CN108685925B - 一种化合物AB-38b在制备治疗糖尿病肾病药物方面的应用 - Google Patents
一种化合物AB-38b在制备治疗糖尿病肾病药物方面的应用 Download PDFInfo
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Abstract
本发明涉及一种化合物AB‑38b在制备治疗糖尿病肾病药物方面的应用,属于医药技术领域。本发明提供的一种药物组合物,以式AB‑38b所示的化合物或其药学上可接受的盐,为活性成分或主要活性成分,辅以药学上可接受的载体。
Description
技术领域
本发明属于医药技术领域,具体涉及一种化合物AB-38b通过抗炎作用在治疗糖尿病肾病药物方面的应用。
背景技术
糖尿病肾病(DN)是糖尿病最常见的并发症之一,目前已成为终末期肾病的主要原因。众多研究表明,高血糖是DN形成过程中的主要始动因子,高血糖引起的持续炎症状态是糖尿病肾病形成的重要病理生理基础,炎症反应参与了DN的发生发展。
Nod样受体蛋白3即NLRP3炎性小体,是胞内危险相关分子模式(DAMPs)的感受器,主要是由NLRP3、凋亡相关的斑点样蛋白(ASC)和天冬氨酸特异性半胱氨酸蛋白酶-1(caspase-1)等相互结合而形成。NLRP3炎性小体的激活以及各种炎性因子的释放能刺激肾小球系膜的肥大和增殖,从而引起系膜外基质的扩张,最终诱发肾小球硬化和肾间质纤维化,导致DN形成。因此,寻找调控NLRP3炎性小体的有效机制,对筛选DN的防治药物具有非常重要的意义。
转录因子NF-E2相关因子2(Nrf2)是细胞内重要的转录因子。高糖情况下,肾脏细胞内高血糖、高尿酸等状态,可代偿性激活Nrf2信号通路,Nrf2转位进入核内,转录表达一系列下游的血红素加氧酶(HO-1)、醌氧化还原酶1(NQO-1)等,Nrf2及其下游信号分子,能够抑制一些免疫介质如白介素(ILs)、细胞粘附因子(ICAM-1)、趋化因子(MCP-1),生长因子等的产生和聚集从而产生抗炎作用,以维持细胞正常的活性。由此可见,Nrf2信号通路在阻止细胞内炎性因子过表达、保证细胞活性和机体正常的代谢水平上发挥重要作用。值得注意的是,高糖引起的Nrf2上调所产生的对细胞与组织的保护作用并不能阻止DN的发生。在糖尿病的初期,代偿性增高的Nrf2能抑制而保护肾脏;但在中后期,Nrf2的作用被饱和,Nrf2被消耗的同时又不能被持续激活,其抗炎的作用远不及持续的高血糖带来的伤害,因此,短暂的保护作用过后,DN的情况将继续恶化。明确Nrf2激活机制,进而寻找Nrf2激动剂,使之在DN发展过程中均能上调Nrf2,从而延缓DN的进展,是开发DN防治药物可行的思路。
发明内容
本发明的目的是在现有技术的基础上,提供化合物AB-38b在治疗糖尿病肾病药物方面的应用。
本发明的另一目的是提供上述化合物AB-38b在制备抗炎药物方面的应用。
本发明的技术方案如下:
式AB-38b所示的化合物或其药学上可接受的盐,在制备治疗糖尿病肾病药物方面的应用,
在一种优选方案中,本发明提供了一种药物组合物,它以本发明的化合物或其药学上可接受的盐为活性成分或主要活性成分,辅以药学上可接受的载体。进一步的,该药物组合物可以制成液体制剂、固体制剂或半固体制剂。更进一步的,该药物组合物可以制成注射剂、片剂、胶囊剂、口服液或外用贴剂。
式AB-38b所示的化合物或其药学上可接受的盐在制备抗炎药物方面的应用。
在一种优选方案中,本发明提供了一种具有抗炎作用药物组合物,它以本发明的化合物或其药学上可接受的盐为活性成分或主要活性成分,辅以药学上可接受的载体。进一步的,该药物组合物可以制成液体制剂、固体制剂或半固体制剂。更进一步的,该药物组合物可以制成注射剂、片剂、胶囊剂、口服液或外用贴剂。
