CN113332275A - 穿心莲内酯在制备促进白色脂肪棕色化的产品中的应用 - Google Patents
穿心莲内酯在制备促进白色脂肪棕色化的产品中的应用 Download PDFInfo
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Abstract
本发明涉及一种天然产物的药物应用技术领域,具体涉及穿心莲内酯在制备促进白色脂肪棕色化的产品中的新用途。本发明中穿心莲内酯将储能组织白色脂肪组织激活转化成能量代谢的棕色脂肪组织,并激活棕色脂肪功能,提高棕色脂肪活性,促进脂肪转化为热能耗散,保持机体能量平衡,并且还能调节机体脂肪分布,减少总体脂肪重量、降低体重的效果。因此,所述穿心莲内酯、含有穿心莲内酯的提取物或其制剂在调节脂肪分布,预防或治疗肥胖症相关代谢性疾病具有广阔的应用前景。
Description
技术领域
本发明涉及一种可激活过氧化物酶体增殖物激活受体α(PPARα),促进白色脂肪棕色化的药物或保健食品或食品添加剂,特别涉及一种中药穿心莲提取成分在促进白色脂肪棕色化的相关产品中的应用。
背景技术
脂肪代谢紊乱是代谢综合征如肥胖,2型糖尿病,脂肪肝等疾病的特征性病理改变。脂肪代谢紊乱的患者其甘油三酯在脂肪组织存储异常,进而导致脂质沉积在身体不同部分,并逐渐导致胰岛素抵抗。随着脂肪的日益增加和较低利用率会使得脂肪组织逐步增大,导致肥胖的形成。随着经济的发展和科技的变革,越来越多国家的肥胖率均呈快速上升趋势。
脂肪组织可通过内皮细胞、成纤维细胞、白细胞和巨噬细胞分泌脂肪细胞因子,包括蛋白质、激素、瘦素,脂联素,抵抗素和炎症因子等,对脑、肝、心脏、骨骼肌和免疫系统等产生影响。脂肪的过度累积将导致脂肪细胞因子在机体中的代谢失衡,诱导低度慢性炎症、糖脂代谢紊乱和心血管疾病的发生。研究证实脂肪的过度堆积(肥胖)与高脂血症、脂肪肝、糖尿病、高血压、心脑血管疾病等多种代谢性疾病和癌症、抑郁症等非代谢性疾病密切相关。肥胖会导致多种并发症,很大程度上会提高个体的健康风险,严重影响人体的身心健康和生活质量。
脂肪组织是重要的新陈代谢器官,根据形态及功能特点主要可以分为白色脂肪组织(White adipose tissue,WAT)和棕色脂肪组织(Brown adipose tissue,BAT)。白色脂肪和棕色脂肪二者不但解剖位置、形态特征完全不同,而且在能量代谢方面的作用截然相反:WAT的主要功能是以脂滴(甘油三酯)的形式储存能量,而BAT则通过消耗能量、产热来维持机体的能量代谢平衡。
白色脂肪组织是构成正常成年人的主要脂肪组织类型,约占健康成年人体重的20%,主要分布在腹部皮下、腹股沟、肠系膜和内脏周围等处,在功能上主要是通过储存多余的甘油三酯和脂肪动员,参与调控人体的物质和能量代谢。白色脂肪细胞有一个大的单房脂滴,其特征是线粒体数量较少,肥胖症主要取决于WAT的数目和体积。而棕色脂肪细胞颜色较深,细胞内含有多房小脂滴,富含线粒体以及解偶联蛋白(uncouple protein 1,ucp1),其主要功能是产生热量来维持体温,主要分布在颈部、锁骨上缘、纵隔、肩胛间区和脊柱两侧的皮下。棕色脂肪细胞可通过CD36、FATP1和FATP4等转运脂肪酸的因子吸收脂肪酸,脂肪酸通过相应的生化反应变成甘油三酯,之后存于脂滴中,冷刺激可激活棕色脂肪组织,促使棕色脂肪对甘油三酯的吸收和利用。棕色脂肪含有丰富的线粒体,可氧化消耗脂肪酸和甘油三脂,大量提供能量,维持体温。随着研究的深入,发现在寒冷刺激或β肾上腺素能受体激动剂的作用下,WAT中会出现散在的棕色化的脂肪细胞,该细胞内线粒体数量增加,氧化磷酸化加快,单一大脂滴变成多个小脂滴。这些细胞的特性介于白色脂肪细胞与棕色脂肪细胞之间,后来将这种细胞命名为米色脂肪细胞(brite or beige cell),这种分化过程称为白色脂肪棕色化。
线粒体是细胞进行能量代谢的核心场所,棕色和米色脂肪细胞中线粒体含量丰富,可以进行适应性产热将脂肪转化为热能耗散,保持机体能量平衡,这一过程需要线粒体内膜上的UCP1参与。UCP1能消除线粒体内膜的质子电化学梯度,过高的质子堆积会致使线粒体呼吸作用中的氧化磷酸化解偶联,抑制ATP的合成。因此白色脂肪组织是机体的主要储能组织,棕色脂肪组织是机体能量代谢失衡的重要调控者,促进机体白色脂肪“棕色化”并激活棕色脂肪功能可促进能量消耗、抑制肥胖。目前亟需寻找新的药物或手段激活体内棕色脂肪活性、促进白色脂肪“棕色化”,从而为肥胖及其相关代谢性疾病的防治提供新的策略。
