技术人员理解。
本实施例中所提供的EDP来源于Selleck官网购买。采用将EDP于3%的DMSO中并用于生物活性实验检测,具体步骤如下:
1、EDP促进L6细胞葡萄糖摄取
首先用L6细胞按1×104~5×104的密度种九十六孔板,每孔100μL,设置空白对照组、阴性对照组、阳性对照胰岛素组以及加药组,每组六个平行。等到用10%FBS的培养基培养细胞长到80%左右,换成2%FBS的α-MEM培养基分化5-7天。用不含血清的培养基饥饿细胞两小时,配药100μL按照浓度梯度为10μM,30μM,50μM,100μM EDP在37℃培养箱培养30分钟。在30分钟内用酶标仪检测波长为505nm的吸收峰,并在4小时后用MTT法检测EDP对细胞的毒性,加150μL的DMSO用酶标仪波长为492nm检测其吸收峰值。
图1为EDP促进L6细胞的葡萄糖摄取和MTT对细胞的毒性检测,如图1所示,图1中左边的图表明:EDP促进了L6细胞的葡萄糖摄取;图1中右边的图表明:EDP对细胞的毒性很小,细胞存活率均在90%以上。
2、激光共聚焦显微镜检测GLUT4表达
在本次实验中,我们利用稳定转染GV348-myc-GLUT4–mOrange腺病毒载体的L6细胞进行实验,当传代细胞长到适合传代密度时,将其接种在盖玻片上于六孔板中,放置在37℃,5%CO2的细胞培养箱。等细胞爬片之后用10%FBS培养基培养成正常形态的细胞,再换用2%FBS培养基培养5-7天使其分化。细胞分化完成后,首先用PSS溶液缓慢清洗两遍,并用PSS溶液饥饿2小时,饥饿完成后,加入10μM EDP作用于30分钟,再用PSS洗两遍;之后将玻片装在激光共聚焦显微镜配套使用的chamber上,观察药物刺激后的GLUT4-mOrange表达变化,同时用激光共聚焦显微镜的555nm滤光片测量细胞mOrange荧光强度,通过观察myc-GLUT4–mOrange L6细胞内荧光强度的变化,从而计算GLUT4在细胞内的表达情况。
图2为激光共聚焦显微镜检测的EDP促进L6细胞GLUT4表达图。如图2A所示,加入10μM EDP作用30分钟后,myc-GLUT4–mOrange-L6细胞的GLUT4表达明显上升;图2B显示,折线图显示GLUT4的表达随着时间呈现依赖性增长。这说明EDP可以促进L6细胞GLUT4表达。
3、Western Blotting
用无血清培养液饥饿细胞2小时,然后加入10μM EDP药物处理30分钟,之后立即将细胞置于冰上用冷的PBS洗三遍。加入蛋白酶抑制剂PMSF,将细胞刮起于上清管子里,接着超声30s,该操作在冰上进行。将细胞在12000g离心15min,取上清加入适量的SDS-PAGEloading buffer,混匀后95℃变性15min。取等量蛋白质样品上样,使用SDS-PAGE凝胶电泳分离相应蛋白2小时左右,并转移至NC膜1.5小时,利用5%脱脂奶粉溶液封闭2小时,之后用TBST洗膜3次,每次10分钟,再加入稀释后的AKT、P-AKT、β-Actin、GLUT4、AMPKα、P-AMPKα和P-PKC(pan)一抗,4℃摇床上孵育过夜。之后再用TBST洗膜3次,每次10分钟,室温摇床孵育稀释的二抗1小时。使用ECL化学发光液显色发光,凝胶成像系统显影及配套软件计算灰度值。
图3为Western Blotting技术检测的EDP促进L6细胞GLUT4表达图。如图3所示,加入10μM,30μM和100μM EDP作用30分钟后,EDP促进了GLUT4蛋白表达水平,其中,浓度为10μMEDP明显促进GLUT4蛋白表达水平。