采用本发明的技术方案,优势如下:
本发明提供的药物组合物AB-38b具备防治糖尿病肾病的作用。通过动物实验,显示AB-38b对2型糖尿病小鼠模型的肾功能状态、肾组织形态具有改善作用;对2型糖尿病小鼠模型引起的炎症反应具有抑制作用;对正常小鼠的肾功能具有保护作用。本发明为开发DN防治药物提供了可行思路。
附图说明
图1为实施例1中各实验组小鼠生化指标与肾功能指标比较图;
其中,A:血糖水平;B:低密度脂蛋白水平;C:高密度脂蛋白水平;D:总胆固醇水平;E:血清中肌酐水平;F:血清中尿素氮水平;
图2为实施例2中各实验组小鼠肾组织形态比较图;
其中,A:糖原染色;B:天狼星红染色;C:层黏连蛋白免疫组织化学;D:四型胶原蛋白免疫组织化学;
图3为实施例3中各实验组小鼠炎性因子表达比较图;
其中,A:白介素-18水平;B:白介素-1β水平;
图4为实施例4中化合物AB-38b对各肾小球系膜细胞组中Nrf2通路的激动作用;
其中,A:Nrf2免疫荧光;B:Nrf2及其下游NQO-1、HO-1蛋白表达水平;
图5为实施例5中各肾小球系膜细胞组NLRP3炎性小体、caspase-1、IL-18、IL-1β蛋白表达水平;
图6为实施例6中各肾小球系膜细胞组层黏连蛋白、四型胶原免疫荧光比较图;
其中,A:层黏连蛋白免疫荧光;B:四型胶原免疫荧光。
具体实施方式
下面结合实施例和附图对本发明进行详细描述,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。
实施例1:化合物AB-38b对2型糖尿病小鼠各生化指标和肾功能指标的影响。
1、实验动物
雌性C57BL/6J小鼠。饲养条件:温度23±2℃、湿度55±5%以及12h的日照。
2、实验动物2型糖尿病模型的建立、分组及给药
小鼠分为空白对照组(NC)、2型糖尿病模型组(DN)、2型糖尿病模型给予化合物AB-38b低剂量组(ABL)、中剂量组(ABM)、高剂量组(ABH)、2型糖尿病模型给予白藜芦醇(RES)阳性对照组、正常给予AB-38b高剂量组(NC+ABH)。小鼠高脂饲养8周后,腹腔注射STZ(100mg/kg)造模。通过灌胃给药AB-38b(10mg/kg、20mg/kg、40mg/kg)、RES(40mg/kg)。
3、血液标本的制备
小鼠称重后摘眼球取血,3000g离心15min,收集血清并分装,-20℃保存,用于后续生化指标的检测。
4、试剂及仪器
试剂:低密度脂蛋白胆固醇(LDL-C)测定试剂盒、高密度脂蛋白胆固醇(HDL-C)测定试剂盒、总胆固醇(T-CHO)测定试剂盒、肌酐(CRE)测定试剂盒、尿素氮(BUN)测定试剂盒,以上均购自南京建成生物工程研究所。
仪器:多功能微孔板读数仪
5、数据处理
采用SPSS16.0分析统计软件,实验数据以均数±标准差(Mean±SD)表示。统计分析采用单因素方差分析(One Way ANOVA),多个实验组与一个对照组比较采用Dunnet t检验,实验组之间两两比较采用q检验,P<0.05表示差异有统计学意义。
6、低密度脂蛋白胆固醇(LDL-C)的测定
充分混合,37℃孵育5min,酶标仪测定波长546nm处的吸光度A2
7、高密度脂蛋白胆固醇(HDL-C)的测定
充分混合,37℃孵育5min,酶标仪测定波长546nm处的吸光度A2
8、总胆固醇(T-CHO)的测定
充分混合,37℃孵育10min,用酶标仪测定波长510nm处的吸光度
9、血肌酐(CRE)的测定(苦味酸法)
在碱性环境中样本中的肌酐与苦味酸生成橙红色的复合物,在510nm波长处测定吸光度值,计算其含量。
操作方法:
(1)样品前处理:取血清9μl加入90μl试剂二(钨酸蛋白沉淀剂)3500转/分,离心10分钟,取上清按下表操作
(2)按照下表测定样本中肌酐含量
充分混合,37℃水浴10min,用酶标仪测定波长510nm处的吸光度OD值。
(3)实验结果的计算方法
10、尿素氮(BUN)的测定(脲酶法)
尿素在脲酶作用下水解产生氨离子和二氧化碳,氨离子在碱性介质中与酚显色剂生成蓝色物质,该物质生成量与尿素含量成正比,在640nm处测OD值。
操作方法:
(1)按照下表测定样本中尿素氮含量
充分混合,37℃水浴10min,用酶标仪测定波长510nm处的吸光度OD值。
(3)实验结果的计算方法
11、实验结果
各生化指标和肾功能指标实验结果如图1A-F所示,与正常组相比,2型糖尿病模型组小鼠的血糖、低密度脂蛋白、高低密度脂蛋白、总胆固醇、肌酐、尿素氮水平均明显增加,给予化合物AB-38b低中高剂量后血糖、高低密度脂蛋白、总胆固醇、肌酐、尿素氮水平较模型组逐渐降低,且有一定的剂量依赖性。由此表明,化合物AB-38b能够改善2型糖尿病小鼠肾功能的损伤状态。与正常组相比,正常给予化合物AB-38b组血糖、高低密度脂蛋白、总胆固醇各生化指标和肌酐、尿素氮肾功能指标没有明显变化,表明化合物AB-38b对正常小鼠的各生化指标和肾功能指标没有毒副影响。
结论:化合物AB-38b能够改善2型糖尿病模型小鼠的肾功能状态。
实施例2:化合物AB-38b对2型糖尿病小鼠各肾组织形态学的影响。
1、小鼠肾脏组织标本的制备
小鼠处死,迅速摘取两侧肾脏并除去包膜,预冷的生理盐水冲洗、滤纸吸干后分别称重。在冰板上去除肾脏髓质部分,并将一侧肾脏分成若干份,一份置于4%多聚甲醛中固定用于后续病理及组织染色;一份切成1mm3左右的立方体并放于2.5%戊二醛溶液中固定,用于后续透射电镜观察;剩余的肾脏组织置于液氮中速冷2h后放于-80℃长期保存。
2、PAS染色
(1)1%过碘酸溶液配制:称量过碘酸(HIO4)1.0g,加蒸馏水100ml,待溶解后置于4℃冰箱避光保存。
(2)1mol/L盐酸配制:37%浓盐酸10ml,加入蒸馏水110ml配制而成。
(3)肾脏组织在4%甲醛中固定24h以上,之后进行石蜡包埋。采用全自动脱水机进行组织脱水,步骤如下:75%乙醇1h→85%乙醇1h→95%乙醇1h→95%乙醇1h→100%乙醇1h→100%乙醇1h→二甲苯1h→二甲苯1h→石蜡1h→石蜡1h→石蜡1h。
(4)染色方法:
①各组大鼠肾皮质进行石蜡包埋,切片4μm,将切片在60℃恒温箱中烘烤2h;
②依次在二甲苯I、二甲苯II中脱蜡各10min,分别于100%,95%,80%,70%乙醇中各5min后进入蒸馏水;
③室温入1%过碘酸溶液,氧化8min;
④流水冲洗5min,再用双蒸水浸洗2次,每次2min;
⑤室温入Schiff试剂,染色15min;
⑥自来水冲洗5min,双蒸水浸洗3min;
⑦放入苏木素中复染核2min;
⑧自来水冲洗5min,换入蒸馏水;
⑨用1%盐酸酒精分化5s;
⑩自来水洗5min;
常规脱水,透明,中性树胶封片。
PAS阳性物质(多糖和糖原)呈红色或紫红色,核呈蓝色。PAS染色法是观察肾脏结构,尤其是肾小球系膜、基底膜以及肾小管结构的主要方法。取PAS染色切片,每例切片观察5个互不重叠肾间质视野,测定每个视野紫红区面积与视野面积比,取其平均值,评价肾小球及肾小管基底膜增厚程度。
3、天狼星红染色
(1)将石蜡包埋的肾组织制成3μm切片;
(2)60℃恒温箱中烘烤2h后依次在二甲苯I、二甲苯II中脱蜡各15min,分别于100%,95%,80%,70%乙醇中各3min;
(3)自来水冲洗3min,冲洗3遍;
(4)天狼星红染色液中染1h;