自19世纪使用药物治疗肥胖开始,大量减肥药物因安全性问题而相继退市。现有的减肥药多是针对限制能量摄入而开发的,如奥利司他通过可逆的抑制胃肠脂肪酶减少脂肪吸收,但长期服用会引起肠胃胀气、腹部疼痛、消化不良。已有一些研究发现可以激活产热性脂肪组织的化合物,如辣椒素及其类似物、黄连素、白藜芦醇、水杨酰水杨酸等,进一步说明了产热性脂肪组织通过化学手段激活,具有作为治疗靶点的可行性。
穿心莲内酯(andrographolide)是从爵床科草本穿心莲(Andrographispanicultata(Burm.f)Nees)全草中提取的主要有效成分,具有抗炎、抗病毒、保肝利胆、免疫调节、抗肿瘤、治疗心脑血管疾病、抗糖尿病、神经保护、中止妊娠等多种药理作用。
目前,穿心莲内酯促进白色脂肪棕色化,调节机体脂肪分布,减少总体脂肪重量,治疗肥胖相关疾病的研究尚未见诸报道。
发明内容
本发明的首要目的是提供一种穿心莲内酯在制备促进白色脂肪棕色化的产品中的应用,即穿心莲内酯作为促进白色脂肪棕色化的新用途。穿心莲内酯将储能组织白色脂肪组织激活转化成能量代谢的棕色脂肪组织,并激活棕色脂肪功能,提高棕色脂肪活性,促进脂肪转化为热能耗散,保持机体能量平衡。内脏脂肪的堆积与炎症反应、代谢性疾病密切相关,穿心莲内酯减少内脏白色脂肪,调节脂肪在机体中的分布,健身和塑形;尤其是调节白色脂肪在内脏中的分布,减轻内脏脂肪组织脂沉积,从而改善脂诱性胰岛素抵抗等代谢性疾病。
穿心莲内酯(3-[2-[decahydro-6-hydroxy-5-(hydroxy-methyl)-5,8a-dimethyl-2-methylenenaphthalenyl]ethylidene]dihydro-4-hydroxyfuran-2(3H)-one),分子式为C20H30O5;分子量为M=350.44;其结构式如下:
为实现本发明的目的,本发明提供一种穿心莲内酯在制备促进白色脂肪棕色化的产品中的应用,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
其中,所述促进白色脂肪棕色化为激活产热性脂肪组织,调节脂肪在体内的分布。
特别是,所述产热性脂肪组织为棕色脂肪组织。本发明的穿心莲内酯将白色脂肪组织转化成棕色脂肪组织,进行适应性产热将脂肪转化为热能耗散,保持机体能量平衡;抑制ATP合成;预防或治疗脂肪代谢紊乱。
穿心莲内酯激活棕色脂肪功能,提高棕色脂肪活性,促进脂肪转化为热能耗散。
穿心莲内酯促进白色脂肪棕色化为激活产热性脂肪组织,调节脂肪在体内的分布。
本发明又一方面提供一种穿心莲内酯在制备用于预防或治疗肥胖症的产品中的应用,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
本发明另一方面提供一种穿心莲内酯在制备用于促进PPARα活性的的药物、食品、保健品、食品添加剂或营养补充剂中的应用。
本发明另一方面提供一种穿心莲内酯在制备用于ATP合成促进剂的药物、食品、保健品、食品添加剂或营养补充剂中的应用。
其中,所述药物由穿心莲内酯和药学上可接受的载体组成。
特别是,所述药物由穿心莲内酯和药学上可接受的载体组成。
尤其是,所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂、乳剂、注射剂、喷雾剂、气雾剂、凝胶型、霜剂、酊剂、巴布剂、橡胶贴膏剂或贴膏剂形式存在。
药学上可接受的载体通常被保健专家认可用于这一目的且作为药剂的非活性成分。有关药学上可接受的载体的汇编可以在《药物赋形剂手册》(Handbook ofPharmaceutical excipients,第2版,由A.Wade和P.J.Weller编辑;AmericanPharmaceutical Association出版,Washington and The Pharmaceutical Press,London,1994)等工具书中找到。
尤其是,所述的载体包括赋形剂,如淀粉、水等;润滑剂,如硬脂酸镁等;崩解剂,如微晶纤维素等;填充剂,如乳糖等;粘结剂,如预胶化淀粉、糊精等;甜味剂;抗氧化剂;防腐剂、矫味剂、香料等;