图4为EDP促进GLUT4表达的信号通路图。如图A所示,200nM的PMA(PKC通路的阳性药物)与10μM,30μM和100μM EDP在刺激30分钟后显著促进PKC磷酸化水平。阳性药物胰岛素促进了Akt的磷酸化,然而浓度为10μM,30μM和100μM EDP在刺激30分钟后的Akt磷酸化水平无影响(图B)。100μM二甲酸胍(AMPK通路的阳性药物)和10μM,30μM和100μM的EDP刺激细胞30分钟后AMPK磷酸化水平随着浓度依赖性显著提高(图C)。在L6细胞中10μM EDP与三种阻断剂AMPK抑制剂(Compound C)、Akt抑制剂(Wortmanin)和PKC抑制剂处理30分钟后GLUT4的表达水平(图D),发现只有AMPK和PKC阻断剂对GLUT4的表达有抑制作用。这说明EDP依赖于AMPK和PKC信号通路促进GLUT4的表达。
4、激光共聚焦显微镜实时监控GLUT4表达与Ca2+
钙离子作为第二信使,细胞溶质中Ca2+的增加会诱导肌肉收缩,进而促进细胞内的GLUT4转位与细胞膜融合。使用Fluo-4 AM荧光染料将细胞内Ca2+染色以确定它在细胞内的含量。在激光共聚焦显微镜下,实时监测EDP刺激下30min内L6细胞中Ca2+的变化。
图5为胞浆Ca2+有利于EDP提高胞内GLUT4易位。如图5A所示,10μM EDP能在Ca2+条件下诱导GLUT4转位明显上升;图5B所示,10μM EDP随着时间梯度促进GLUT4的表达;图5C所示,10μM EDP随着时间梯度促进胞内Ca2+的上升。
5、细胞内不同Ca2+水平下的葡萄糖摄取与免疫荧光:
在本次实验中,我们利用稳定表达的myc-GLUT4-mOrange L6细胞系中GLUT4蛋白的构造特点进行免疫荧光实验,由于myc为GLUT4蛋白的膜外结构域,所以在免疫荧光实验中使用myc的特异性一抗与FITC标记的二抗来标记myc结构域,在激光共聚焦显微镜下,GLUT4会显示两种荧光,一种是mOrange红色荧光,指示总GLUT4,一种是绿色荧光FITC指示细胞膜上的GLUT4,二者的共定位merge指示转运至细胞膜上的GLUT4。在此结果中,我们可以清晰地看见这两种荧光,表明GLUT4在10μM EDP的刺激下,从细胞内转运至细胞膜上。因此可以利用监测myc-GLUT4–mOrange来监控L6细胞内GLUT4的转运情况。首先将myc-GLUT4–mOrange L6用2mM Ca2+、0mM Ca2+与0mM Ca2++BAPTA分别饥饿2小时,加入药物10μM EDP作用半小时后在激光共聚焦显微镜下观察myc-GLUT4–mOrange L6细胞的荧光并检测细胞膜上的荧光强度,从而检测细胞GLUT4的转运情况。细胞培养等步骤与葡萄糖摄取以及检测GLUT4转运相同,用2mM Ca2+、0mM Ca2+与0mM Ca2++BAPTA种空白对照组(无细胞)、阴性对照组(有细胞组)、阳性对照组(Insulin)以及加药组(10μMEDP)的九十六孔板,饥饿细胞与配药用各自对应的相关钙离子进行。其中不同钙离子条件下的葡萄糖摄取与实验方法1一样。
图6为Ca2+参与EDP促进L6细胞的葡萄糖摄取和插入细胞质膜的过程。在EDP显著提高。在正常2mM细胞外钙条件下,10μM EDP作用30min后葡萄糖摄取明显上升,而0mM细胞外钙降低葡萄糖的摄取,更一步0mM钙离子被螯合以后降低更明显,表明钙离子在刺激骨骼肌细胞摄取葡萄糖是有明显的影响的(图6B)。接着对上一步实验用免疫荧光技术进行探讨关于EDP诱导GLUT4易位是否与Ca2+有影响。在通过与对照2mM细胞外钙相比,2mM细胞外钙的阳性药物胰岛素明显增强了GLUT4表达与细胞质膜的融合(即FITC的荧光明显增强),GLUT4表达增强(即mOrange荧光增强),然而在0Ca2++10μM BAPTA-AM环境中,Insulin或EDP促进GLUT4表达与细胞质膜融合的能力被显著抑制(图6A)。