(5)自来水洗5min;
(6)Mayer苏木素复染细胞核1min;
(7)自来水洗10min;
(8)乙醇脱水,封片。
天狼星红染色肾间质红染区主要为胶原成分,包括I型胶原、III型胶原等。取天狼星红染色切片,每例切片观察5个互不重叠的肾间质视野,测定每个视野胶原红染区面积与视野面积比,取其平均值,以对肾间质胶原的分布和程度进行定量分析
4、Laminin和Collagen IV的免疫组织化学染色
(1)将石蜡包埋的肾组织制成4μm切片;
(2)60℃恒温箱中烘烤2h后依次在二甲苯I、二甲苯II中脱蜡各10min,分别于100%,95%,80%,70%乙醇中各5min后,PBS冲洗3次,每次3min;
(3)3%H2O2室温孵育10min,以消除内源性过氧化物酶的活性,PBS冲洗3次,每次3min;
(4)0.4%胃蛋白酶修复30min,PBS冲洗3次,每次3min;
(5)2%BSA室温封闭20min,除去多余液体;
(6)将抗Laminin与Collagen IV抗体按照1:100比例用一抗稀释液进行稀释,4℃孵育过夜;
(7)37℃复温30min,PBS冲洗3次,每次3min;
(8)滴加试剂一室温20min,PBS冲洗3次,每次3min;
(9)滴加试剂二室温25min,PBS冲洗3次,每次3min;
(10)DAB显色:取1ml试剂二(DAB底物缓冲液),加入1滴(约50μl)试剂一(DAB浓缩液),混合均匀(现用现配),配后避光保存,30min内使用。拭去组织周围多余残留液,加50μlDAB显色液铺满组织片,在显微镜下观察显色程度,及时终止反应,PBS冲洗;
(11)将组织片加苏木素复染5min,双蒸水冲洗;
(12)1%盐酸酒精分化3s,双蒸水冲洗;
(13)常规脱水,透明,中性树胶封片。
免疫组化染色主要是观察Laminin和Collagen IV在肾脏中表达,阳性着染可呈棕黄色、褐色。取免疫组化染色切片,每例切片观察5个互不重叠的肾间质视野,使用计算机图像分析软件Image-pro plus 6.0以积分光密度(IOD)为指标对各组免疫组化阳性染色进行定量分析。平均积分光密度即IOD与图片面积(Area)的比值,其大小反映了免疫反应物的表达强度。
5、实验结果
糖原染色结果如图2A所示,与正常组相比,模型组小鼠肾皮质中,尤其是在肾小管基底膜以及肾小管间质区域,糖原沉积增多,伴随层黏连蛋白表达增多。低、中、高剂量的化合物AB-38b组以及白藜芦醇对照组的小鼠都表现出这些区域中糖原与层黏连蛋白沉积的减少,正常给予AB-38b与正常组相比没有明显差异。表明化合物AB-38b可以改善糖尿病所致的大鼠肾间质纤维化。
天狼星红染色如图2B所示,运用肾脏切片的天狼星红染色评估各组小鼠的肾纤维化水平。结果显示,与正常组小鼠相比,模型组小鼠肾皮质中肾小球及肾间质区域天狼星红染色阳性区域面积显著增多,表明糖尿病小鼠肾脏中胶原蛋白沉积增加。与模型组相比,给予低、中、高剂量的化合物AB-38b组以及白藜芦醇对照组可以显著减少胶原蛋白沉积的现象,正常给予AB-38b与正常组相比没有明显差异。由此可知,化合物AB-38b能够使小鼠肾皮质中胶原的过度表达得到了抑制,从而减轻了肾纤维化的程度。
层黏连蛋白(Laminin)和IV型胶原的免疫组化结果如图2C-D所示,IV型胶原和层黏连蛋白是细胞外基质的重要组成成分,运用层黏连蛋白的免疫组化来评估糖尿病大鼠肾皮质中细胞外基质的水平。与正常组相比,模型组小鼠肾皮质中,尤其是在肾小管基底膜以及肾小管间质区域,IV型胶原和层黏连蛋白表达增多。低、中、高剂量的化合物AB-38b组以及白藜芦醇对照组的小鼠都表现出这些区域中IV型胶原和层黏连蛋白沉积的减少。综上所述,化合物AB-38b可以改善糖尿病小鼠的肾间质纤维化。