其中,所述药物是通过经胃肠道给药和非经胃肠道给药途径给药。
特别是,所述的非经胃肠道给药途径选择注射给药、呼吸道给药、皮肤给药、粘膜给药或腔道给药。
其中,非经胃肠道给药药剂选择注射剂、喷雾剂、气雾剂、贴剂等。
特别是,所述的经胃肠道给药制剂选择片剂、胶囊剂、散剂、颗粒剂、丸剂、溶液剂或糖浆剂等。
穿心莲内酯作为PPARα激活剂的应用。
在制备用于预防、缓解和/或治疗由PPARα活性受到抑制所引起的病理性病症的药物、食品、保健品、食品添加剂或营养补充剂中的应用。
PPARα活性受到抑制所引起的病理性病症或症状为腹型肥胖、代谢综合征及相关并发症中的一种或多种;调整脂肪在机体内的分布,还能对健身、体型塑造起作用。
本发明再一方面提供一种促进白色脂肪棕色化的产品,含有穿心莲内酯,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
其中,所述穿心莲内酯的重量与所述产品的总重量之比为0.01~10:100。
本发明又一方面提供一种促进白色脂肪棕色化的方法,包括给予有效量的穿心莲内酯或其药学上可接受的盐。
本发明中,穿心莲内酯的体内治疗有效量为10~90mg/kg·d。
除非另外说明,本文所用的术语“治疗有效量”为需要产生有效作用的药物的用量;“治疗有效量”是可以调整和变化的,最终由医务人员确定,其所考虑的因素包括给药途径和制剂的性质、接受者的体重、年龄等一般情况以及所治疗疾病的性质和严重程度。
与现有技术相比,本发明具有如下的明显优点:
1、本发明对已知化合物穿心莲内酯发掘了新的药用价值,基于促进白色脂肪棕色化这一作用机理,穿心莲内酯表现出降低体重的效果,制备成治疗、预防肥胖相关代谢疾病的药物或保健品。
2、穿心莲内酯将储能组织白色脂肪组织激活转化成能量代谢的棕色脂肪组织,并激活棕色脂肪功能,提高棕色脂肪活性,促进脂肪转化为热能耗散,保持机体能量平衡,并且穿心莲内酯调节脂肪在机体内的分布。
3、本发明的产品原料来源丰富、价廉、临床使用安全,制备工艺简单,可制成各种剂型,且服量小,使用方便,因此易于推广。
附图说明
图1为不同浓度的非诺贝特对PPARα转录活性的影响结果图;
图2为PPARα特异性阻断剂对非诺贝特激活的PPARα转录活性的影响结果图;
图3为不同浓度的穿心莲内酯对PPARα转录活性的影响;
图4为PPARα特异性阻断剂对穿心莲内酯激活的PPARα转录活性的影响;
图5A为3T3-L1脂肪细胞油红O染色结果图,空白对照组;
图5B为穿心莲内酯大剂量(15umol/L)处理3T3-L1脂肪细胞油红O染色结果图;
图5C为穿心莲内酯小剂量(5umol/L)处理3T3-L1脂肪细胞油红O染色结果图;
图6A为正常空白对照组小鼠附睾白色脂肪组织UCP1免疫组织化学染色结果图;
图6B为模型组肥胖小鼠附睾白色脂肪组织UCP1免疫组织化学染色图;
图6C为穿心莲内酯治疗组肥胖小鼠附睾白色脂肪组织UCP1免疫组织化学染色影响图;
图7为穿心莲内酯对肥胖小鼠附睾白色脂肪组织UCP1蛋白表达的影响,A为正常对照组,B为模型组,C为穿心莲内酯治疗组。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
以下通过试验例来进一步阐述本发明所述药物的有益效果,这些试验例包括了本发明药物的药效学试验。
试验例1:穿心莲内酯具有激活PPARα受体的作用
当PPARα受体被相应激动剂激活后,会直接激活PRDM16、白色脂肪组织中UCP-1mRNA及脂联素的表达,促进白色脂肪的棕色化改变。当PPARα基因缺失时,β3肾上腺素能受体激动剂诱导的脂肪组织褐变减弱。UCP1启动子包含PPRE,即PPAR家族分子的共有序列,激活PPARα转录活性可激活UCP1表达。
1.1、细胞质粒
HEK293细胞系从北京协和医学院细胞资源中心(其为细胞系资源的国家基础设施,NSTI的总部)购得。通过PCR和培养检查细胞系没有支原体污染,用PCR鉴定其种源,用STR谱(FBI,CODIS)鉴定细胞系的鉴定结果,所有结果均可在网站(http://cellresource.cn)上查看。
pcDNA3.1(+)-PPARα、pGL4.23-PPRE-luc及pRL-CMV由北京合生基因科技有限公司构建。
