CN116139145A - 尼达尼布乙磺酸盐在防治二型糖尿病中的新用途 - Google Patents
尼达尼布乙磺酸盐在防治二型糖尿病中的新用途 Download PDFInfo
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Abstract
本发明提供了一种尼达尼布乙磺酸盐在预防和/或治疗二型糖尿病中的新用途。本发明实验发现尼达尼布乙磺酸盐主要通过激活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。
Description
技术领域
本发明属于医疗领域，具体的说，涉及一种尼达尼布乙磺酸盐在防治二型糖尿病中的新用途。
背景技术
二型糖尿病(Type 2 Diabetes Mellitus，T2DM)是一种以长期血糖水平升高为特征的代谢紊乱，它通常是由于热量摄入超过能量消耗，加上分泌胰岛素(insulin)的胰腺细胞功能障碍导致胰岛素分泌不足。热量过剩会引发机体新陈代谢的负担，机体对热量代谢的能力在整个进化过程中一直是保守的，所以热量过剩会抑制肌肉、脂肪组织和肝脏对能量基质的进一步摄取，导致胰岛素抵抗的临床表现。胰岛素信号通路是糖代谢与脂肪代谢关键，长期的胰岛素抵抗会让机体骨骼肌功能衰退，脂肪积累增加，进而引起肥胖。二型糖尿病发病机制复杂，且伴随着糖尿病肾病和多种心血管疾病的并发症，所以糖尿病的治疗需要从多个角度进行，例如增强骨骼肌对葡萄糖的消耗和抑制脂肪生成。
尼达尼布乙磺酸盐(Nintedanib Ethanesulfonate Salt，NES)是一种小分子酪氨酸激酶抑制剂，靶向与血小板衍生生长因子(platelet-derived growth factor，PDGF)受体α和β，FGFR1-3和血管内皮生长因子(vascular endothelial growth factor，VEGF)受体1-3的ATP口袋竞争性结合，从而达到抑制生长因子表达的作用。美国与欧洲将尼达尼布乙磺酸盐作为治疗特发性肺纤维化(idiopathic pulmonary fibrosis，IPF)的特效药。目前临床上尼达尼布乙磺酸盐还被应用于肺癌，结肠癌和乳腺癌等癌症治疗。尼达尼布乙磺酸盐在肺鳞状细胞癌中通过抑制Akt-ERK通路抑制了癌细胞的增殖。在我们的研究中，NES通过磷酸化AMPK和PKC通路增强了L6细胞中GLUT4的表达和转运，从而增强了L6细胞的葡萄糖摄取和改善胰岛素抵抗。因此NES有治疗二型糖尿病的潜力。
发明内容
本发明提供了一种尼达尼布乙磺酸盐在预防和/或治疗二型糖尿病中的新用途。
本发明还提供了一种尼达尼布乙磺酸盐在制备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尼达尼布乙磺酸盐在制备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尼达尼布乙磺酸盐在制备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一种用于治疗二型糖尿病的药，该药包含尼达尼布乙磺酸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：本发明发现了尼达尼布乙磺酸盐在防治二型糖尿病中的新用途。实验发现尼达尼布乙磺酸盐主要通过激活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.尼达尼布乙磺酸盐促进L6细胞的葡萄糖摄取；
图2.尼达尼布乙磺酸盐促进L6细胞GLUT4转运；
图3.尼达尼布乙磺酸盐促进L6细胞的GLUT4转运为钙离子依赖性的。
图4.尼达尼布乙磺酸盐通过磷酸化AMPK通路与PKC通路促进L6细胞GLUT4表达；
图5.尼达尼布乙磺酸盐促进L6细胞GLUT4的转运被AMPK信号通路抑制剂CompoundC抑制。
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。
将selleck公司购买的尼达尼布乙磺酸盐溶解于DMSO中并用于生物活性实验检测，具体步骤如下：
1、细胞葡萄糖摄取实验：
将L6细胞按1×104～5×104的密度种于96孔板内。用含2％FBS的α-mem培养液将L6细胞分化5～7天。然后将细胞饥饿2小时，并将同一孔板内细胞划分组别，分别加入浓度梯度的尼达尼布乙磺酸盐。使用葡萄糖氧化酶测试每个组别中溶液中葡萄糖浓度，该试验进行了3次独立重复实验，并设立了对照组和胰岛素组。实验结果检测图为图1。