结论:化合物AB-38b可以改善糖尿病小鼠的肾间质纤维化。
实施例3:化合物AB-38b对糖尿病小鼠炎性因子的影响。
1、试剂
小鼠白介素18试剂盒Mouse IL-18 ELISA Kit、小鼠白介素1beta ELISA试剂盒/Mouse IL-1βELISA Kit,均购于杭州联科生物。
2、IL-18ELISA Kit操作步骤
(1)准备好所有需要的试剂及工作浓度标准品。
(2)将不需要的板条拆卸下来,放回装有干燥剂的铝箔袋,重新封好封口。
(3)加入300μl 1×洗液静置浸泡30秒。为了获得理想的实验结果浸泡是必须的。弃掉洗液之后,在吸水纸上将微孔板拍干。洗板完成之后,请立即使用微孔板,不要让微孔板干燥。
(4)复孔加入50μl 1×检测缓冲液和50μl 2倍倍比稀释的标准品。空白孔复孔加入50μl 1×检测缓冲液和50μl标准品稀释液。
(5)样本孔加入80μl 1×检测缓冲液和20μl样本。
(6)每孔加入50μl稀释的检测抗体。保证步骤4、5、6连续加样,不要间断。加样过程在15分钟内完成。
(7)使用封板膜封板。300转/分钟振荡,室温孵育2小时。
(8)弃掉液体,每孔加入300μl洗液洗板,洗涤6次。每次洗板,在吸水纸上拍干。为获得理想的实验性能,必须彻底移除残留液体。
(9)每孔加入100μl显色底物TMB,避光,室温孵育5至30分钟。
(10)每孔加入100μl终止液。颜色由蓝色变为黄色。如果颜色呈现绿色或者颜色的变化明显不均匀,请轻轻叩击板框,充分混匀。
在30分钟之内,使用酶标仪进行双波长检测,测定450nm最大吸收波长和570nm或630nm参考波长下的OD值。校准后的OD值为450nm的测定值减去570nm或630nm的测定值。仅使用450nm测定会导致OD值偏高,并且准确度降低。
3、IL-1βELISA Kit操作步骤
(1)取250μl浓缩的小鼠IL-1β标准品,加入250μl标准品稀释液,作为标准曲线的最高浓度(1000pg/ml)。在每一个试管中加入250μl标准品稀释液。使用高浓度标准品做1:1系列稀释。每次移液时,请确保充分混匀。以标准品稀释液作为标准曲线的零浓度。
(2)准备好所有需要的试剂及工作浓度标准品。
(3)将不需要的板条拆卸下来,放回装有干燥剂的铝箔袋,重新封好封口。
(4)加入300μl 1×洗液静置浸泡30秒。为了获得理想的实验结果浸泡是必须的。弃掉洗液之后,在吸水纸上将微孔板拍干。洗板完成之后,请立即使用微孔板,不要让微孔板干燥。
(5)复孔加入100μl 2倍倍比稀释的标准品。空白孔复孔加入100μl标准品稀释液。样本孔加入80μl 1×检测缓冲液和20μl样本。
(6)每孔加入50μl稀释的检测抗体。保证步骤4、5、6连续加样,不要间断。加样过程在15分钟内完成。
(7)使用封板膜封板。300转/分钟振荡,室温孵育1.5小时。
(8)弃掉液体,每孔加入300μl洗液洗板,洗涤6次。每次洗板,在吸水纸上拍干。为获得理想的实验性能,必须彻底移除残留液体。
(9)每孔加入100μl稀释的辣根过氧化物酶标记的链霉亲和素。
(10)使用新的封板膜封板。300转/分钟振荡,室温孵育30分钟。
(11)重复步骤8。
(12)每孔加入100μl显色底物TMB,避光,室温孵育5至30分钟。
(13)每孔加入100μl终止液。颜色由蓝色变为黄色。如果颜色呈现绿色或者颜色的变化明显不均匀,请轻轻叩击板框,充分混匀。
(14)在30分钟之内,使用酶标仪进行双波长检测,测定450nm最大吸收波长和570nm或630nm参考波长下的OD值。校准后的OD值为450nm的测定值减去570nm或630nm的测定值。仅使用450nm测定会导致OD值偏高,并且准确度降低。