1.2药品及主要试剂
穿心莲内酯(Andrographolide,AG):批号:190402;检验编号:C-01-19013,四川文龙药业有限公司。
非诺贝特,上海源叶生物科技有限公司,货号49562-28-9;GW6471,MCE,美国,货号HY-15372;Lipofectamine3000,赛默飞,美国,货号L3000015;双荧光素酶报告基因检测系统,promega,美国,货号E1910
1.3主要仪器
SynergyII酶标仪,bio-tek,美国
2、试验方法
2.1、分组及给药
实验分别设空白对照组,阳性药物组,穿心莲内酯实验组,其中,空白对照组为0.1%DMSO;阳性药物组中阳性药物非诺贝特的浓度分别为5、10、20、30、40uM;穿心莲内酯实验组中穿心莲内酯的浓度分别为5、10、15、20、25uM。阳性药物、穿心莲内酯用0.1%DMSO溶解,制成上述相应的浓度。
2.2质粒瞬时转染HEK293细胞
2.2.1取生长旺盛的HEK293细胞,转染前18-24小时传代,接种至24孔板,细胞密度为2×105/孔;
2.2.2接种24小时后换液,至于培养箱中30min-1h;
2.2.3制备脂质体-DNA复合物,按每孔转染样品量描述:
(A)将0.75ul lipo3000加入至25ul无血清无抗生素的培养基中,充分混匀;
(B)将pcDNA3.1(+)-PPARα、pGL4.23-PPRE-luc、pRL-CMV、P3000按每孔0.2μg、0.2μg、0.02μg、1ul的比例加入至25ul无血清无抗生素的DMEM培养基中,充分混匀;
(C)将B液加入A液中,室温静置15min。
(D)按每孔50ul将脂质体-DNA复合物加入到24孔板培养体系中,轻摇培养板使其混匀,37℃培养24h后给药干预。
2.2.4加药干预:
(A)PPARα特异性激动剂及抑制剂干预
转染24h后,吸弃培养基,加入不同浓度的非诺贝特,浓度设置为0.1%DMSO、5uM、10uM、20uM、30uM、40uM;选择激动PPARα作用最强的浓度,加入PPARα特异性抑制剂GW64710.1uM,每个浓度4个复孔,重复3次。
(B)穿心莲内酯及PPARα特异性抑制剂干预
转染24h后,吸弃培养基,加入不同浓度的穿心莲内酯,浓度设置为0.1%DMSO、5uM、10uM、15uM、20uM、25uM;选择激动PPARα作用最强的浓度,加入PPARα特异性抑制剂GW6471 0.1uM,每个浓度4个复孔,重复3次。
2.2.5荧光素酶报告基因检测:除去培养基,用预冷的1×PBS清洗2遍,不要接触贴壁细胞,尽可能多地将漂洗用PBS吸出,每孔加100μl的1×被动裂解液,室温下震摇15min,将裂解液转移至96孔板中,每孔20ul。应用双荧光素酶活性表达测定试剂盒测定荧光素酶活性,预先将100ul荧光素酶测定试剂(LARII)加入孔板,酶标仪测定萤火虫荧光素活性(F),再加入100ul终止液后测定海肾荧光素活性(R),萤火虫荧光素与海肾荧光素光强的比值(F/R)即为PPARα转录表达强度。
3.检测指标
3.1非诺贝特对PPARα转录活性的影响
共转染48h后,非诺贝特以浓度依赖的方式增加HEK293细胞PPARα的转录活性,非诺贝特30uM时荧光素酶活性达到峰值(图1);其中与空白对照组比较,*p<0.05,**p<0.01;PPARα特异性拮抗剂GW6471与30uM非诺贝特共同孵育后可显著抑制PPARα的转录活性(图2),其中与30uM非诺贝特比较,**p<0.01。以上说明该筛选体系成立且灵敏。
3.2穿心莲内酯对PPARα活性的影响
共转染48h后,穿心莲内酯以浓度依赖的方式增加HEK293细胞荧光素酶的活性,穿心莲内酯15uM时荧光素酶活性达到峰值(图3),其中与空白对照组比较,**p<0.01;GW6471与15uM穿心莲内酯共同孵育后可显著抑制荧光素酶活性(图4),其中与15uM穿心莲内酯比较,**p<0.01。
该实验利用双萤光素酶报告基因分析方法,建立PPARα激动剂体外筛选体系,采用不同浓度的PPARα激动剂非诺贝特干预,PPARα的转录活性随浓度增加而增强,加入特异性阻断剂GW6471后转录活性被抑制,表明该筛选体系成立。穿心莲内酯以浓度依赖的方式增加PPARα的转录活性,并可以被GW6471抑制。以上结果表明,穿心莲内酯具有通过激活PPARα发挥生物学作用,诱导并促进白色脂肪向褐色脂肪组织转化。