实验结果
数据以平均值±标准误差(X±SE)表示,组间显著性差异检验用t检验及相关分析。
图1为EDP促进L6细胞的葡萄糖摄取图,如图1所示,图中左图显示:EDP显著促进了L6细胞的葡萄糖摄取,而且EDP对L6细胞葡萄糖摄取的促进效果与胰岛素(insulin)相当。图1中右图显示,EDP对细胞的无毒,细胞存活率高。
图2为EDP促进L6细胞GLUT4转运图,如图2所示,加入10μM EDP后,myc-GLUT4–mOrange L6细胞膜的荧光强度明显增强。说明EDP可以促进L6细胞GLUT4转运。
图3为EDP促进L6细胞GLUT4表达图,如图3所示,加入10μM EDP促进了L6细胞GLUT4表达。
图4为EDP依赖于AMPK和PKC信号通路促进GLUT4的表达。如图4所示,EDP主要通过激活AMPK与PKC通路来促进GLUT4表达和易位,而Akt信号通路不是药物介导GLUT4易位过程的主要用途。
图5为胞浆Ca2+有利于EDP提高胞内GLUT4易位。如图5所示,EDP能在Ca2+条件下诱导GLUT4转位明显上升。
图6为Ca2+参与EDP促进L6细胞的葡萄糖摄取和插入细胞质膜的过程。在EDP显著提高。在正常2mM细胞外钙条件下,10μM EDP作用30min后葡萄糖摄取明显上升,而0mM细胞外钙降低葡萄糖的摄取,更一步0mM钙离子被螯合以后降低更明显,表明钙离子在刺激骨骼肌细胞摄取葡萄糖是有明显的影响的(图6B)。接着对上一步实验用免疫荧光技术进行探讨关于EDP诱导GLUT4易位是否与Ca2+有影响。在通过与对照2mM细胞外钙相比,2mM细胞外钙的阳性药物胰岛素明显增强了GLUT4表达与细胞质膜的融合(即FITC的荧光明显增强),GLUT4表达增强(即mOrange荧光增强),然而在0Ca2++10μM BAPTA-AM环境中,Insulin或EDP促进GLUT4的表达与细胞质膜融合的能力被显著抑制(图6A)。图6结果说明EDP促进葡萄糖摄取以及GLUT4转位都是钙离子依赖性的。
最后说明的是,以上优选实施例仅用于说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
Claims (9)
1.雌二醇二丙酸酯或其药学上可接受的盐在预防和/或治疗二型糖尿病中的新用途。
2.雌二醇二丙酸酯或其药学上可接受的盐在制备GLUT4活性促进剂中的用途,且该GLUT4活性促进剂用于治疗和/或预防二型糖尿病。
3.雌二醇二丙酸酯或其药学上可接受的盐在制备PKC激活剂中的用途,且该PKC激活剂用于治疗和/或预防二型糖尿病。
4.雌二醇二丙酸酯或其药学上可接受的盐在制备AMPK激活剂中的用途,且该AMPK激活剂用于治疗和/或预防二型糖尿病。
5.一种用于治疗二型糖尿病的药,其特征在于:该药包含雌二醇二丙酸酯或其药学上可接受的盐和一种或多种药学上可接受的药物载体。
6.根据权利要求5所述的一种用于治疗二型糖尿病的药,其特征在于:该药的剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
7.根据权利要求5所述的一种用于治疗二型糖尿病的药,其特征在于:所述药物载体包括:表面活性剂、润滑剂、吸收促进剂、稀释剂。
8.根据权利要求2所述的GLUT4活性促进剂,其剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
9.根据权利要求3或4所述的激活剂,其剂型为片剂、散剂、汤剂、丸剂、胶囊剂。
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