4、实验结果
IL-18与IL-1β实验结果如图3A-B所示,与正常组相比,模型组小鼠IL-18与IL-1β炎性因子明显增加,给予化合物AB-38b低、中、高剂量后炎性因子IL-18与IL-1β较模型组逐渐降低,且有一定的剂量依赖性。结果表明,化合物AB-38b能够改善2型糖尿病小鼠肾炎症状态。与正常组相比,空白给予化合物AB-38b组IL-18与IL-1β炎性因子没有明显变化,表明,化合物AB-38b对正常小鼠的炎性因子的表达没有影响。
结论:化合物AB-38b能够改善2型糖尿病模型小鼠的肾炎症状态。
实施例4:化合物AB-38b对肾小球系膜细胞中Nrf2信号的激动作用。
1、细胞培养、分组
细胞分为正常组、过氧化氢组(400μM H2O2)、高糖组(30mM)、AB-38b低剂量组(2.5μM)、AB-38b中剂量组(5μM)、AB-38b高剂量组(10μM)、白藜芦醇组(10μM RES)培养24h。
2、免疫荧光实验检测Nrf2的入核情况
(1)盖玻片灭菌预处理后,放入12孔板中。盖玻片上种肾小球系膜细胞,置于细胞培养箱中培养;
(2)24h后,吸掉培养液,每孔加入1mL 1×PBS液洗细胞,弃去PBS。分为NG组、400μMH2O2组、30mM高糖组、30mM高糖分别加2.5μM、5μM、10μM浓度的AB-38b组、30mM高糖加10μM浓度的白藜芦醇组继续培养;
(3)24h后,吸掉培养液,每孔加入1mL 1×PBS液洗3次细胞,然后每孔用1mL 4%多聚甲醛-20℃固定15min;
(4)固定完成后,吸掉4%多聚甲醛,用1×PBS缓冲液洗细胞3次,每次5min,后用0.1%Triton-100处理15min;
(5)用1×PBS缓冲液洗细胞3次,每次5min。PBS洗完以后,加入1mL含3%BSA的1×PBS溶液封闭;
(6)30min后,加入一抗(1:200),4℃孵育3h,然后用1×PBS缓冲液洗3次,每次5min;
(7)加入二抗(1:100)于37℃孵育,1h后用1×PBS缓冲液洗3次,每次5min;
(8)洗完以后在载玻片上轻轻加上1滴DAPI染2min后,用1×PBS缓冲液洗3次,然后把盖破片取出,将有细胞的一面与加了一滴抗荧光淬灭剂的载破片贴在一起,排除气泡后,避光置于4℃待测。
3、蛋白质免疫印迹实验
(1)定蛋白浓度
将收取细胞用裂解液于冰上充分裂解30min,每10min对其涡旋混匀一次。然后于4℃条件下,12000rpm高速离心15min后尽可能吸取上清,之后用BCA工作液定浓度。
(2)蛋白样品加5×蛋白上样缓冲液,于95℃金属浴变性10min。进行SDS-聚丙烯酰胺凝胶电泳,将蛋白转印到NC膜上;
(3)转膜完成后将NC膜置于5%的脱脂牛奶中封闭60min。将含目的蛋白条带的NC膜置于自封袋中,一抗用一抗稀释液1:1000稀释至工作液浓度,4℃条件下过夜或室温摇床孵育3~4h,再用TBST洗膜3次,5min/次。
(4)用二抗稀释液稀释二抗至工作浓度,按前法封袋,室温摇1h后弃二抗,TBST溶液洗2次,5min/次;TBS溶液洗1次,5min/次;
(5)奥得赛近红外双色荧光成像系统对条带进行扫描,软件Image J对条带进行定量分析,
4、实验结果
如图4A免疫荧光结果表明,与正常组相比,在高糖和过氧化氢处理条件下,由于代偿作用Nrf2表达有一定的增加,加入化合物AB-38b后,Nrf2的表达比高糖组的增加更明显,且Nrf2的入核情况明显增加。由此表明,化合物AB-38b能够促进Nrf2的表达及入核。如图4B蛋白表达结果,与正常组相比,在高糖和过氧化氢处理条件下,由于代偿作用Nrf2以及下游NQO-1、HO-1蛋白表达有一定的增加,与高糖组相比,AB-38b能够进一步促进Nrf2以及下游NQO-1、HO-1的蛋白表达水平,表明化合物AB-38b对肾小球系膜细胞中Nrf2及其下游靶基因有一定激动作用。