试验例2:穿心莲内酯对3T3-L1脂肪细胞脂滴的影响
1、实验材料
1.1、细胞
3T3-L1前脂肪细胞系从北京协和医学院细胞资源中心(其为细胞系资源的国家基础设施,NSTI的总部)购得。通过PCR和培养检查细胞系没有支原体污染,用PCR鉴定其种源,用STR谱(FBI,CODIS)鉴定细胞系的鉴定结果,所有结果均可在网站(http://cellresource.cn)上查看。
1.2、药品
穿心莲内酯同试验例1;
3T3-L1细胞诱导液A(分化诱导液组成为:0.5mmol/L IBMX(sigma:I7018)、1μg/mL胰岛素(sigma:I9278)、0.25μmol/L地塞米松(sigma:D1756),底液为完全培养基)
3T3-L1细胞诱导液B,(1μg/mL胰岛素,底液为完全培养基)
油红O粉末,购自美国sigma公司(货号:O0625)。
2、实验方法
2.1、分组及给药
实验分别设对照组,穿心莲内酯低剂量组,穿心莲内酯高剂量组。
将复苏培养的3T3-L1前脂肪细胞以1×106接种于24孔细胞培养板内,于完全培养基(DMEM培养基,10%FBS(胎牛血清))内培养,接触抑制48h后,各组细胞先加入3T3-L1细胞诱导液A诱导分化48h,然后换3T3-L1细胞诱导液B继续诱导分化,分化48h后可见脂滴形成,吸出处理液后,加入完全培养基、相应的处理药物,对各组细胞进行如下处理:
对照组:DMEM培养基(10%FBS),不添加任何处理药物;
穿心莲内酯低剂量组:DMEM培养基(10%FBS)+5μM穿心莲内酯;
穿心莲内酯高剂量组:DMEM培养基(10%FBS)+15μM穿心莲内酯。
2.2染色
培养12天后,向各组细胞中加入细胞油红O进行染色:
2.2.1.油红O染色液的配制
油红O染色液分储备液和工作液,①油红O储备液的配方:100%异丙醇100mL+0.5g油红O干粉,充分溶解,过滤。②油红O工作液的配方:实验前,将储备液与蒸馏水按3:2稀释,过滤。
2.2.2. 60%异丙醇的配制
60%异丙醇的配方:100%异丙醇60mL+蒸馏水40mL,4度保存。
2.2.3.细胞吸掉培养液,PBS润洗两次,多聚甲醛固定20min,PBS润洗2次。
2.2.4. 60%异丙醇分化1min,油红O染色15min(避光)。
2.2.5. 60%异丙醇润洗一次(30s),PBS洗两次,显微镜下观察并拍照。
3.结果
显微镜下观察并拍照,图5A为对照组,图5B为大剂量穿心莲内酯组,图5C为小剂量穿心莲内酯组。3T3-L1脂肪细胞经油红染色后显示,与对照组比较,穿心莲内酯组脂滴明显较小,多为多房脂滴,而对照组中脂滴较大,表明穿心莲内酯使脂滴呈多房脂滴,诱导和促进白色脂肪组织向褐色脂肪组织转化。
试验例3:穿心莲内酯对LDLR-/-小鼠体重,附睾脂肪体重比和血脂调节作用
1.实验材料
1.1实验动物及饲养
选取40只6周龄LDLR-/-雄性小鼠,体质量(20±2)g,购自江苏集萃药康实验动物有限公司(许可证号SCXK(苏)2018-0008);10只C57BL/6J雄性小鼠,体质量(20±2)g,购自北京维通利华实验动物有限公司(许可证号SCXK(京)2018-0006)。
适应性喂养(SPF级)1周后开始实验。在广安门医院Ⅱ级动物房饲养,每笼5只,给予规律光照(昼夜各12小时),饮用无菌水,环境温度为20±2℃,相对湿度为45%-50%,良好的通风条件。
模型组及治疗组小鼠以高脂饲料饲料喂养6月;空白对照组小鼠以普通饲料喂养6月;高脂饲料购于北京华阜康生物科技有限公司,普通饲料由斯贝福(北京)生物技术有限公司提供。
高脂饲料(又称西方饮食)的配方:酪蛋白、蛋氨酸、玉米淀粉、麦芽糖糊精、蔗糖、纤维素、玉米油、无水奶油、矿物质混合物、碳酸钙、维生素混合物、酒石酸氢胆碱、胆固醇、抗氧化剂等,营养成分如表1。
维持饲料(即普通饲料)的原料组成:玉米、豆粕、鱼粉、面粉、麸皮、食盐磷酸铵钙、石粉、多种维生素、多种微量元素、氨基酸等,营养成分如表1。
表1小鼠标准饲料和高脂饲料的组成表
1.2主要药品及试剂
穿心莲内酯(Andrographolide,AG):同试验例1.