图1为尼达尼布乙磺酸盐促进L6细胞的葡萄糖摄取图，如图1所示，尼达尼布乙磺酸盐显著促进了L6细胞的葡萄糖摄取。
2、免疫荧光实验检测GLUT4转运：
本发明使用稳定表达myc的L6细胞(myc-GLUT4-mOrange)作为研究对象。myc是一种跨膜小分子蛋白，目前研究已证明myc与GLUT4存在高度共定位关系。因此可以利用观测myc–mOrange来监控L6细胞内GLUT4的转运情况。首先将myc-GLUT4-mOrange L6细胞接种于圆玻片上培养并分化，用无血清培养液饥饿2小时，再加入尼达尼布乙磺酸盐作用30min，用PBS清洗细胞，再用3％多聚甲醛固定细胞30min，之后用PBS清洗细胞2次，每次5min。再用50mM甘氨酸溶液作用20min去除背景杂质。用PBS清洗细胞2次，每次5min。2％BSA封闭1h，枪头吸干封闭液，吸水纸吸干。anti-myc抗体1：200孵育1h，每片玻片滴加50μL。2％BSA清洗3次，每次5min。以下操作皆避光操作，二抗孵育1h，2％BSA清洗3次，每次5min，再用PBS洗2次，每次5min。用镊子将圆玻片夹出，滴10μL封片剂在圆玻片有细胞的一面，将有细胞那面倒置贴紧载玻片，将指甲油滴在玻片边缘，封闭。等待指甲油完全干透，将玻片置于共聚焦激光扫描显微镜下观察细胞荧光。将玻片置于共聚焦激光扫描显微镜下观察细胞荧光。实验结果检测图为图2。
图2为尼达尼布乙磺酸盐促进L6细胞GLUT4转运图，如图2所示，m-Orange为myc-GLUT4-mOrange L6细胞的GLUT4标记荧光，指示细胞中总GLUT4蛋白，FITC为myc抗体携带的FITC荧光，指示细胞膜上的GLUT4，merge则为两组荧光的共定位，加入尼达尼布乙磺酸盐后。myc-GLUT4-mOrange细胞膜的FITC荧光强度明显增强。说明尼达尼布乙磺酸盐可以促进L6细胞GLUT4转运。
3、myc-GLUT4-mOrange L6细胞的钙离子免疫荧光实验：
首先将myc-GLUT4-mOrange L6细胞接种于圆玻片上培养并分化，将细胞分别置于0mM Ca2++BAPTA(一种细胞内钙离子螯合剂)PSS溶液、0mM Ca2+PSS溶液和2mM Ca2+PSS溶液中饥饿2h，用PBS清洗细胞，再用3％多聚甲醛固定细胞30min，之后用PBS清洗细胞2次，每次5min。再用50mM甘氨酸溶液作用20min去除背景杂质。用PBS清洗细胞2次，每次5min。2％BSA封闭1h，枪头吸干封闭液，吸水纸吸干。anti-myc抗体1：200孵育1h，每片玻片滴加50μL。2％BSA清洗3次，每次5min。以下操作皆避光操作，二抗孵育1h，2％BSA清洗3次，每次5min，再用PBS洗2次，每次5min。制片后，将玻片置于共聚焦激光扫描显微镜下观察细胞荧光。实验结果检测图为图3A和3B。
4、钙离子葡萄糖摄取实验:
将L6细胞按1×104～5×104的密度种于96孔板内。用含2％FBS的α-mem培养液将L6细胞分化5～7天。然后分别使用0mM Ca2++BAPTA(一种细胞内钙离子螯合剂)PSS溶液、0mMCa2+PSS溶液和2mM Ca2+PSS溶液中饥饿2h，加入30μM尼达尼布乙磺酸盐作用30min后，使用葡萄糖氧化酶测试每个组别中溶液中葡萄糖浓度，该试验进行了3次独立重复实验，并设立了对照组和胰岛素组。实验结果检测图为图3C。
图3为尼达尼布乙磺酸盐在L6细胞中所引起的GLUT4的转运是钙离子依赖性的，如图3所示，为了验证NES引起的L6细胞中的GLUT4转运与葡萄糖摄取是否需要Ca2+参与，我们使用了三种细胞生理溶液，0mM Ca2++BAPTAPSS溶液中BAPTA为细胞内钙螯合剂，使细胞处于失去内钙外钙的状态，0mM Ca2+PSS溶液让细胞处于失去外钙的状态，2mM Ca2+PSS溶液则为细胞维持钙稳态的状态。免疫荧光实验表明，在L6细胞处于内钙外钙失衡的情况下，GLUT4的转运作用明显收到了抑制，在2Ca2+PSS溶液中NES明显促进了GLUT4的转运(图3A和3B)。为了进一步验证该实验的可信度，我们使用葡萄糖摄取实验验证了尼达尼布需要在钙离子的作用下才能促进L6细胞的葡萄糖摄取(图3C)。
3、Western Blotting：