结论:化合物AB-38b对肾小球系膜细胞中Nrf2及其下游靶基因具有激动作用。
实施例5:化合物AB-38b对高糖诱导的肾小球系膜细胞中NLRP3炎性小体及炎性因子的抑制作用。
1、细胞培养、分组
细胞分为正常组、过氧化氢组(400μM H2O2)、高糖组(30mM)、AB-38b低剂量组(2.5μM)、AB-38b中剂量组(5μM)、AB-38b高剂量组(10μM)、N-乙酰半胱氨酸组(10mM NAC)培养24h。
2、实验方法
蛋白质免疫印迹实验检测相关蛋白的表达,具体步骤同实施例4。
3、实验结果
图5结果显示高糖和氧化应激条件下肾小球系膜细胞中NLRP3炎性小体蛋白表达水以及炎性因子caspase1、白介素18、白介素1β蛋白表达水平均增加,表明高糖刺激和氧化应激能引起细胞的炎症反应。给予低、中、高三组剂量的AB-38b后,以上炎性标志物的蛋白表达下降,表明化合物AB-38b对高糖诱导的肾小球系膜细胞中NLRP3炎性小体及炎性因子有抑制作用。
结论:化合物AB-38b对高糖诱导的肾小球系膜细胞中NLRP3炎性小体及炎性因子具有抑制作用。
实施例6:化合物AB-38b对高糖诱导的肾小球系膜细胞中IV型胶原和层粘连蛋白的抑制作用。
1、细胞培养、分组
细胞分为正常组、过氧化氢组(400μM H2O2)、高糖组(30mM)、AB-38b低剂量组(2.5μM)、AB-38b中剂量组(5μM)、AB-38b高剂量组(10μM)、N-乙酰半胱氨酸组(10mM NAC)培养24h。
2、实验方法
免疫荧光实验检测相关蛋白的表达,具体步骤同实施例4。
3、实验结果
结果见图6A-B,与正常组相比,在高糖和过氧化氢处理条件下,IV型胶原和层粘连蛋白的蛋白表达明显增加,加入低、中、高剂量的化合物AB-38b后,IV型胶原和层粘连蛋白的蛋白表达逐渐降低,表明AB-38b对高糖诱导的肾小球系膜细胞中IV型胶原和层粘连蛋白的蛋白表达增加有抑制作用。表明AB-38b可以改善糖尿病肾病细胞外基质堆积,抑制肾小球硬化和肾间质纤维化,具备防治糖尿病肾病作用。
结论:化合物AB-38b可以改善糖尿病肾病细胞外基质堆积,抑制肾小球硬化和肾间质纤维化,具备防治糖尿病肾病作用。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (8)
1.式AB-38b所示的化合物或其药学上可接受的盐,在制备治疗糖尿病肾病药物方面的应用,
2.一种药物组合物,它以权利要求1所述的式AB-38b化合物或其药学上可接受的盐为活性成分或主要活性成分,辅以药学上可接受的载体。
3.根据权利要求2所述的药物组合物,其特征在于所述药物组合物制成液体制剂、固体制剂或半固体制剂。
4.根据权利要求3所述的药物组合物,其特征在于所述药物组合物制成注射剂、片剂、胶囊剂、口服液或外用贴剂。
5.式AB-38b所示的化合物或其药学上可接受的盐,在制备抗炎药物方面的应用,
6.一种具有抗炎作用的药物组合物,它以权利要求5所述的式AB-38b化合物或其药学上可接受的盐为活性成分或主要活性成分,辅以药学上可接受的载体。
7.根据权利要求6所述的药物组合物,其特征在于所述药物组合物制成液体制剂、固体制剂或半固体制剂。
8.根据权利要求7所述的药物组合物,其特征在于所述药物组合物制成注射剂、片剂、胶囊剂、口服液或外用贴剂。
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