2、实验方法
2.1动物分组
所有小鼠适应性喂养1周后按体重随机分组,分组如下:
正常(空白)对照组:10只C57BL/6J小鼠,普通饮食+生理盐水灌胃。
模型对照组:10只LDLR-/-小鼠,高胆固醇饮食+生理盐水灌胃。
穿心莲内酯高剂量组:10只LDLR-/-小鼠,高胆固醇饮食+穿心莲内酯(90mg/kg/d)灌胃。
穿心莲内酯中剂量组:10只LDLR-/-小鼠,高胆固醇饮食+穿心莲内酯(30mg/kg/d)灌胃。
穿心莲内酯低剂量组:10只LDLR-/-小鼠,高胆固醇饮食+穿心莲内酯(10mg/kg/d)灌胃。
每周使用小鼠体重计测定体重。AG配制使用研磨钵研细后使用生理盐水配成悬浊液,每次混匀后按体重给药,给药容积为10ml/kg。对照组及模型组每日灌服同容积的生理盐水。共造模及给药8周。所有小鼠均为自由摄食及摄水。
2.2取材
取材前禁食12小时以上,对小鼠进行异氟烷气体麻醉。沿腹中线剪开腹部及胸腔,注射器针尖插入左心室,同时剪开右心耳,用预冷的冰生理盐水灌流,完整取下两侧附睾脂肪,生理盐水中清洗血渍,滤纸吸干多余水分,称重。部分放入多聚甲醛固定液中待病理检测。其余组织保存于-80℃备用。
3、实验结果
3.1AG对小鼠体重的影响
比较各组小鼠灌胃前以及取材前称重,结果见表2。
表2LDLR-/-小鼠体重
注:与模型组比较,*P<0.05
3.2AG对小鼠附睾周围脂肪的影响
高脂饮食对小鼠脂肪组织影响较大,经6个月的高脂饮食喂养后,小鼠附睾周围脂肪重量明显增加。给予穿心莲内酯后,相比模型组,中、高剂量穿心莲内酯治疗组脂肪重量明显下降(P<0.05)。见表3
表3LDLR-/-小鼠附睾脂肪质量与体重比值
注:与模型组比较,*P<0.05
由上表2、3可见,穿心莲内酯组小鼠体重明显低于模型组,附睾脂肪比重降低,提示本品可能有调节机体脂肪分布的作用,减少脂肪重量的作用。
3.3甘油三脂检测
用贝克曼AU5822自动生化仪检测血清甘油三酯(TG)水平,结果如表4。
表4穿心莲内酯对LDLR-/-小鼠模型甘油三酯的影响
注:与模型组比较,*P<0.05
本发明的穿心莲内酯具有降低甘油三酯作用。可用于预防和治疗高甘油三脂血症以及控制继发心血管疾病等。
试验例4:穿心莲内酯对肥胖小鼠附睾UCP1蛋白的影响
1.实验材料
1.1实验动物及饲养
选取30只5月龄ICR雄性小鼠,体质量(43±3)g,购自北京维通利华实验动物有限公司(许可证号SCXK(京)2016-0002)。适应性喂养(SPF级)1周后开始实验。在广安门医院Ⅱ级动物房饲养,每笼2只,给予规律光照(昼夜各12小时),饮用无菌水,环境温度为20±2℃,相对湿度为45%-50%,良好的通风条件。
模型组及治疗组小鼠以高脂饲料喂养24周;对照组小鼠以普通饲料喂养24周;高脂饲料购于北京华阜康生物科技有限公司,普通饲料由斯贝福(北京)生物技术有限公司提供。
高脂饲料(又称西方饮食)、普通饲料的配方同试验例3。
1.2主要药品及试剂
穿心莲内酯(Andrographolide,AG):同试验例1。
重组Anti-UCP1抗体(ABCAM,ab234430);goat anti rabbit IgG(CW0103S,康为世纪);高效RIPA裂解液(Cat#R0010,索莱宝);BCA蛋白浓度测定试剂盒(PC0020-50T,索莱宝);PVDF(YA1701,索莱宝)。
1.3主要仪器
YB502精密电子天平(上海精密仪器仪表有限公司);3-18K高速冷冻离心机(Sigma,Germany);IX70光学显微镜(Olympus,Japan);-80℃超低温冰箱(Sanyo,Japan);BCD-190TMPK 4℃冰箱(中国海尔公司);Milli-Q超纯水净化装置(MerckMillipore,USA);SIM-F140AY65自动制冰机(Sanyo,Japan);BioRad转膜仪(Bio-Rad Laboratories,Inc.,USA)。
2.2.分组及干预方法
2.1动物分组
所有小鼠适应性喂养1周后按体重随机分组,分组如下:
正常(空白)对照组:10只ICR小鼠,普通饮食+生理盐水灌胃。
模型对照组:10只ICR小鼠,高胆固醇饮食+生理盐水灌胃。