用无血清培养液饥饿细胞2小时，然后加入相应药物处理，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。将细胞置于冰水中超声15s，该操作在冰上进行。将细胞在500g离心10min，取上清加入SDS-PAGE loading buffer，混匀后95℃变性10min。使用SDS-PAGE快速凝胶快速制备试剂盒制备聚丙烯酰胺凝胶，加入1×running buffer浸过凝胶后，将蛋白质样品按总克数相等的原则进行点样，然后进行电泳，使目的蛋白条带与其它条带分离。然后准备转膜仪，包括转膜夹和滤纸，每个转膜夹的每一面至少要垫三层滤纸，将含有目的条带的凝胶切下，放置于略大于凝胶的NC膜(硝化纤维膜)上，用玻璃棒驱赶气泡，按照黑胶白膜的原理，正确地将NC膜放置于转膜夹的白色面，与此同时，凝胶对着转膜夹黑色面，将转膜夹放入转膜仪中，加入1×trans buffer直至淹过转膜夹后，300mA电流转膜1h。转膜完毕后，使用TBST配制5％脱脂奶粉或BSA封闭液，NC膜在室温下封闭2h，轻轻吸去NC膜上多余的封闭液，事先配制的一抗溶液中(一抗与TBST的体积比为1：1000)，4℃过夜。将一抗孵育完毕的NC膜在TBST中洗三次，每次10min，然后用以1:10000的比例用TBST稀释的二抗室温孵育NC膜1h。最后用TBST洗膜三次，将膜用镊子夹放于干净的容器中，避光的条件性均匀撒上显影液，在凝胶成像系统中显影。实验结果检测图为图4和图5。
图4为尼达尼布乙磺酸盐通过促进AMPK和PKC通路的磷酸化来促进GLUT4的表达(图4A)，结果表明尼达尼布乙磺酸盐不能磷酸化AKT通路(图4B)，但能显著增强AMPK和PKC的磷酸化(图4C和4D)。显然，NES通过促进GLUT4表达来促进葡萄糖摄取的机制是独立于胰岛素通路的。
图5为尼达尼布乙磺酸盐促进L6细胞GLUT4的转运被AMPK信号通路抑制剂Compound C抑制图。根据图5所示，为了确定AMPK通路与PKC通路中作为增强GLUT4表达的主要通路，分别于使用AKT通路抑制剂Wortmannin、PKC通路抑制剂
和AMPK通路抑制剂Compound C处理L6细胞30min后，再添加30μM NES继续作用30min。Western Blot结果表明只有Compound C完全抑制了GLUT4的表达，说明NES主要通过磷酸化AMPK通路进而使GLUT4表达增加，PKC通路可能由激活的AMPK通路通过级联反应激活。
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.尼达尼布乙磺酸盐在预防和/或治疗二型糖尿病中的新用途。
2.尼达尼布乙磺酸盐在制备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.尼达尼布乙磺酸盐在制备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.尼达尼布乙磺酸盐在制备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：该药包含尼达尼布乙磺酸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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| WO2015179436A1 (en) * | 2014-05-19 | 2015-11-26 | Sanford-Burnham Medical Research Institute | Inflammation therapy using mekk3 inhibitors or blocking peptides |
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| WO2015179436A1 (en) * | 2014-05-19 | 2015-11-26 | Sanford-Burnham Medical Research Institute | Inflammation therapy using mekk3 inhibitors or blocking peptides |
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