穿心莲内酯组:10只ICR小鼠,高胆固醇饮食+穿心莲内酯(90mg/kg/d)灌胃。
2.2取材
同试验例3
2.3石蜡切片步骤
①固定:脂肪组织在4%多聚甲醛固定液中固定48h后,流水冲洗30min,脱水浸蜡;
②脱水:按照70%-80%-95%-100%-100%浓度乙醇各1h;
③透明:二甲苯I、II各20min;
④浸蜡:60℃石蜡I、II、III中各1h;
⑤石蜡包埋:包埋机包埋横切面;
⑥切片:包埋后的蜡块按4μm厚度切片,45℃水展片,充分展平切片后,捞到载玻片的中下1/3处,微晾干后,放入70℃恒温箱内烘片1h。
2.4免疫组化
2.4.1脱蜡、水化:将切片依次浸入二甲苯Ⅰ10min,二甲苯Ⅱ(10min)。再次浸入无水乙醇Ⅰ5min)-无水乙醇Ⅱ(5min)-95%酒精(5min)-80%酒精(5min)-70%酒精(5min),然后去离子水冲洗2次,每次2min。
2.4.2抗原修复:将组织切片放入修复盒,然后加入适量0.01M枸橼酸缓冲液(pH6.0)或EDTA修复液(pH 9.0),液面要浸没组织。微波中档修复10min(液体沸腾时开始计时),此过程中勿使组织干片。将修复盒从微波炉中拿出,自然冷却降温,当修复液降至室温后取出玻片,PBS(pH 7.4)冲洗3遍,每次3min(冲洗过程中切勿对着组织冲洗,以免弄破组织)。
2.4.3灭活:将配制好的3%的过氧化氢(去离子水稀释30%过氧化氢)滴加于切片组织上以阻断内源性过氧化物酶,室温孵育15min,PBS冲洗3次,每次3min。
2.4.4抗体孵育:吸水纸吸干PBS,在玻片上滴加5%的正常血清(与二抗种属来源一致或相似),37℃封闭30min。用吸水纸擦干玻片组织周围的液体,用油性笔在组织周围画圈,然后滴加稀释好的一抗,如果做阴性对照实验,就在对照组的组织上滴加PBS。加完一抗后于4℃湿盒中孵育过夜。(抗体的最佳稀释比应预先通过实验确定,时间控制在15h以上)。PBS冲洗切片3次,每次3min,吸水纸擦干切片后滴加辣根过氧化物酶标记二抗,37℃孵育30min。
2.4.5显色:PBS冲洗切片4次,每次3min,甩去PBS液体后吸水纸擦干切片,每张切片滴加新鲜配制的DAB显色液,显微镜下观察,阳性信号为棕黄色或棕褐色,时间要控制好,切勿显色过深。用自来水冲洗切片终止显色。Harris苏木素复染,一般30s-1min左右,水洗后用1%的盐酸酒精分化,再用自来水冲洗返蓝。
2.4.6脱水固定封片:将切片置于水中冲洗后,将切片依次放入:70%酒精-80%酒精-90%酒精-95%酒精-无水乙醇Ⅰ-无水乙醇Ⅱ-二甲苯Ⅰ-二甲苯Ⅱ中脱水透明,每个试剂中放置2min,最后在通风橱中风干切片。中性树胶滴在组织旁边,再用盖玻片盖上。先放平一侧,然后轻轻放下另一侧,以免产生气泡,封好的切片平躺置于通风橱中晾干。晾干的切片可以在显微镜下观察并采集图像。
2.5Western Blot
2.5.1脂肪组织蛋白的提取
在1ml的冷裂解液中各加入10ul的磷酸酶抑制剂、蛋白酶抑制剂和PMSF;混匀,放置在冰上备用。称取0.1g脂肪组织,放置于玻璃匀浆器的入口缓冲处,用眼科剪将组织块尽可能的剪碎,然后加入1ml的新配置的裂解液,冰上匀浆;将组织匀浆液移入1.5ml的EP管中,4℃12000g离心30min;吸取上清至新的管中;
2.5.2.BCA法测定蛋白浓度
配制BCA工作液:根据标准品和样品数量,按50体积BCA试剂加1体积Cu试剂(50:1)配制成BCA工作液,充分混匀。
稀释标准品:取10微升BSA标准品用PBS稀释至100微升(样品一般可用PBS稀释),使终浓度为0.5mg/ml。将标准品按0,2,4,6,8,12,16,20微升加到96孔板的蛋白标准品孔中,加PBS补足至20微升。
将样品作适当稀释,加20微升到96孔板的样品孔中。各孔加入200微升BCA工作液,37℃放置15-30分钟。用酶标仪测定A562nm,根据标准曲线计算出蛋白浓度。上清与5x上样缓冲液(含琉基乙醇)混匀后煮沸5分钟。
2.5.3.蛋白印迹
样品首先在80V恒压下电泳至染料接近分离胶顶端;然后12OV恒压电泳至溴酚蓝刚出胶底部;滤纸在转移缓冲液中至少浸泡30秒;pvdf膜浸入电转液中浸泡1分钟;从玻璃板上取下凝胶,去除所有浓缩胶;将凝胶浸入电转液中浸泡30分钟;按照泡沫、滤纸、凝胶、转印膜、滤纸、泡沫的顺序组装转移夹层;将转移夹放入转移槽中,凝胶侧面向阴极(-)而膜侧面向阳极(+);加预冷电转液,1OOV恒压转膜50min;lxTBST加入5%w/v封闭奶粉,4℃封闭过夜;洗膜液洗膜3-4次,每次5分钟;一抗为UCP-1抗体(1:1000稀释于1%BSA),室温振荡孵育膜lhr;洗膜液洗膜3-4次,每次5分钟;HRP标记的山羊抗兔lgG二抗,加入膜上,室温1小时振荡孵育;洗膜液洗膜3-4次,每次5分钟;以1:1比例混合eECL溶液A和溶液B,避光;将膜平铺在工作液上,至完全覆盖工作液,室温孵育5分钟;用镊子从工作液中取下膜(避免用手触碰),以塑料膜将膜封好,不留气泡,曝光显影;用β-actin做内参。
2.6数据分析
实验数据采用SPSS22.0软件包进行统计学分析,计量资料用均数±标准差表示:组间比较,符合正态分布时用单因素方差分析,p<0.05为差异有统计学意义;非正态分布时,采用非参数检验,p<0.05为差异有统计学意义。
3指标测定
3.1AG对小鼠体重的影响
比较各组小鼠灌胃前以及取材前称重,结果见表5。
表5ICR小鼠体重
注:与模型组比较,*P<0.05
3.2AG对小鼠附睾周围脂肪的影响
高脂饮食对小鼠脂肪组织影响较大,经24周的高脂饮食喂养后,小鼠附睾周围脂肪重量明显增加。给予穿心莲内酯后,与模型组比较,穿心莲内酯组脂肪重量明显下降(P<0.05)。见表6
表6ICR小鼠附睾脂肪质量与体重比值
注:与模型组比较,*P<0.05,与对照组比较,#p<0.05
由表5,6可知,穿心莲内酯可以明显减轻高脂饮食诱导的肥胖小鼠体重,减少内脏白色脂肪占比。
3.2穿心莲内酯对小鼠白色脂肪UCP1蛋白表达的影响
3.2.1免疫组化
附睾脂肪为内脏白色脂肪,已有研究证明高脂饮食会诱导小鼠附睾脂肪UCP1表达,镜下可见棕黄色颗粒出现于模型组及穿心莲内酯组白色脂肪细胞胞质中,穿心莲内酯组UCP1表达较模型组显著,观察结果如图6A-6C,图中穿心莲内酯对肥胖小鼠附睾白色脂肪组织UCP1免疫组织化学染色的影响(×200)。说明穿心莲内酯促进了小鼠白色脂肪棕色化,即穿心莲内酯能诱导并促进白色脂肪组织向褐色脂肪组织褐变。
3.2.2Western blot
用Western blot法对小鼠附睾脂肪UCP1蛋白表达量进行检测。检测结果如图7,检查结果表明穿心莲内酯组UCP1蛋白表达量高于模型组。
穿心莲内酯激动PPARα后可直接激活白色脂肪组织中UCP-1表达,促进脂肪的褐变。穿心莲内酯作为PPARα的激动剂,可通过降低肥胖小鼠体重、血清中甘油三酯和白色脂肪重量/体重比值,减少白色脂肪堆积,同时增加白色脂肪组织UCP1蛋白的表达,促进肥胖小鼠白色脂肪棕色化,增加热量消耗,调节脂肪分布。
Claims (10)
1.穿心莲内酯在制备促进白色脂肪棕色化的产品中的应用,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
2.穿心莲内酯在制备用于促进PPARα活性的的药物、食品、保健品、食品添加剂或营养补充剂中的应用。
3.穿心莲内酯在制备用于预防或治疗肥胖症的产品中的应用,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
4.根据权利要求1-3任一所述的应用,其特征是,所述药物由穿心莲内酯和药学上可接受的载体组成。
5.根据权利要求1-3任一所述的应用,其特征是所述药物以片剂、胶囊剂、丸剂、散剂、颗粒剂、糖浆剂、溶液剂或乳剂形式存在。
6.穿心莲内酯作为PPARα激活剂的应用。
7.一种促进白色脂肪棕色化的产品,其特征是,含有穿心莲内酯,其中所述产品为药物、制剂、食品、保健品、食品添加剂或营养补充剂。
8.根据权利要求7所述的产品,其特征是,所述穿心莲内酯的重量与所述产品的总重量之比为0.01~10:100。
9.一种促进白色脂肪棕色化的方法,其特征是,包括给予有效量的穿心莲内酯或其药学上可接受的盐。
10.根据权利要求9所述的方法,其特征是所述穿心莲内酯的含量为1%~98%。
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